Mesenchymal Stem Cell in Veterinary Sciences: Mudasir Bashir Gugjoo Amar Pal Editors
Mesenchymal Stem Cell in Veterinary Sciences: Mudasir Bashir Gugjoo Amar Pal Editors
Bashir Gugjoo
Amar Pal Editors
Mesenchymal
Stem Cell in
Veterinary
Sciences
Mesenchymal Stem Cell in Veterinary
Sciences
Mudasir Bashir Gugjoo • Amar Pal
Editors
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore
Pte Ltd. 2020
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Preface
Tissues and/or organs are constituted by building blocks, the cells. These cells are
either undifferentiated stem cells or differentiated tissue-specific cells. The minis-
cule number of available undifferentiated stem cells act as a reserve to maintain
normal cell turn-over and thereby, tissue homeostasis. The differentiated cells per-
form the tissue-specific function. For a substantial and effective cellular therapy, the
cells have to be sufficient in quantity and compatible with the recipient immune
system, besides expressing as per the tissue requirements. The differentiated cells
taken from the particular tissue source often are limited in number, have little or no
proliferation potential and elicit the immune response in the recipient upon alloge-
neic or xenogeneic transplantation. Contrarily, the stem cells especially mesenchy-
mal stem cells (MSCs) can be harvested from almost all the body tissue sources
including the foetal membranes and have good proliferation and trans-differentiation
potential. MSCs tend to have little tendency to elicit an immune response in the
recipient even upon allogeneic or xenogeneic applications. These cells have mini-
mal teratogenic and ethical issues normally associated with the pluripotent stem
cells. However, MSCs unlike that of pluripotent stem cells tend to have limited
proliferation potential.
The advancement of stem cell biology is increasing by each passing day and has
found its way into the clinical applications under regenerative medicine, a branch of
tissue engineering. Due to the limited understanding in the cellular characteristics,
ipso facto MSCs therapeutic applications currently come under the advanced ther-
apy medicinal products (ATMPs). The role of microenvironment in cellular fate
remains to be understood. Such an understanding shall aid in their definitive utiliza-
tion in regenerative medicine.
There are many books that discuss stem cells and their features and have invari-
ably focused on their applications in humans. However, the book on stem cells
especially mesenchymal stem cells and their applications in veterinary species is
very much lacking. The pressing need for a book that provides a comprehensive
introduction to the field and summarizes its recent progress in veterinary sciences
has given rise to this book. This current work discusses the mesenchymal stem cell
features and their applications in veterinary species. The book starts with introduc-
tion to the stem cells. In the introductory chapters, the stem cell properties and types
are discussed followed by the details of the mesenchymal stem cell sources, isola-
tion, culturing and characterization, and preservation. This is followed by a chapter
v
vi Preface
on the effect of different cues on mesenchymal stem cells properties. Thereafter, the
species-specific mesenchymal stem cell features including the complexities in their
characteristics, besides, their potential applications have been discussed. Finally,
the book is concluded with the summary and future research avenues of MSCs.
We thank all the contributors for their hard work and valuable contributions.
With all the humility I thank almighty “Allah” the most Beneficent, the most
Merciful, who blessed me with limitless strength and favourable circumstances, to
face and pass through all odds successfully at this juncture.
I feel a great sense of elation and fulfilment to submit this tiny drop of work in
the ocean of human knowledge to benefit the differently abled creatures surround-
ing and helping us in many ways. The completion of this work would not have been
possible without the help and support of many people.
I would like to appreciate the contribution of all those teachers from childhood
till now who played an important role in my life to reach this place without which
the realization of the present work into practice would have been a dream. Apart
from the teaching faculty, schools/institutes that I attended like Miranda Public
High School, Srinagar, SP Higher Secondary School, Srinagar, SKUAST-Jammu
and Indian Veterinary Research Institute, Izatnagar, during the course have been the
ocean that taught me to swim in all the spheres of life and reach to the stage. A spe-
cial thanks go to all the stem cell scientists who burn themselves and provide the
literature that makes the material in the book.
I am highly thankful to Science and Engineering Research Board (SERB),
Government of India that provided the financial support and infused zeal in me to
conduct the stem cell research and come out with this piece. I really feel elated to
convey my gratitude to the SKUAST-Kashmir under which I am currently working.
This institute has provided me the platform to express my potential to the best of my
capacities. The faculty members of the institute have provided great company
through the journey.
Literary skills grope in the dark when it comes to express what I owe to my fam-
ily. These mean achievements to which I lay claim today have all precipitated from
the sincere prayers and silent sacrifices of my Pappa and Mummy. The blessings,
love, guidance and affection provided by my sister and brother-in-law deserve a
special mention. I admit myself being incapable to pay regards in words to the affec-
tion, blessings, encouragement and dedication of my wife that made many difficult
tasks easier for me. In recent times, the happiest moment and encouragement of my
life has probably been my daughter.
I am highly thankful to Dr Bhavik Sawhney, Editor-Biomedicine, Springer
Nature for his guidance and timely help in giving final shape to this book. I admit
the affections and encouragement provided to me by my extended family. This list
vii
viii Acknowledgement
is obviously incomplete but allows me to submit that the omissions are inadvertent
and I once again record my deep-felt gratitude to all those associated with me in this
endeavour.
Finally, I supplicate to Allah (SWT), the One who listens and responds to sup-
plications, “Oh Allah, guide me to the right path, the path of those on whom You
have bestowed Your favors, not of those who have incurred Your wrath, nor of those
who have gone astray and let not me die except in the state of pure submission to
You alone”. Aameen.
About the Book
During the last few years, a large volume of information has been generated on stem
cell types, their biological nature, in vitro and in vivo behaviour and potential appli-
cation in research and therapy. This book makes a systematic collection of the sci-
entific information scattered in the literature on different aspects of stem cell
research. The focus of the book is on mesenchymal stem cells as they have been
investigated most extensively for therapeutic application in animals. The contents of
the book have been designed to include all the relevant information about the mes-
enchymal stem cells of animal origin. The initial chapter covers the historical aspect
and chronology of developments in the field of stem cell research. The details about
the basic properties and nature of the stem cells are provided. A comprehensive
account of the materials and methods used for isolation of mesenchymal stem cells
from different sources, culture expansion, characterization and long-term storage
has been given in the subsequent chapters. The book also provides a logical appraisal
of the mechanisms involved in therapeutic efficacy, immunomodulation and anti-
inflammatory properties of mesenchymal stem cells. The book also highlights
knowledge about the interaction between the stem cells and their environment and
factors responsible for the maintenance of stemness. A chapter in the book has been
dedicated to genetic engineering relevant to stem cell research for their better
exploitation for research and therapy in animals. The major section of the book is
devoted to the recount of the research efforts and their outcomes towards the poten-
tial application of mesenchymal stem cells in regenerative research and therapy in
important species of domestic and pet animals including sheep, goat, cattle, buffalo,
cat, dog and horse. The book also presents an abridgement of challenges and future
prospects of stem cell research and application in medicine in general and veteri-
nary sciences in particular.
ix
Contents
xi
xii Contents
xiii
xiv Editors and Contributors
international journals and more than 380 abstracts in seminar and conference pro-
ceedings. He has also authored two books and contributed several book chapters.
Dr Amarpal is Editor-in-Chief of the Indian Journal of Veterinary Surgery and
Associate Editor of the Indian Journal of Canine Practice, and is a referee for
numerous international journals.
Contributors
xv
xvi Abbreviations
Abstract
The stem cell concept is as old as the understanding of the origin of life. The
concept of origin of life has developed from initial ‘spontaneous generation’ to
the current concept of development through ‘zygote formation’. Understanding
the concept of the development of life has paved way for the ‘stem cell’ concept.
There are various types of stem cells that develop right from early phase of life
to the adult life though with variable potentialities. An overview of the develop-
ment of stem cell concept has been attempted in the current chapter.
1.1 Introduction
The first and foremost attempt to solve mystery around the origin of life and of
human development may be attributed to Aristotle (384–322 BC) (Arey 1974). His
observation made him to believe that embryonic development occurs in the uterus
and that the mother’s menstrual blood is the basis, supporting then previous hypoth-
esis of ‘spontaneous generation’. Until 1800, the concept behind development of an
individual was of a pre-formed individual who exists in the egg or sperm, known as
homunculus. The homunculus (egg), however, requires an activation to form an
individual, which occurs through sperm. The sperm, thus, stimulates homunculus in
the uterus that ultimately leads to the formation of an individual. Leydig later in
1855 hypothesized that life originates from an already existing life, omne vivum
ex vivo. This hypothesis was extended by Rudolf Virchow with a view that all new
cells develop from existing cells, omnis cellula e cellula (Oberling 1944). Later on
M. B. Gugjoo (*)
Division of Veterinary Clinical Complex, FVSc & AH, SKUAST-Kashmir,
Jammu and Kashmir, India
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 1
Springer Nature Singapore Pte Ltd. 2020
M. B. Gugjoo, A. Pal (eds.), Mesenchymal Stem Cell in Veterinary Sciences,
https://doi.org/10.1007/978-981-15-6037-8_1
2 M. B. Gugjoo
the concept of fertilization was developed, wherein an egg after fusion with the
sperm leads to the formation of zygote (Needham 1959). The zygote forms the
actual basis of life as it gives rise to all the cells required together to make an indi-
vidual (totipotency) (Sell 2004a, b). Thus, the existence of cell, now popularly
known as ‘stem cell’, carries the potential to develop into the complete individual.
The term ‘stamzelle or stem cell’ was initially used by a German biologist Ernest
Haeckel in 1868 for a fertilized egg that develops into a complete organism (Reimer
1868). The term was also used for the single-celled organism that is considered as
an ancestor to all the living organisms. The stem cell research actually started with
the process of learning the basis of development and regeneration. In 1930, nuclear
transplantation was first carried out in Amoeba (Comandon and deFonbrune 1930).
In 1950s Briggs and King applied the same technique to replace nucleus of a frog’s
egg with other cell (Briggs and King 1952). Gurdon later cloned a frog from an egg
and the nucleus of small intestine (Gurdon 1974). The experiments confirmed that
the developmental changes that occur are through the environmental factors rather
than the permanent changes to the genetic code and in the present case factors were
present in oocyte of the frog (Bio-Rad Lab Inc.n.d.). This somatic cell nuclear trans-
fer (SCNT) technique, however, proved to be comparably harder in mammals, and
for long time the mammalian cells were considered to be far specialized and, thus,
cannot be reverted to totipotent state. But as the research in the field advanced, the
findings were proved to be wrong and different mammalian clones were developed.
The most noted one was in the form of Dolly, developed by fusion of mammary
gland epithelial cell with the enucleated oocyte (Wilmut et al. 1997), besides, the
others (Forsberg et al. 2002; Gao et al. 2003; Walker et al. 2002). However, the
frequency of successful transplants appears to be very less, <1–2% (Sell 2004a, b),
giving rise to the question, why? Thus, not all adult cells are capable to give rise to
successful transplants and the cell that give rise could actually be the ‘stem cell’ or
‘tissue progenitor cell’ (Van der Kooy and Weiss 2000). In 1945 the initial experi-
mental basis of the stem cell was recognized (Owen 1945). Since then stem cell
properties are being discussed (Lajtha et al. 1964; Lajtha 1967) with in vivo stem
cell governing factors yet to be fully comprehended. The historical aspect of various
stem cell types is briefed below.
The blood stem cell concept was first introduced in Berlin Haematological Society
by Russian academician Alexander Maximow on 1 June 1909. He delivered a theo-
retical lecture that all the blood cells come from same ancestral cell and, thus, intro-
duced the concept of multipotent nature of blood stem cell (Kalra and Tomar 2014).
The stem cell search in blood had begun after the bombings in Hiroshima and
1 Introduction to Stem Cells 3
Nagasaki in 1945 when radiation exposure led to the haematopoietic failure in resi-
dents. It was found that the shielding of the spleen with lead (Jacobson et al. 1950)
and transplantation of bone marrow or spleen cells (Lorenz et al. 1951; Jacobson
et al. 1951) prevent such a failure. However, it was confirmed later in 1963 when
Ernest McCulloch and James Till (Canadian scientists) carried out experiments on
mice bone marrow. It was demonstrated that various blood cells originate from a
specific class of cells bearing self-renewal and differentiation properties (Till et al.
1964; Till and McCulloch 1961). Even before actual laboratory confirmation of
blood stem cell, the human bone marrow transplant was first attempted by E. Donnall
Thomas in 1957 that later fetched him Nobel prize in 1990. The first successful
transplant of bone marrow, however, was conducted by Robert A. Good in 1968. He
transplanted bone marrow of a sibling to an immune-deficient child who later devel-
oped into a healthy adult (Thomson et al. 1998a, b). Since then the blood transfusion
has become a common procedure in case of leukaemia patients.
The embryonic stem cell understanding has started with the development of sponta-
neous testicular tumour in a terato-carcinoma study. In 1970s, it was observed that
such spontaneous tumours, composed of differentiated cell types and undifferenti-
ated stem cell, may be produced following ectopic grafting of these tumour cells in
the adults (Solter et al. 1970; Stevens 1970; Stevens 1983; Papaioannou and Rossant
1983). Those undifferentiated stem cells (embryonal carcinoma cells) were corre-
lated to the pluripotent stem cells of an early embryo. This paved the way to harvest
stem cells directly from embryos. It was in 1981 when independent researchers,
Marlin Evans and Kaufmann of University of Cambridge and Garl Martin of
University of California, could successfully derive embryonic stem cells (ESCs)
from mouse blastocysts (Evans and Kaufman 1981). Trophoectoderm, however,
was demonstrated to harbour stem cells with limited differentiation potential called
trophoblast stem cells (Tanaka et al. 1998). ESCs could grow on a feeder layer
formed by mitotically inactivated mouse embryonic fibroblast cells. Later on ESCs
were also derived from primates (Thomson et al. 1995, 1996). All these studies
along with the research conducted on culturing human in vitro fertilized (IVF)
embryos (Gardner et al. 1998) paved the way for successful cultivation and mainte-
nance of ESCs into an undifferentiated state (Thomson et al. 1998a, b). It was dem-
onstrated that feeder cell layer for mouse ESCs could be employed for human
counterparts, but variability in biochemical pathways and humoral factors exist. In
the same year, cultured human ESCs were differentiated into different tissues, viz.
gut epithelium, cartilage, bone, muscle, neurons, etc. (Thomson et al. 1998a, b;
Shamblott et al. 1998). Considering these factors ESC line maintenance and ability
to trans-differentiate has led to the tremendous research in the field especially in
relation to the development and repair of damaged or lost tissue (Donovan and
Gearhart 2001). Attempts to create ESCs from veterinary species like cattle, sheep,
dog and others too started immediately afterwards (Telugu et al. 2010). Due to the
4 M. B. Gugjoo
The presence of mesenchymal stem cells within the bone marrow may be docu-
mented from the work of Goujon (1869). An osteogenic potential of bone marrow
was demonstrated after its heterotopic transplantation. The results were confirmed in
the transplantation experiments by Biakow (1870). Later on the osteogenesis was
shown to be due to the whole bone marrow and not by the chemo-attractants (Danis
1960). These experiments were also conducted by other researchers who demon-
strated the same results and found that bone and cartilage formation does occur
(Tavassoli and Crosby 1968; Friedenstein et al. 1966; Bruder and Caplan 1990).
Friedenstein and co-workers observed that the heterotopic bone formation post-bone
marrow cells transplantation was associated with minor subpopulation of such cells
that can be differentiated from haematopoietic cells based on their specific features,
viz. plastic adherence and fibroblast-like appearance. The fibroblast-like appearance
indicated their origin from the stromal compartment of the bone marrow. The same
authors also observed that seeding of bone marrow cell suspension had resulted in
the establishment of discrete colonies. These colonies being initiated by single cell
are known as colony-forming unit fibroblastic (CFU–Fs) (Friedenstein et al. 1970).
The concept of stem cells in bone marrow was formally presented by Owen in
1978 (Owen 1978). It was hypothesized that bone marrow harbours a lineage that is
1 Introduction to Stem Cells 5
analogous to haematopoietic lineage (Till and McCulloch 1980). The in vivo exper-
imental transplantation of such cells had resulted in the formation of numerous
skeletal tissues, viz. bone and cartilage. The cells were named as osteogenic stem
cell (Friedenstein et al. 1987) or bone marrow stromal stem cell (Owen and
Friedenstein 1988). The non-haematopoetic stem cell hypothesis though well-
established previously, its importance, however, was felt in 1999 when similar work
was published by a commercial company, Osiris Therapeutics Inc. (Pittenger
et al. 1999).
The term ‘mesenchymal stem cell’ has been coined by Caplan who showed that
these cell types carry the potential to differentiate into other phenotypes including
adipogenic, tenogenic, ligamentous, dermal or others (Caplan 1991, 1994). The term
‘mesenchymal stem cell’ although widely used has restricted acceptability (Sipp
et al. 2018) and shall henceforth be called by that name in this chapter. The stem cell
has ‘stemness’ characteristics depicted by ‘self-renewal and differentiation’ proper-
ties. Mesenchymal stem cell (MSC) most often undergoes culture-dependent senes-
cence (Wagner et al. 2009), which also diminishes its ability to differentiate (Bonab
et al. 2006; Lo Surdo et al. 2013). It is also unclear whether ectopically placed MSCs
can actually differentiate in vivo or not. The stem cell label is, thus, questionable with
respect to MSCs (Bianco 2014). As such these cells are also termed as stromal stem
cells (Owen and Friedenstein 1988), mesenchymal progenitor cells (Dennis et al.
1999), skeletal stem cells (Bianco and Gehron 2000), multipotent adult progenitor
cells and mesodermal progenitor cells (Jiang et al. 2002). In 2006, International
Society for Cellular Therapy that gave specific guidelines for characterization of
these cells recommended the name to be given as ‘Multipotent Mesenchymal Stromal
Cells’ (Dominici et al. 2006). The latest work of Caplan, however, recommends these
cells to be named as “Medicinal Signaling Cells” based on their ability to modulate
host systems. It may, thus, be pointed out that more emphasis on the mechanism of
their therapeutic effects may be laid (Caplan 2017).
A huge literature has been piled on MSCs including their properties, isolation pro-
tocols and their applications in diverse ailments both under preclinical experimental
studies and in the clinical trials. In veterinary science, MSCs have been harvested
from numerous adult tissue sources and foetal membranes (Gugjoo and Amarpal
2018; Gugjoo et al. 2018a, 2019). Mesenchymal stem cells (MSCs) have also been
harvested from iPSCs (Chow et al. 2017; Xu et al. 2019). Currently, there are wide
variations in their tri-lineage differentiation potential, especially in relation to a
marked reduction in their chondrogenic and adipogenic lineages as compared to the
bone marrow-derived (BM) MSCs (Xu et al. 2019). MSCs in veterinary sciences
like human are characterized as per the criteria laid down by International Society
for Cellular Therapy (ISCT) (Dominici et al. 2006). The early mesenchymal stem
cell in vivo applications had employed non-specific cell fractions. A heterogeneous
cell fraction containing tissue cells, leukocytes, endothelial cells, fibroblasts and a
small subset of self-renewing multipotent cells had been utilized (Tiwary et al.
2014). However in recent time culture-expanded MSC preparations are being
increasingly studied containing good number of MSCs. Further, advancement like
specific cell selection methods need to be studied to make roadways to a more
6 M. B. Gugjoo
purified cell preparation (Devireddy et al. 2017). The cells are even being cryopre-
served for their future applications (Gugjoo and Amarpal 2018; Gugjoo et al. 2018a,
2019). Among various veterinary species, MSC clinical studies have been con-
ducted mainly in dog, cat and horse (Quimby and Borjesson 2018; Gugjoo et al.
2019a, b). In horse, the therapeutic applications of stem cells have mainly been
aimed at the issues of musculoskeletal system. An initial attempt was made to repair
the superficial digital flexor tendon (Smith et al. 2003). In sheep and goat MSCs,
in vivo experimental evaluations have been conducted mostly as human transla-
tional studies (Gugjoo and Amarpal 2018; Gugjoo et al. 2020). Cattle and buffalo
MSCs have been evaluated in experimental studies mainly to address reproductive
problems (Gugjoo et al. 2018b). It may be noted that all MSC studies have so far
failed to provide any conclusive results.
The cancer stem cell (CSC) concept was initially given by Cohnheim and Durante
about one-and-a-half century back. The proliferation and differentiation potential of
CSC was demonstrated to be comparable to that of the embryonic tissues and can-
cers. Thus, a concept was proposed that embryonic cells present in adults upon
stimulation may lead to cancer. More curiosity in this concept was developed with
the work of Furth and co-workers (Furth et al. 1937). It was demonstrated that acute
myeloid leukaemia (AML) develops in a mouse after transplantation of single
undifferentiated leukaemia cell (Furth et al. 1937; Al-Hajj et al. 2003). After a long
gap of three decades, in vitro studies supported AML cells’ ability to self-renew,
proliferate and generate new tumours (Sell 2004a, b). Initially, such studies were
performed on tumours in liquid (blood) phase. The first evidence of these stem cells
in solid tumours came in 2003 and was reported from breast cancer (Al-Hajj et al.
2003). Thereafter, CSC concept was validated in a wide variety of solid tumours
including lung cancer (Kim et al. 2005), melanoma (Fang et al. 2005), prostate can-
cer (Collins et al. 2005), brain cancer (Piccirillo et al. 2006), colon cancer (O’Brien
et al. 2006) and ovarian cancer (Szotek et al. 2006). The oncogenic therapy on the
basis of CSC concept is also currently being researched extensively (Gugjoo et al.
2015; Atashzar et al. 2020).
1.3 Conclusion
The stem cell concept dates back to the origin of life. The concept of development
of life has changed from initial ‘spontaneous generation’ to the current concept of
development through ‘zygote formation’. Understanding the development of life
has itself given rise to stem cell concept. Various stem cell types including MSCs
have been identified with variable differentiation potential. Overall stem cell con-
cept is still in its infancy, and there are lot of intricacies involved. Among these cell
types, mostly MSCs are being evaluated in clinical trials. The stem cell research
1 Introduction to Stem Cells 7
though is being conducted at a very rapid pace; however, the understanding is yet to
reach to the level that can harvest the fruitful results.
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Mesenchymal Stem Cell and Its
Properties 2
M. B. Gugjoo, Amar Pal, and G. T. Sharma
Abstract
Stem cell (SC) concept is one of the important research areas seen to bring new
vistas in the biological science and/or medicine. Various stem cell types have
been identified including mesenchymal stem cells (MSCs). MSCs are present in
almost all the body parts of an individual and carry the specialized stemness
property, in addition to the immuno-modulation, plasticity and homing. These
cells are studied for their therapeutic applications due to lack of any ethical issue
together with a limited ability to develop tumour. However, there is dearth of
understanding in their basic specialized physiological properties and as such
definitive clinical applications are restricted. The current chapter details various
stem cell types and discusses the characteristic properties of the stem cells with
special focus on MSCs.
2.1 Introduction
The stem cell (SC) concept is one of the most thrilling scientific research areas in
twenty-first century. Stem cell research is seen and hoped to bring new dawn in the
field of biology and medicine. An individual in their lifespan undergoes through
M. B. Gugjoo
Division of Veterinary Clinical Complex, FVSc & AH, SKUAST-Kashmir,
Jammu and Kashmir, India
A. Pal
Division of Surgery, Indian Veterinary Research Institute, Izatnagar, UP, India
G. T. Sharma (*)
Division of Physiology & Climatology, Indian Veterinary Research Institute,
Izatnagar, UP, India
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 13
Springer Nature Singapore Pte Ltd. 2020
M. B. Gugjoo, A. Pal (eds.), Mesenchymal Stem Cell in Veterinary Sciences,
https://doi.org/10.1007/978-981-15-6037-8_2
14 M. B. Gugjoo et al.
Stem cells are classified in many ways but most commonly based upon the degree
of differentiation and source of isolation.
Totipotent Cell The cell differentiates into any cell type of the body including the
foetal membrane like zygote up to morula stage of the embryogenesis.
Pluripotent Cell The cell differentiates into all the cell types of the body barring
foetal membranes like embryonic stem cells (ESCs) and induced pluripotent stem
cells (iPSCs).
Multipotent Cell The cell gives rise to limited cell types of the body, e.g. mesen-
chymal stem cells (MSCs).
2 Mesenchymal Stem Cell and Its Properties 15
Embryonic Stem Cell The cell is derived from an embryo and may be totipotent
(cells up to the morula stage of embryogenesis) or pluripotent (inner cell mass of the
blastocyst of embryo).
Adult Stem Cell It is an unspecialized cell present in all the tissues/organs of the
body for internal repair or replacement of damaged cells. The self-renewal and the
differentiation capacity are restricted in these cells as compared to pluripotent cells.
Among various stem cell types, MSCs currently form an important constituent of
the regenerative medicine. This can be attributed to their diverse and numerous
accessible sources, and the simplicity of isolation procedures with better success
rates. These cells have minimal teratogenicity usually seen with the pluripotent
stem cells and bear limited ethical issues commonly associated with human ESCs
(Zuk et al. 2001; Cardoso et al. 2017). Additional features like ability to immuno-
modulate and provide anti-inflammatory response, thereby regeneration of tissue
without scar formation, make it more suitable for regenerative medicine (Zuk et al.
2001; Stewart and Stewart 2011; Gugjoo et al. 2019). However, MSCs in contrary
to ESCs have limited culture lifespan, and after certain culture passages these cells
become senescent (Caplan 2012). Even upon in vivo implantation, MSCs (autolo-
gous/allogeneic) survive only for certain period (ranging from few hours to as long
as 90 days) but not indefinitely (Guest et al. 2010; McDuffee et al. 2012). This ques-
tions their self-renewal property (Lopez and Jarazo 2015). In order to extend their
lifespan, vector viruses like simian virus 40 (SV40-T) and human papillomavirus
have been used to extend their culture lifespan (Klingelhutz et al. 1994). The
SV40-T retrovirus may immortalize dog adipose tissue-derived MSCs (Ayala-
Cuellar et al. 2018) by reducing the growth inhibition on retinoblastoma protein
(pRb) and tumour protein (p53) (DeCaprio et al. 1988; Pipas 2009). These are pre-
liminary studies that need further extensive research in the area to confirm their
clinical safety and efficacy.
2.4.1.1 Stemness
The prime stemness property, represented by self-renewal and differentiation, of
MSCs is poorly understood and has even been questioned. Only few genes support
16 M. B. Gugjoo et al.
MSCs stemness markers. There have to be deep insights into its molecular basis
especially through the main transcription factors. So far, not a single key transcrip-
tion factor to MSCs has been determined unlike pluripotent genes like Oct4, Nanog
and Sox2 being attributed to the embryonic stem cells. Several highly expressed
genes including nine transcription factors in undifferentiated MSCs have been
detected after comparing undifferentiated and tri-lineage-differentiated MSCs.
However, individual knockdown of such genes could only partially decrease the cell
proliferation or their differentiation (Kubo et al. 2009). Several reasons that may be
underlined to such poor understanding are as follows:
1. The cellular heterogeneity with respect to the species, age, sources, culture con-
ditions and number of culture passages (Mareddy et al. 2010; Menicanin
et al. 2010).
2. MSCs have finite culture lifespan characterized by ageing and senescence after
certain passages. MSCs exhibit morphological abnormalities, attenuated expres-
sion of specific surface markers, reduced differentiation potential and prolifera-
tion arrest (Baxter et al. 2004; Wagner et al. 2008; Liu et al. 2013).
3. Poor understanding of microenvironment/niche that controls MSCs features.
There is need to study the environmental effects. Among various niche cues stud-
ied include hypoxia (Drela et al. 2014; Hu et al. 2014; Fotia et al. 2015), avail-
able extra-cellular matrix (ECM) (Xiong et al. 2015; Wong et al. 2017), scaffolds
(Cesarz and Tamama 2016; Obara et al. 2016), mechanical factors and/ humoral/
growth factors (Terraciano et al. 2007; Bottagisio et al. 2015; Hu et al. 2015).
Such cues, however, have been studied in isolation while in vivo microenviron-
ment/niche presents an intricate system.
Self-Renewal and Differentiation
The self-renewal property keeps the stem cell pool intact, which makes it possible
to repair the damaged cells or to replace the non-functioning cells throughout an
individual’s life (Fig. 2.1). This feature of stem cell differentiates it from progenitor
cell as the latter cell although able to differentiate but is unable to self-renew. Stem
cell self-renewal occurs through either symmetric or asymmetric divisions. Through
symmetric divisions, the parent cell is able to produce identical copies, that is,
daughter cell copies entire genome including the epigenetic changes. In asymmetric
divisions, two variable cells are produced either through divisional asymmetry or
environmental asymmetry (Wilson and Trumpp 2006; He et al. 2009). In divisional
asymmetry, cell fate determinants get reorganized prior to division and after divi-
sion two cells are produced, one identical and other non-identical to the parent cell.
In environmental asymmetry, the cell gets exposed to multiple signals from the local
environment (micro-environment), which initially leads to symmetric division pro-
ducing identical copies. One of the daughter stem cell then undergoes asymmetric
cell division generating non-identical cells to perform tissue-specific function
(Spradling et al. 2001; Ohlstein et al. 2004). This process is known as differentiation
and is governed by available micro-environment. The growth factors and cytokines
like IGF-1, TGF-β1, BMP-1, etc. may facilitate stem cell proliferation or inhibition
2 Mesenchymal Stem Cell and Its Properties 17
Fig. 2.1 Diagrammatic representation of development of mesenchymal stem cells (MSCs), their
self-renewal, differentiation, and mobilization and homing properties
depending upon the tissue specific requirements (Fuchs et al. 2004; Potten et al.
2009; Spees et al. 2016). The microenvironment/niche tends to play an essential
role in maintaining the stem cell self-renewal property. The cells lose their self-
renewal potential upon loss of the niche (Xie and Spradling 2000; Mackenzie and
Flake 2001; Li and Neaves 2006; Lilly et al. 2011).
The self-renewal potential varies among stem cell types. In culture environment,
embryonic stem cells are able to self-renew almost indefinitely while MSCs self-
renew only for few generations. The mechanism behind this wonderful property is
still ambiguous though some information has been gained in recent times. One of
the mechanisms reported is of telomerase, an enzyme required to maintain DNA
regions at the chromosomal ends, telomeres, by preventing accumulation of DNA
damage due to replication (Greenwood and Lansdorp 2003). It has been seen that
telomerase absence decreases self-renewal capacity of haematopoietic stem cells
(HSCs) while forced expression is insufficient to transplant such cells indefinitely
(Allsopp et al. 2003a, b). The cells that are able to express elements of different
signalling pathways like Wnt/ fzd/beta-catenin, sonic, Hedgehog, etc. usually
undergo self-renewal divisions (Watt and Hogan 2000; Willert et al. 2003). Besides,
over-expression of several proteins like homeobox 4 genes (Hoxb4) and Cdx4
(Kyba et al. 2002; Wang et al. 2005) may also favour self-renewal divisions. All
these signalling pathways maintain pleuripotency of the cells and may not be appli-
cable to unipotent or oligopotent cells.
18 M. B. Gugjoo et al.
2.4.2 Plasticity
Differentiation capability of stem cell type varies. Embryonic stem cell is able to
give rise to all the cell types of the three germinal layers while adult stem cells usu-
ally have restricted potential. Recent studies have shown an extended differentiation
potential of adult stem cells. Mesenchymal stem cells (MSCs) differentiation poten-
tial earlier restricted to osteogenic, chondrogenic and adipogenic lineages (Gugjoo
et al. 2015) has now been demonstrated to extend to other lineages like myocyte-/
cardiomyocyte-like cells (Orlic et al. 2001; Ferrari et al. 1998; Vieira et al. 2010;
Wang et al. 2018), neural-like cells (Oh et al. 2011; Ryu et al. 2012; Yan et al. 2015;
Kriston-Pál et al. 2017; Li et al. 2017; Zhang et al. 2019) and germ cell-like cells
(Ghasemzadeh-Hasankolaei et al. 2014, 2015), among others. This phenomenon is
known as trans-differentiation. As such the stem cell fate is not fixed rather change-
able or plastic (plasticity) in nature depending upon the available local microenvi-
ronment (Pelagalli et al. 2018).
Tissue-specific cells continuously undergo the process of wear and tear and ulti-
mately die. The cells if not replaced may lead to malfunctioning of the tissue system
and finally death. Such damaged cells are, thus, being replaced by an internal repair
system formed by the stem cells. However, in extensive cases an intrinsic system
fails to maintain such homeostasis. In an organ failure case (kidney, heart failure),
donors are required who are hardly available that too at right time. Besides, some
highly specialized tissues like nervous tissue have very limited internal repair sys-
tem, which demands the medical support for damage repair. Currently, stem cells
are seen to carry the potential to fill this gap in medical field. Further, stem cell dif-
ferentiation if controlled can be used to produce specific tissue/organs. Such tissue/
organs can be used as disease models to study the course of disease and the thera-
peutic options.
2.6 Conclusion
MSCs are important stem cell types that are considered to provide therapeutic
effects in regenerative medicine through self-renewal, differentiation, immuno-
modulation, migration and homing. However, there are various queries that need to
be addressed before stem cell application can be made successful.
1 . How do stem cells proliferate without differentiation while non-stem cells do not?
2. What are the in vivo factors and how do they regulate stem cell self-renewal,
proliferation and differentiation?
3. Why pluripotent stem cells are almost immortalized while adult stem cells like
MSCs have limited lifespan?
4. How immuno-modulation, anti-inflammatory and homing properties of MSCs
can be controlled?
The answers if discovered may pave the way for the following potential uses of
stem cells:
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Mesenchymal Stem Cell Isolation,
Culture, Characterization 3
and Cryopreservation
Abstract
The characteristic features of mesenchymal stem cells (MSCs) have made them
an important subject of the regenerative medicine. These cells are being har-
vested, isolated and characterized from numerous adult body tissues/organs, in
addition to the foetal membranes. The cells in donor tissues are present in a very
limited and variable concentrations making it imperative to culture expand them
for effective utilization. The variations in tissue types and/in culture techniques
of the cells make it difficult to understand their features and clinical applications.
For ready to use application, the cells are being banked, mostly through cryo-
preservation. The details of isolation, culture and characterization, in addition to
cryopreservation, of MSCs are detailed in this topic.
3.1 Introduction
Initially, mesenchymal stem cells (MSCs) were identified by Friedenstein and co-
workers from bone marrow. The typical fibroblast-like morphology and ability to
adhere to the plastic were basis of their characterization (Friedenstein et al. 1976;
M. B. Gugjoo (*)
Division of Veterinary Clinical Complex, FVSc & AH, SKUAST-Kashmir,
Jammu and Kashmir, India
A. Pal
Division of Surgery, Indian Veterinary Research Institute, Izatnagar, UP, India
V. Chandra · G. T. Sharma
Division of Physiology & Climatology, Indian Veterinary Research Institute,
Izatnagar, UP, India
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 27
Springer Nature Singapore Pte Ltd. 2020
M. B. Gugjoo, A. Pal (eds.), Mesenchymal Stem Cell in Veterinary Sciences,
https://doi.org/10.1007/978-981-15-6037-8_3
28 M. B. Gugjoo et al.
Phinney and Sensebe 2013). Subsequently, it had been shown that these cells are
present in almost all the adult tissues of the body, in addition to the foetal mem-
branes (Gugjoo and Amarpal 2018; Gugjoo et al. 2019). These cells represent very
small population in a particular tissue and are mixed with other cell types. In bone
marrow less than 0.1% of the mononuclear cells are MSCs and such a concentration
decreases with ageing (Caplan 2007). The therapeutic applications of MSCs require
much higher doses (Gugjoo et al. 2020a, b), making it imperative to harvest and
culture expand them. The culture expansion of MSCs may change their phenotype
making their regulations necessary. Bone marrow mononuclear cells and stromal
vascular fraction that are minimally manipulated and applied directly are being used
unrestrictedly (Gugjoo et al. 2018; Gugjoo et al. 2020a, b).
For development of any therapeutic agent comprehensive standardized proce-
dures are mandatory in good manufacturing practices (GMPs). Likewise MSCs
must be harvested, isolated, culture expanded and characterized as per the GMP
norms for their successful utilization (Gugjoo et al. 2015; Akram et al. 2017). The
techniques employed for MSCs processing, however, involve variable procedures.
MSCs harvested from liquid source like bone marrow/peripheral blood are cultured
from mononuclear cell (MNC) fraction being separated by density gradient method
(Gugjoo et al. 2015). In contrast, MSCs harvested from solid tissue like adipose tis-
sue or cord matrix, among others are harvested from stromal vascular fraction
(SVF) or MNC fraction. MNC fraction conforms to the mixture of red blood cells,
granulocytes, platelets and myeloid immature precursors, in addition to the MSCs
obtained from peripheral blood or bone marrow. SVF refers to the nucleated cell
fraction of heterogeneous mixture of endothelial, muscle, fibroblastic and mast
cells, and pericytes, pre-adipocytes and MSCs (Mamidi et al. 2012; Salehinejad
et al. 2012; Otsuru et al. 2013). The main difference in the procedure to harvest
MSCs from these tissue types is that the solid tissues require an extra step of enzy-
matic digestion while such a step is not required for liquid sources (Fig. 3.1). For
MSCs clinical applications, GMPs and good clinical practices (GCPs) must be
ensured during the whole procedure of cell harvesting, isolation, culturing proce-
dures and therapeutics (Gugjoo et al. 2019). Fortunately, GMP-compliant media and
chemicals are available to process and culture expand MSCs (Bieback et al. 2008).
MSCs may be harvested from liquid sources by various methods involving classical
and density gradient method. Density gradient solutions utilized are percoll (silica
particle colloidal solution, density of 1.084–1.088) and ficoll (polysaccharide solu-
tion; density of 1.077) (Kadiyala et al. 1997; Smith et al. 2003; Hegewald et al.
2004; Vidal et al. 2006; Pacini et al. 2007; Wilke et al. 2007; Colleoni et al. 2009;
Gugjoo et al. 2015). After bone marrow centrifugation, the supernatant is aspirated
and pellet obtained therefrom is washed with phosphate buffer solution (PBS), fol-
lowed by addition of growth medium in culture flask or plate. In case of density
gradient methods, MNC fraction is obtained from bone marrow after addition of
3 Mesenchymal Stem Cell Isolation, Culture, Characterization and Cryopreservation 29
Fig. 3.1 Mesenchymal stem cell isolation protocols and their culture expansion
MSCs from solid tissues are usually derived either by explant culture or enzymatic
protocol. Explant culture method is one of the earliest protocol, in which well rinsed
and mechanically split tissue (<few millimetres tissue pieces have better gas and
nutrient diffusion towards the cells) is directly placed into the culture flask contain-
ing the growth medium (Atala 2002). The smaller fraction of MSCs comes out from
these smaller tissue pieces and adheres to the culture dish surface. The residual tis-
sue pieces are washed off after the visible attachment of the cells. In enzymatic
protocol, an extra step of adding the enzymatic solution like collagenase to degrade
the extracellular matrix (ECM) is performed and the assembly is kept in shaking
30 M. B. Gugjoo et al.
incubator for proper digestion. The resultant SVF or MNC fraction obtained after
centrifugation is transferred to culture dishes containing growth media (detailed
below). The culture flasks/plates are maintained at 37 °C incubation temperature
with 5% carbon dioxide and under humid conditions. The cells are harvested after
attaining 70–80% confluency in flasks (Gugjoo et al. 2015).
There is a dearth of extensive literature comparing the isolation protocols.
However, few studies have compared different density gradient media, and classi-
cal/explants methods to the enzymatic protocol. In an equine study, bone marrow
MSCs (BM-MSCs) isolated by various density gradient solutions had shown match-
ing differentiation potential, but yield and properties like self-renewal were more
desirable in MSCs isolated by percoll solution (Bourzac et al. 2010). Comparison of
the enzymatic method to the explant method had shown that the latter method pro-
vided little heterogeneous cell population that exhibited higher proliferation rates
and better viability (Salehinejad et al. 2012; Hilkens et al. 2013; Yoon et al. 2013).
The explant methods keep tissue pieces intact and their extracellular matrix un-
dissociated and thus, the cells might remain shielded from proteolytic action and
mechanical stress (Hynes 2009; Hendijani 2017). The release of the cytokines and
growth factors from the local tissue may additionally provide cell growth promoters
(Hynes 2009; Jing et al. 2011; Shah et al. 2013). However, the explant method is
more cumbersome. In enzymatic methods, type of enzyme and its concentration
play an important role in cell yield and their viability (Can and Karahuseyinoglu
2007; Mitchell et al. 2003; Wang et al. 2004). Dissociation of extracellular matrix
(ECM) tends to give lower cell yield (Karahuseyinoglu et al. 2007; Seshareddy
et al. 2008) and also increases time of cell adherence (Han et al. 2013). To improve
the cell yield to about 70% or more, a combination of enzymatic method and
mechanical dissociation can be performed (Tong et al. 2011).
Contrary to embryonic stem cells (ESCs), the cell feeder layer remains dispensable
in MSCs culture. MSCs are plated directly into the plastic culture dishes/flasks sup-
plemented with growth media. This is due to their characteristic property of plastic
adherence. MSCs expand and proliferate in growth medium in the culture flasks.
Growth medium includes basal medium, foetal bovine serum (FBS) and antibiotics
and/antimycotics. The commonly used basal culture media for MSCs culture is
Dulbecco’s Modified Eagle’s Medium (DMEM) and/or alpha minimal essential
medium (Sampaio et al. 2015). Other media like TCM199/DMEM (1:1) (Colleoni
et al. 2005), Aminomax-II complete medium (da Silva et al. 2016) and Mesencult
medium (Hepsibha et al. 2011) have also been used. One of the studies had demon-
strated that mesencult medium had favoured better growth and proliferation of buf-
falo adipose tissue derived MSCs (AD-MSCs) as compared to the DMEM. The
results however, were obvious only at passage 1 as cells at passage 3 had better
colony formation even in DMEM (Hepsibha et al. 2011). In general, the cells are
allowed to attach for 4–8 days depending upon the culture ingredients, cell density
3 Mesenchymal Stem Cell Isolation, Culture, Characterization and Cryopreservation 31
and species involved. Most of the cells in MNC or SVF fraction that lack plastic
adherence properties (including haematopoietic cells) are removed after decanting
the initial culture medium. After achieving 70–80% confluency in about 10–14 days,
the cells are detached by various enzymes like trypsin, trypsin-ethylene diamine
tetra acetic acid (EDTA), collagenase I or accutase (Fortier et al. 1998; Arnhold
et al. 2007; Li et al. 2015a) and passaged for further proliferation. The buffalo amni-
otic membrane MSCs (bufAm-MSCs) extracted with the combined use of trypsin-
EDTA and collagenase I had been reported to prove better than trypsin-EDTA alone
(Deng et al. 2018). Between accutase and trypsin, immediate viability of human
neural stem cells (NSCs) after trypsin disassociation had been lower as compared to
that with accutase; however, subsequent apoptosis of NSCs caused by trypsin was
demonstrated to be lower than that caused by accutase (Li et al. 2015a).
FBS is one of the commonly used supplementation medium to culture MSCs (Vidal
et al. 2008; Toupadakis et al. 2010; Gugjoo et al. 2015). FBS has been used at
changeable concentration between 5 and 25%. Buffalo BM-MSCs had shown better
growth at 15% FBS (Gade et al. 2013) and for cattle AD-MSCs and BM-MSCs 10%
FBS concentration had been sufficient (Lu et al. 2014a, b). Although 20% FBS had
also been used with good results (de Moraes et al. 2016; Deng et al. 2018). Goat-
and equine-MSCs derived from adipose tissue, bone marrow and liver had acceler-
ated cell confluency at higher FBS concentration (20%) than at its lower concentration
(10%) (Heidari et al. 2013; Spaas et al. 2013; Martins et al. 2017). FBS furnishes
nutrients, hormones/growth factors and cell adhesion plasma proteins for cellular
growth and proliferation (Seo et al. 2013). GMP-compliant FBS is available and is
being used to produce clinical-grade MSCs (Li et al. 2015a). However, there are
associated limitations like potential threat of disease transmission, and xenogeneic
protein induced change in cell behaviour and non-uniform cell preparations. FBS
tends to hamper effective clinical applications of MSCs due to the potential immu-
nogenic reactions that can arise from internalized FBS proteins (McIntosh
et al. 2009).
To overcome these problems, FBS alternatives for culturing MSCs are being
studied extensively. Among various FBS alternatives, autologous serum, allogeneic
serum and platelet lysate (Del Bue et al. 2008; Toupadakis et al. 2010; Schwarz
et al. 2012; Bieback 2013) and commercially available serum substitutes (Schwarz
et al. 2012) have been evaluated. Overall, these alternatives have failed to provide
consistent MSCs adhesion, morphology, growth patterns (Seo et al. 2013; Eydt
et al. 2016; Schubert et al. 2018), proliferation rate (Toupadakis et al. 2010; Schwarz
et al. 2012; Glynn et al. 2013) and immune-modulatory properties (Russell et al.
2015; Clark et al. 2016; Enciso et al. 2018) in animals. However, autologous serum
had supported the replacement of foetal calf serum (FCS) to culture ovine
BM-MSCs. The morphology, stem cell markers, osteogenic differentiation and pro-
tein expression of ovine BM-MSCs supplemented with autologous serum had been
32 M. B. Gugjoo et al.
comparable to that obtained with FBS supplementation (Weigand et al. 2017).
Similarly, a dog study had shown that plasma rich in growth factors was able to
improve adipose tissue MSCs (AD-MSCs) survival and had promoted their prolif-
eration, migration but had induced their differentiation (Mellado-López et al. 2017).
Serum-free media had failed to maintain equine MSCs consistency in morphol-
ogy and immunophenotype (Schubert et al. 2018). Contrarily, a study had demon-
strated that bovine umbilical cord MSCs (UC-MSCs) can be cultured in serum-free
medium for longer periods. The normal karyotype cells had good proliferation with
an ability to differentiate into neuron-like cells, in addition to tri-lineages up to pas-
sage 60 (Cardoso et al. 2012). In absence of the serum, application of various
growth/ humoral factors like bovine fibroblast growth factor (bFGF), epidermal
growth factor (EGF), platelet derived growth factor (PDGF), fibroblast growth fac-
tor-2 (FGF-2), insulin growth factor − 1 (IGF-1) and most significantly transform-
ing growth factor-α (TGF-α) had supported MSCs proliferation especially of ovine
and bovine sources (Mccarty et al. 2009; Lu et al. 2014a, b). Combined use of these
growth factors may also affect MSCs proliferation, migration and invasion.
Combined use of PDGF/TGF-β and PDGF/IGF-I had promoted proliferation; EGF/
bFGF and EGF/bFGF/PDGF had promoted migration and EGF/bFGF/TGF-β had
promoted BM-MSCs invasion (Somers et al. 2012). These growth factors however,
tend to favour MSCs differentiation. However, further studies for better understand-
ing in FBS alternatives are desired.
The conventional culture expansion of MSCs follows static mechanism. This sys-
tem is labour intensive, bears low reproducibility, poses contamination problem and
above all is less economical for large-scale expansion. In order to make MSCs pro-
duction economical and reduce chances of contamination various modifications are
being tried. Among various modified techniques, the application of static magnetic
field has proven to be useful to some extent. Application of static magnetic field to
equine adipose tissue MSCs (eAD-MSCs) had accelerated their population dou-
bling potential with high colony-forming potential. An enhanced secretory potential
of growth factor rich microvesicles with no compromise on morphology was also
demonstrated (Marędziak et al. 2015; Iacono et al. 2017).
markers, lack haematopoietic marker expression and are able to show tri-lineage
(adipogenic, chondrogenic and osteogenic) differentiation. MSCs population must
express ≥95% of the CD73 (5’-Nucleotidase), CD90 (Thy1) and CD105 (Endoglin)
as measured by flow cytometry. There should be ≤2% of the CD11b (Integrin-α) or
CD14, CD19, CD34, CD45, CD79α and HLA Class II. Multipotent tri-lineage dif-
ferentiation potential is confirmed by staining these in vitro differentiated cells
(Dominici et al. 2006).
These cells from various animal species generally meet the norm of plastic
adherence and multipotency (Guest et al. 2008; Ranera et al. 2011; De Schauwer
et al. 2012) but consensus on surface marker expression is debatable (Kato et al.
2004; Knippenberg et al. 2005; Kang et al. 2008; Vieira et al. 2010; Lyahyai et al.
2012; Corradetti et al. 2013; Caminal et al. 2014; Mediano et al. 2015; Khan et al.
2016; De Cesaris et al. 2017; Ghaneialvar et al. 2018). In some species like rabbit
markers including CD90 have been demonstrated to be transcribed by MSCs but
their protein translation has failed (Kovac et al. 2017). Variable surface marker
expression of MSCs from various sources may be explained by donor tissue differ-
ences, variability in enzyme harvesting methods and specific reactivity of antibod-
ies used (Ranera et al. 2011; De Schauwer et al. 2012; Colleoni et al. 2009; Radcliffe
et al. 2010). The cell detaching agents like trypsin and accutase may vitiate cell
surface receptors (Ranera et al. 2011; De Schauwer et al. 2012). All antibodies are
not reactive to interspecies molecules (Radcliffe et al. 2010). MSCs dynamic
immune-phenotype may additionally promulgate such differences (Huss et al. 2000;
Mosna et al. 2010; Strioga et al. 2012). The differences in surface markers expres-
sion especially of CD44 and CD90 may impact MSCs ability to migrate (Cabezas
et al. 2018). Recent mice study had demonstrated that MSCs chromatin accessibil-
ity signatures and gene expression programs may be useful modes to provide
insights into tissue specific cell differences, though chromatin signature molecules
had been more accurate. This demands extensive MSCs epigenome analysis studies
to characterize them on the basis of tissue source (Ho et al. 2018).
In order to avoid such donor tissue specific cell characteristics, a uniform source
like induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) are being
evaluated. MSCs from these sources, although in preliminary studies, demand exten-
sive studies so as to harvest sufficient and phenotypically stable cell number so as to
utilize them clinically (Chan et al. 2018; Gugjoo et al. 2018; Steens and Klein 2018).
Apart from basic characterization protocol, MSCs must be viable, free of microbes
and/or endotoxin and pyrogen and finally dose titrated to conform to the standards of
good-manufacturing and clinical practices (GMPs and GCPs) (Bieback et al. 2008).
MSCs have the potential to be utilized as allogeneic therapy and as such may be
stored for future use and for distant transport. Excipient medium (like PBS) can be
retained along with the cells while ancillary medium (like bone marrow aspirate,
plasma, serum and cryogenic medium) must be removed prior to the in vivo
3 Mesenchymal Stem Cell Isolation, Culture, Characterization and Cryopreservation 35
3.8.1.1 Cryopreservation
Cryopreservation of cells is achieved either by fast or slow freezing rate. During this
process, various steps are ensured to avoid cell dehydration and damage. During fast
cell freezing rate solute and electrolyte imbalances can do the damage while during
slow freezing process intra- and extracellular ice crystal formation can have adverse
effects on the cell viability (Mazur 1977). During these processes, incorporation of
cryoprotectant is an important step. To prevent cell damage during such a delicate
process cryoprotectant is being added to reduce overall freezing point of the system.
The combined mixture of the cells, medium and the cryoprotectant makes a eutectic
system that has lower freezing temperature as compared to the individual compo-
nents. It is important to note that freeze-thaw method directly affects cell viability.
One of the studies had reported an unacceptable post-thaw viability of cells and
might have been due to the faulty thaw process (Espina et al. 2016).
Another way of cryopreservation that involves an extremely rapid (>−1000 °C/s)
cooling process known as vitrification has also been used for cell preservation
(Fowler and Toner 2005). In this process, the cells and potentially cytotoxic concen-
tration of cryoprotectant are immersed in open storage vessels. However, require-
ment of higher concentration of cryoprotectant, continuous maintenance of cells at
cryogenic temperature and need of open containers (that act as a potential source of
contamination) makes vitrification least opted technique for MSCs cryopreserva-
tion (Taylor et al. 2004; Fowler and Toner 2005).
• MSCs concentration and characteristics vary with the source and are affected by
the feeding, age and health status of the donor (Han et al. 2010; Pillai et al. 2016).
Allogeneic therapy can avoid such variations and assure uniform applications in
clinical cases and/or trials. Besides, the studies can effectively be studied and
compared for the results.
• MSCs viability during long distance can be safeguarded in cryopreservation state.
3.8.1.3 Cryoprotectant
MSCs cryopreservation is done in cryogenic media containing cryoprotectant that
prevents cell damage during freezing and thawing (Fuller 2004; Thirumala et al.
2010). Variable concentration of cryoprotectant has been used. The concentration of
cryoprotectant varied among species and sources, in addition to the freezing tech-
nique used (Fuller 2004; Thirumala et al. 2005). Cryoprotectants are of two types
including cell membrane permeable and impermeable (Fuller 2004), with highly
permeable cryoprotectants tend to be more toxic (Gao and Critser 2000). To prevent
such a toxicity permeable cryoprotectants like DMSO, ethylene glycol, etc. are
combined with less permeable ones like hydroxyethyl starch (HES), dextran and
polyethylene glycol (Gao and Critser 2000; Fuller 2004). In veterinary practice
cryoprotectants like DMSO and FBS are most commonly used (Martinello et al.
2010; Marx et al. 2015). The ancillary cryoprotectants like DMSO exhibit carcino-
genic properties (Quimby et al. 2013). The xenogeneic proteins in FBS may alter
cellular behaviour (Duan and Lopez 2016) and need to be removed before cell
implantation.
post-thaw cellular viability at –20 and –80 °C, citing importance of freeze-thaw
method (Espina et al. 2016).
The cryopreserved MSCs viability remains same irrespective of their sources
(Knippenberg et al. 2005). Goat foetal adnexa derived MSCs had revived success-
fully upon thawing. The revived cells could express surface antigens and pluripo-
tency markers and had shown tri-lineage differentiation (Somal et al. 2017). The
population doubling time (PDT) too had remained the same (Somal et al. 2016,
2017) as had also been reported for ovine BM-MSCs (Rhodes et al. 2004). Storage
of dog MSCs for a year had not affected fibroblast-like morphology, alkaline phos-
phatase (AP) activity, surface marker expression and tri-lineage differentiation
potential. The cryopreserved cells however, had reduced proliferation ratio and
telomerase activity in comparison to the fresh cells (Martinello et al. 2011).
Apart from the cells, cryopreservation of tissue engineered (TE) constructs may
provide an off-the-shelf TE constructs. In one such study cryopreserved tissue engi-
neered scaffold and goat BM-MSCs (gBM-MSCs) had good viability (Costa et al.
2012). Although other factors like scaffold design and structural properties might be
affected by cryopreservation and should be studied in detail.
3.9 Conclusion
Culture expansion of MSCs is imperative for their effective utilization. The isola-
tion of MNCs from liquid tissues can be achieved with conventional or density
gradient separation methods while SVF from solid tissues can be separated either
by direct explant method or by prior enzymatic digestion. MNCs and/SVF cells are
culture expanded in growth media containing basal culture medium, FBS and
antibiotic-antimycotic solution being kept under standard culture conditions. FBS
poses various impediments for clinical applications and as such its alternatives are
being studied. The plastic adhered cells are harvested by enzymatic digestion and
characterized as per ISCT recommendations. The cells that are plastic adherent
express specific surface markers while lacking haematopoietic markers and are able
to differentiate into at least tri-lineages are termed as MSCs. For large-scale and
cost-effective production of MSCs, stirred suspension bioreactor process is being
tried and seems useful. The culture expanded cells are stored for longer period to be
utilized as ready to use supply. The cells are preserved in cryopreservation media
under liquid nitrogen.
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Mesenchymal Stem Cell Immuno-
Modulatory and/Anti-Inflammatory 4
Properties
M. B. Gugjoo and Amar Pal
Abstract
Abbreviations
ConA Concanavalin A
COX2 Cyclooxygenase 2
FASL Fas ligand
HGF Hepatocyte growth factor
HMOX1 Heme oxygenase 1
M. B. Gugjoo (*)
Division of Veterinary Clinical Complex, FVSc & AH, SKUAST-Kashmir,
Jammu and Kashmir, India
A. Pal
Division of Surgery, Indian Veterinary Research Institute, Izatnagar, UP, India
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 47
Springer Nature Singapore Pte Ltd. 2020
M. B. Gugjoo, A. Pal (eds.), Mesenchymal Stem Cell in Veterinary Sciences,
https://doi.org/10.1007/978-981-15-6037-8_4
48 M. B. Gugjoo and A. Pal
4.1 Introduction
et al. 2018; Taguchi et al. 2019; Kafarnik et al. 2020), horse (Carrade and Borjesson
2011; Deuse et al. 2011; Peroni and Borjesson 2011; Carrade et al. 2012; Carrade
et al. 2014; Colbath et al. 2017; Cassano et al. 2018; Cortés-Araya et al. 2018;
Lepage et al. 2019; Longhini et al. 2019), sheep (Mrugala et al. 2008), chickens
(Khatri et al. 2009), pig (Poncelet et al. 2007; Cho et al. 2008), rabbit (Liu et al.
2006; Moreno et al. 2010), goats (Somal et al. 2016), cattle (Cardoso et al. 2012;
Jacca et al. 2014; Cardoso et al. 2017; de Moraes et al. 2017; Lara et al. 2017) and
cat (Mumaw et al. 2015; Zajic et al. 2016; Chae et al. 2017; Clark et al. 2017; Parys
et al. 2017). In general, MSCs immuno-modulation and/or anti-inflammatory activi-
ties occur through their interaction with immune cells. These activities are mediated
through secretome or through cell-cell contact. MSCs lack direct cytotoxic or
humoral defence activities unlike that of the immune cells. These cells fail to secrete
granzymes or perforins, lack antibody production ability and are devoid of phago-
cytic activity (Tso et al. 2010).
et al. 2016). MSCs microvesicle production varies with respect to their source.
Among various equine foetal adnexal sources, Wharton’s jelly derived MSCs
(WJ-MSCs) had copious micro-vesicles as compared to the other adnexal sources
(Iacono et al. 2017).
lymphocytes (Lee et al. 2011) and even if produced levels were insufficient to
inhibit lymphocyte proliferation. Contrarily, dog AD-MSC, and feline AD-MSCs
and rabbit BM-MSCs had shown increased TGF-β1 production after exposure to
lymphocytes (Kang et al. 2008) and IFN-γ pre-treatment (Liu et al. 2006; Kang
et al. 2008; Clark et al. 2017), respectively. TGF-β1 production of feline AD-MSCs
had also increased after stimulation with TNFα while combined stimulation (INFγ
and TNFα) of feline AD-MSCs had resulted in decreased TGF-β1 production (Parys
et al. 2017).
through indoleamine 2,3-dioxygenase (IDO) while later cell type had activated
inducible nitric oxide synthase (iNOS) (Somal et al. 2016). The effect of MSCs on
different immune cells is detailed below:
4.4.1.3 Neutrophils
MSCs cultured with neutrophils tend to decrease their reactive oxygen species
(ROS) potential. The inhibition potential of MSCs varies with respect to the source
of cell. Feline BM-MSCs had quite significantly inhibited neutrophilic ROS poten-
tial in comparison to the feline AD-MSCs (Mumaw et al. 2015).
4.4.1.4 Macrophages
Macrophages based upon the available environment are polarized into two types:
M1 possesses antimicrobial activity while M2 alleviates inflammation and expe-
dites tissue repair. The anti-inflammatory activity and repair process of M2 may be
ensured by secretion of IL10 and VEGF and IGF trophic factors (Mosser and
Edwards 2008). MSCs induce production of M2 macrophages from M1. M2
increases the phagocytic activity and secretion of interleukin 10 and decreases
inflammatory cytokines (Zhang et al. 2010; Selleri et al. 2016). Further, MSCs
4 Mesenchymal Stem Cell Immuno-Modulatory and/Anti-Inflammatory Properties 57
4.4.2.1 T Cells
MSCs are considered to repress T-cell proliferation either through cell based or
nonspecific mitogenic stimuli (Di Nicola et al. 2002). The action is mediated
through secretome being responsible for effective T-cell suppression and apoptosis
(Ren et al. 2008; Ren et al. 2010). The secretome involved in proliferation inhibition
include PGE2, IDO, NO and TGF-β (Gao et al. 2016). The cells may prevent T-cell
activation and proliferation by decreasing the expression of activation markers like
CD25, CD38 and CD69 (Le Blanc et al. 2003; Groh et al. 2005). Furthermore,
MSCs may promote apoptosis of activated T-cells via the Fas/Fas ligand pathway
(Akiyama et al. 2012). Allogeneic and autologous cells may comparably suppress
proliferation and IFN-γ production of T-cells (Colbath et al. 2017).
Despite all these reports, it is worth mentioning here that MSCs immunosuppres-
sive potential may not be activated every time. Such a response is affected and
determined by the inflammatory mediators and their strength (Renner et al. 2009).
Besides, MSCs activities to inhibit T-cell proliferation may not be sufficient in pres-
ence of pathogen associated molecules. Toll-like receptors (TLR3 and TLR4) which
damage Notch signalling may help in T-cells recuperation (Rashedi et al. 2017).
Blocking activity of human BM-MSC produced TGF-β1 had failed to decrease
T-regulatory cell production (English et al. 2009).
4.4.2.2 B Cells
MSCs suppress B cell proliferation, activation, differentiation, chemokine receptor
expression either through cell-cell contact and/ or secretome (IDO) (Augello et al.
2005; O’Connor et al. 2006). However, MSCs ability to suppress B cell antibody
production is dependent upon inflammatory stimulation strength, and MSCs:B cell
ratio (Krampera et al. 2006a, b; Bernardo et al. 2009).
58 M. B. Gugjoo and A. Pal
4.5 Conclusion(s)
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Mesenchymal Stem Cell Differentiation
Properties and Available 5
Microenvironment
M. B. Gugjoo and Amar Pal
Abstract
5.1 Introduction
Mesenchymal stem cells (MSCs) due to their characteristic features are considered
to have an all-in-one solution for diverse ailments (Gugjoo and Amarpal 2018;
Gugjoo et al. 2019a). The characteristics of MSCs are believed to be controlled by
the available microenvironment/or niche. The “microenvironment or niche” com-
prises cells, various chemical/mechanical and topographical cues. Such an
M. B. Gugjoo (*)
Division of Veterinary Clinical Complex, FVSc & AH, SKUAST-Kashmir,
Jammu and Kashmir, India
A. Pal
Division of Surgery, Indian Veterinary Research Institute, Izatnagar, UP, India
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 67
Springer Nature Singapore Pte Ltd. 2020
M. B. Gugjoo, A. Pal (eds.), Mesenchymal Stem Cell in Veterinary Sciences,
https://doi.org/10.1007/978-981-15-6037-8_5
68 M. B. Gugjoo and A. Pal
environment serves as source of cues that determine the cell migration and homing,
local proliferation, secretome, and ultimately their fate towards particular lineage
(Place et al. 2009). Understanding the mechanisms involved in MSCs fate based on
these signals or cues becomes imperative for their fruitful applications. To confirm
proof of principle, in vitro studies are initially being conducted which allows a wide
range of permutations and combinations without any kind of involved risk. Such a
system, however, has limitations of being less extensive and is more controlled. In
vivo system in contrast presents an uncontrolled system subjected to numerous
simultaneous cues from various quarters (Hwang et al. 2011).
The scientific field that studies such cell–microenvironment/niche interactions is
tissue engineering. The tissue engineering (TE) concept has been introduced by
Professor Robert Nerem in 1988 and was defined as “the application of life sciences
and engineering to develop a basic understanding of the functional and structural
relationships of natural and pathologic mammalian tissues and the development of
bio-substitutes that can be utilized to restore, maintain, or improve tissues damaged
or lost by various disease conditions” (Meyer et al. 2009; Lanza et al. 2011). To
utilize tissue engineering and stem cells to the service of medicine, William
Haseltine in 1999 has coined the term regenerative medicine (Atala and Lanza
2001). It is a translational research branch of tissue engineering and molecular biol-
ogy and is aimed to develop technologies/therapeutics that can remove/provide
relief in sufferings of diseased/malfunctional/non-functional tissues/organs (Gregor
et al. 2017; Gugjoo et al. 2019a).
TE employs various ingredients like cells, signaling factors, and the bio-
fabricated scaffold to develop the tissue graft (Gugjoo et al. 2018). In vivo implanta-
tion exposes cells to the unfavorable environment. To prevent such adverse effects
and keep the cells viable and effective for a prolonged period, the biocompatible
scaffold is being incorporated. To promote the cell specific expression various sig-
naling factors and scaffolds play an important role. The role played by these critical
factors in relation to the MSCs characteristics is of utmost importance for latter’s
effective clinical applications. The current chapter provides an overview of the
ex vivo effects of such cues on the MSCs activities including their differentiation
(Fig. 5.1).
MSCs availability in donor tissues is very limited and as such their culture expan-
sion becomes imperative. These cells are cultured in a specific medium being
renewed after certain intervals. An extensive culturing exhausts the culture ingredi-
ents and needs to be replenished. Accumulation of higher lactate and ammonia con-
centration affects goat BM-MSCs growth and proliferation under in vitro conditions.
To prevent the adverse effects of these accumulated noxious ingredients, the culture
medium is being replaced after some intervals. Prolonged proliferation of goat
MSCs had been maintained by replacing about 30% of medium after every 3 days.
5 Mesenchymal Stem Cell Differentiation Properties and Available Microenvironment 69
Fig. 5.1 Pictorial representation of role of different in vitro cues in determining MSCs fate
MSCs chondrogenesis has generally been studied under pellet or micromass sys-
tem, although 3D culture system is now increasingly being studied. MSCs in pellet
culture system had undergone spontaneous chondrogenesis without the addition of
any chondrogenic factor. At the center of bovine BM-MSCs pellet, cartilage specific
matrix (collagen type II) had accumulated with its progressive increase as culturing
continued (Bosnakovski et al. 2004). To achieve desired and specific cartilaginous
matrix formation, chondrogenic factors like growth factors, scaffolds, cells, or
mechanical factors are being evaluated (Gugjoo et al. 2019b, c, d). There are various
types of commercially available chondrogenic differentiation media that contains
basic ingredients required for chondrogenesis.
(Knippenberg et al. 2006; Zhang and Jiang 2006; Goldman and Barabino 2016).
This shows that combined utilization of growth factor (BMP-2) and scaffold (algi-
nate beads) may promote MSCs chondrogenesis, while BMP-2 alone may favor
their osteogenesis, though further studies are desired. The role of BMP-7 in MSCs
differentiation is again controversial. BMP-7 may promote chondrogenic
(Knippenberg et al. 2006), osteogenic (Elkhenany et al. 2016) differentiation of goat
adipose tissue derived MSCs (gAD-MSCs) and adipogenic differentiation of human
BM-MSCs (Katja et al. 2007). gAD-MSCs short (15 min) incubation under BMP-7
(10 ng/mL) had been sufficient to induce their chondrogenic differentiation while
long term incubation had no additional effect on such differentiation (Knippenberg
et al. 2006). Thus, MSCs may or may not undergo chondrogenesis under the influ-
ence of these above mentioned humoral factors. It is worth mentioning here that
biologic activities of these recombinant growth factors are much lower than their
analogues being synthesized endogenously and as such ectopic incorporation of
these factors may be required in higher concentrations under in vivo conditions
(Wang et al. 2010; Gugjoo et al. 2017; Gugjoo et al. 2020).
that delayed differentiation may not be the reason behind their diminished chondro-
genic capacity (Mauck et al. 2006). Despite these properties of chondrocytes, their
culture expansion deleteriously affects their chondrogenic potential. Culture
expanded chondrocytes had secreted mechanically inferior (more Col I and less Col
II) matrices that lacked integration with native tissue. Contrarily, cBM-MSCs had
secreted cartilage specific matrices of superior mechanical properties (Rackwitz
et al. 2014). MSCs chondrogenic potential may be further promoted in the presence
of chondrocytes. Co-culture of cBM-MSCs and fibrochondrocytes had favored lat-
ter’s chondrogenic expression. The cBM-MSCs:fibrochondrocytes ratio in co-
culture, however, may affect MSCs differentiation. In monoculture a significant
GAG content was produced in a cell ratio of 100:0, while highest secretory activity
had been demonstrated at equal cellular concentrations (50:50). Such a combined
co-culture chondrogenesis may also prevent hypertrophic chondrocyte formation
(type X collagen) (McCorry et al. 2016). Similarly, infrapatellar fat pad derived
gMSCs in the presence of chondrocytes had shown improved secretion of cartilage
specific matrices (Arora et al. 2017). In vitro mono and co-cultures too had sup-
ported these results (McCorry and Bonassar 2017). It may be inferred that MSCs
chondrogenesis improves in the presence of chondrocytes and as such results may
be more encouraging in less damaged cartilage than extensively damaging one. To
enhance MSCs in vitro chondrogenic matrices synthesis, co-culture may be an
effective option (McCorry and Bonassar 2017).
MSCs osteogenic lineage differentiation may occur in the presence of relevant cues
eliciting from available environment. In vitro MSCs osteogenic studies have uti-
lized commercially available medium (containing growth factors). However, vari-
ous other factors, either alone or in combination, including matrices or growth
factors or cells, in addition to mechanical forces also direct such differentiation. It
may be emphasized that β-glycerolphosphate appears to be unsuitable phosphate
ion source for sheep adipose tissue MSCs (sAD-MSCs) and sBM-MSCs. These
cells had failed to show alkaline phosphatase activity (Kalaszczynska et al. 2013).
Human MSCs although do show such activity and thus, MSCs mineralization status
may sometimes be misleading.
MSCs neural cell like trans-differentiation has been achieved by various factors,
although the actual differentiation is questioned. Cattle umbilical cord MSCs (cUC-
MSCs) had differentiated into the neural like cells under low glucose Dulbecco’s
modified Eagle’s medium (L-DMEM), fetal bovine serum (FBS), and basal eagle
medium (BME) (Xiong et al. 2014). Neural induction medium for dog AD-MSCs
included FBS, dimethyl sulfoxide, butylated hydroxyanisole, hydrocortisone, insu-
lin, 3-isobutyl-1-metylxanthine, and adenosine 3′5- cyclic monophosphate sodium
salt monohydrate (Oh et al. 2011). Likewise, other specific medium had promoted
neurogenesis in cattle Wharton’s jelly (cWJ-MSCs). Neural differentiation was
characterized by their ability to express neuronal/glial markers like neurofilament
200 (N200), neuronal microtubule associated protein 2 (MAP2), neurotrophin 3
(NT3), Tau, glial fibrillary acidic protein (GFAP) (Cardoso et al. 2012). Neural like
cell trans-differentiation of gAD-MSCs had also been achieved with BIX-01294, a
specific inhibitor of methlytransferase G9a. gAD-MSCs trans-differentiation
achieved with methlytransferase inhibitor relies on Nanog regulatory network
(Wang et al. 2018). The neural cell like differentiation of bufAm-MSCs had been
promoted by glycogen synthase kinase 3 inhibitor (kenpaullone) (Deng et al. 2018).
The neurogenic markers are variably expressed at different time intervals. Nestin
and Map-2 expression mostly occurs at 24 hr. culture period while tropomyosin
receptor kinase A (TRKA) and PRion Protein Cellular (PrPc) expression is seen at
144 hrs (Dueñas et al. 2014). Therefore, at different time intervals MSCs neural
expression profile is variable making it important to study their differentiation as per
the developmental phase of an individual.
Bovine umbilical cord MSCs (cUC-MSCs) differentiate into the islet like cells upon
supplementation with media like H-DMEM, HGF, mercaptoethanol, and niacinamide.
The trans-differentiation had been demonstrated through pancreatic and duodenal
homeobox 1 (PDX-1) and insulin secretion (Xiong et al. 2014). Similarly, a culture
medium containing nicotinamide, extendin-4, glucose, and poly-D-lysine had pro-
moted cattle placental MSCs islet like cell differentiation. The differentiated cells had
exhibited special characteristics including zinc ion staining, secretion of insulin, gluca-
gon, paired box protein (Pax-4), Pax-6, homeobox protein Nkx6.1 and Forkhead box
protein (Fox) (Peng et al. 2017). During MSCs islet-like cell differentiation, glucose
promotes growth of pancreatic β-cells through phosphatidylinositol 3-kinase pathway
(Wu et al. 1997). During islet like cells trans-differentiation of MSCs poly-D-lysine
acts an important ingredient. It provides an intercellular connection and support, water
retention, compression resistance, cell migration, cell physiology, and finally cell dif-
ferentiation (Peng et al. 2017). These in vitro studies need to be translated under in vivo
applications to evaluate actual feasibility and utility.
78 M. B. Gugjoo and A. Pal
cUC-MSCs tend to differentiate into the hepatocyte like cells upon culturing in
L-DMEM, FBS, hepatocyte growth factor (HGF), fibroblast growth factor 4
(FGF-4), dexamethasone, and ITS (Xiong et al. 2014). FGF-4 acts as an important
growth and development factor for hepatocytes, while HGF increases their mitosis
and resists their apoptosis (Jiang et al. 2002). cBM-MSCs upon culture supplemen-
tation with α-fetoprotein, albumin, α1-antitrypsin, connexin 32, tyrosine amino-
transferase, and cytochrome P450 had differentiated to hepatogenic lineage. MSCs
hepatogenesis was characterized on the basis of their significant increase in the
expression of genes like cytochrome P34A4 (CYP3A4), ALB, α1-AT, CNX32, and
TAT. CYP3A4 mRNA expression was higher as early as day 7 up to day 28 (Scott
and Halpert 2005; Dueñas et al. 2014). α1AT and TAT, late markers of the hepato-
cyte lineage, play the role of tyrosine degradation and inhibit proteases (Rehman
et al. 2004; Jedicke et al. 2014). CNX32 accounts for majority of liver connexin
proteins (90%) (Nakashima et al. 2004) whose expression occurs at the later stage
of differentiation. It points out that MSCs must be exposed to hepatogenic factors
for longer time to trigger them towards the hepatic line.
Myogenic differentiation of bovine fetal BM-MSCs has been achieved with various
protocols like incorporation of 5-Aza-2′-deoxycytidine (5-Aza), myoblast-secreted
factor Galectin-1 (Gal-1), and/or myoblast culture medium SkGM-2 BulletKit (Oh
et al. 2011; Okamura et al. 2017). Variable concentration of 5-Aza has bearing on
cell viability. Higher concentration (100 μM) may increase cellular differentiation
but may be more toxic to cells. It had been concluded that cBM-MSCs were myo-
genically differentiated to some extent (Okamura et al. 2017). Similarly, bezafibrate
had promoted myogenesis of cBM-MSCs (Ramirez-Espinosa et al. 2015). Muscle
derived cells (MDCs) had differentiated gBM-MSCs through their fusion producing
myotubes (Kulesza et al. 2016). Sheep fetal derived BM-MSCs supplemented with
reversine and 5-azacytidine had differentiated into cardiomyocyte like cells (Soltani
et al. 2016). Arterial matrix supplementation to culture had directed sheep AD-MSCs
differentiation towards endothelial like cells (Zhang et al. 2016).
In vitro cultured sheep MSCs and bovine MSCs under the influence of TGF-β1,
BMP-4, BMP8b, and retinoic acid transdifferentiate into the germ cells
(Ghasemzadeh-Hasankolaei et al. 2014; Ghasemzadeh-Hasankolaei et al. 2015).
Dog adipose tissue and ovarian derived MSCs were transdifferentiated into primor-
dial germ cell like cells and male germ cell like cells under the influence of insulin,
epidermal growth factor (EGF), glial derived nerve factor (GDNF), basic fibroblast
growth factor (bFGF), and BMP-4 (Wei et al. 2016; Trindade et al. 2017).
5 Mesenchymal Stem Cell Differentiation Properties and Available Microenvironment 79
5.5 Conclusion(s)
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Mesenchymal Stem Cell Genetic
Engineering and Regenerative Medicine 6
M. B. Gugjoo, E. Rasool, and Amar Pal
Abstract
6.1 Introduction
M. B. Gugjoo (*)
Division of Veterinary Clinical Complex, FVSc & AH, SKUAST-Kashmir,
Jammu and Kashmir, India
E. Rasool
Division of Surgery & Radiology, FVSc & AH, SKUAST-K,
Srinagar, Jammu and Kashmir, India
A. Pal
Division of Surgery, Indian Veterinary Research Institute, Izatnagar, UP, India
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 89
Springer Nature Singapore Pte Ltd. 2020
M. B. Gugjoo, A. Pal (eds.), Mesenchymal Stem Cell in Veterinary Sciences,
https://doi.org/10.1007/978-981-15-6037-8_6
90 M. B. Gugjoo et al.
2018). MSCs supposedly work via trophic effects through plethora of compounds,
immune-modulatory/anti-inflammatory and/or by cell replacement activities. Their
trophic action and differentiation is, however, determined by the available microen-
vironment upon which a limited control can be exerted (Gugjoo et al. 2019, 2020).
MSCs show poor in vivo survival and weak host tissue engraftment especially after
local implantation (Wagner and Ho 2007). To realize MSCs full therapeutic poten-
tial and tailor them to the specific diseases, the cells are being genetically engi-
neered (Bersenev and Levine 2012, Gage 2012; Roís et al. 2012; Lu et al. 2013; Yan
et al. 2015). This avenue has generated much interest among researchers, and the
initial reports demonstrated are also promising (Kode et al. 2009; Griffin et al. 2010;
Hang et al. 2012).
Genetic engineering/genetic modification/genetic manipulation involves a set of
technologies to alter genetic makeup of cells. It is aimed to deliver proteins to neigh-
bouring cells, kill cancer cells, reduce immune rejection, secrete regulatory and
pro-healing factors and increase survival of engrafted cells. In genetic engineering,
a specific gene cassette is formed to be loaded into a carrier/vehicle (vector). Vectors
are the carriers to deliver gene cassette (containing transgene) to the cells (Phillips
and Tang 2008; Li et al. 2017; Zhang et al. 2019). These include viruses like retro-
viruses (Boura et al. 2014), DNA plasmids (Marquez-Curtis et al. 2013; Watkins
et al. 2014), mRNA (Rejman et al. 2010), miRNA (Meng et al. 2015) and siRNA
(Roís et al. 2012), constructs of minimalistic, immunologically defined gene expres-
sion (MIDGE) (Mok et al. 2012; Mählmann et al. 2015) and transposons construct
like the Sleeping Beauty System (Martin et al. 2014). Viral vector transduction
directly transduces cells while non-viral transfection requires specific methods like
lipofection or electroporation to cross the cell membrane (Papanikolaou et al. 2011).
Viral vectors may be genome integrating like lentivirus and adeno-associated
virus or non-integrating like adeno and Sendai virus vectors. The integrating viral
vectors tend to wield transgene for longer period, while non-integrating vectors
express the gene of interest transiently. The genome-integrating viral vectors though
may have long-lasting effects but tend to compromise the safety concerns, espe-
cially in relation to the insertional mutagenesis and associated potential malignancy
(Hacein-Bey-Abina et al. 2003a, b). Inside the cells, the transgene may overexpress
unphysiologically (constitutive synthesis) or can express within the physiological
limits (controlled by a gene switch). The constitutive transgene expression leads to
constant unphysiological oversynthesis of specific proteins. This down-regulates
receptors and renders gene expression ineffective. Gene switch transgene expres-
sion can be well controlled. The cell responds to the physiological stimulus like
oxygen level, concentration of hormones, drugs or chemical agents (Phillips and
Tang 2008; Nowakowski et al. 2013). These techniques have their pros and cons and
need to be fully weighed before any in vivo application.
MSCs are also being virally transduced but due to the above-mentioned con-
cerns, a safer approach is desired. MSCs characteristic properties including the one
of homing make them a suitable non-viral vector candidate. Add-on genetic engi-
neering if well controlled and understood can make MSCs application a game
changer in regenerative medicine. The genetically engineered MSCs currently
6 Mesenchymal Stem Cell Genetic Engineering and Regenerative Medicine 91
remain to be approved for clinical trials (Nowakowski et al. 2015). The main aim
currently remains to develop genetic engineering technique that does not change
basic stem cell properties, induce cytotoxicity and should be rescuable after in vivo
transplantation (Papanikolaou et al. 2011). The current chapter focuses on the
genetically engineered MSCs of veterinary species and their utility in regenerative
medicine.
Non-viral transfected MSCs in comparison to the viral transduced ones may offer a
plausibly safe option in regenerative medicine. The current gene transfection effi-
ciency of MSCs is not at par to that of the MSCs viral vector transduction. One of
the reasons that might affect their efficiency is transfection technique. Transfection
of cattle bone marrow MSCs (cBM-MSCs) by the electroporation had yielded bet-
ter (58%) gene transfer efficiency (Colleoni et al. 2005) in comparison to the lipo-
fection (15%) (Díaz et al. 2015) demanding further incites.
GE-MSCs should be ensured to maintain their basic properties (Pinel and Pluhar
2012). MSCs in comparison to other stem cell types have limited culture lifespan.
The cell proliferation may cause telomere shortening, and as such the extended
culture passaging might lead to genetic changes. In order to improve MSCs sur-
vival, genetic engineering has been tried. Sheep bone marrow MSCs (sBM-MSCs)
transfected with telomerase reverse transcriptase (TERT) gene had been cultured up
to 40 passages. These cells had normal karyotype rate of 88.24% with unaffected
differentiation properties (Zhu et al. 2017). Thus, further MSC studies may be con-
ducted to determine the reasons behind their limited self-renewal properties and
finally the possible addressal.
Furthermore, it is imperative to confirm MSCs properties remain unaffected with
genetic engineering. In a mongrel dog study, VEGF165-transfected BM-MSCs have
been demonstrated to express the surface markers as per International Society of
Cellular Therapy (ISCT) (Hang et al. 2012). A comparison had been made through
the characterization of dog AD-MSCs (dAD-MSCs) and SV40-T retrovirus-
transfected dAD-MSCs (tdAD-MSCs). Both these cell lines had characteristic
properties as laid down by ISCT. tdAD-MSCs though had higher colony forming
potential; pluripotency markers (Sox2 and Nanog) were expressed in both cell lines
with stable karyotype. In conclusion, tdAD-MSCs and dAD-MSCs had comparable
characteristics (Ayala-Cuellar et al. 2019).
In veterinary science, the genetically engineered MSC studies are very limited.
However, the available literature favours their utilization under in vivo conditions.
Below are described some of the in vivo GE-MSCs studies conducted in animals.
92 M. B. Gugjoo et al.
Stem cell therapy has a significant potential to regenerate the udder tissues (Sharma
et al. 2017; Gugjoo et al. 2019). Mammary stem cells are currently difficult to har-
vest and as replacement MSCs can be utilized. One of the studies had created bovine
lactoferricin (LFcinB) transgene to confirm feasibility of creating genetically engi-
neered cattle or buffalo MSCs. These modified cells had variable expression of the
LFcinB, an antibacterial peptide (Sharma et al. 2017). Such LFcinB-cloned MSCs
may be injected into the mammary gland for their higher antibacterial properties
especially against Staphylococcus aureus and Escherichia coli (Sharma et al. 2017).
et al. 2017). Thus, the combined use of genetic and tissue engineering may provide
an innovative way to treat bone defects/fractures. Although these studies have dem-
onstrated improved bone healing results, however, the dose titration of these factors
is required. An excess bone formation leading to tumour formation may arise in
case higher dose of BMPs is dissipated by these transfected cells (Pinel and Pluhar
2012). Similarly, transplantation of VEGF-transduced autologous BM-MSCs in
experimentally induced dog osteonecrotic femoral head had led to better healing in
comparison to the simple BM-MSCs (Hang et al. 2012).
(Araujo et al. 2012). In peripheral nerve regeneration experimental study, the BDNF-
transfected AD-MSCs were engaged in nerve regeneration. The cells had differenti-
ated into various cell types including Schwann cells and had encouraged functional
recovery of the rat sciatic nerve (Tseng and Hsu 2014). Adipose-derived mesenchy-
mal stem cells (AD-MSCs) transfected with BDNF and heme oxygenase-1 (HO-1)
were utilized had led to a significant improvement in hind limb functions of spinal
cord-injured dogs. Co-transfection of AD-MSCs had been more effective. An
increase in clinical evaluation scores and a robust increase in neuronal regeneration
were demonstrated. Thus, MSCs transfection with multiple factors may be more
effective in promoting the healing of spinal cord injury (SCI) (Khan et al. 2018).
Stem cell therapy in myocardial infarction offers an exciting research area. The cur-
rent issues like stem cell retention and their efficacy in the heart remains to be
solved. MSCs genetic modification has been achieved with various factors like
glucagon-like peptide-1 (GLP-1), and VEGF and hypoxia-inducible factor 1-α
(HIF1-α) are considered to favour better cardiac repair and function. Glucagon-like
peptide-1 (GLP-1) is an incretin hormone with cardio-protective properties (Wright
et al. 2012), VEGF improves angiogenesis (Locatelli et al. 2015) and hypoxia-
inducible factor 1-α is considered to mediate overexpression of erythropoietin, vas-
cular endothelial growth factor, angiopoietin-1 and inducible nitrous oxide synthase
(Hnatiuk et al. 2016).
Transfection of human BM-MSC with GLP-1 has been demonstrated as benefi-
cial adjunct in pig myocardial infarction model. GLP-1-transfected human MSCs
encapsulated in alginate (bead-GLP-1 MSC) after coronary artery injection had led
to the better-left ventricular function and a decrease in infarct area as compared to
control. The GLP-1-transfected cell-treated infarcts had reduced inflammation and
apoptotic cells together with an increased collagen deposition and fewer myofibro-
blasts (Wright et al. 2012). In sheep acute myocardial infarction (AMI) model, vas-
cular endothelial growth factor (VEGF)-transfected MSCs had been demonstrated
as useful. An increased angiogenesis together with decreased infarct size and
improved left ventricular (LV) function was demonstrated with intracardiac implan-
tation of these cells (Locatelli et al. 2015). Similarly, HIF1-α-transfected allogeneic
BM-MSCs as compared to the simple BM-MSCs had led to significant reduction of
infarct size in a sheep acute myocardial infarction (AMI) model. BM-MSCs too had
decreased infarct size although lower than transfected cells but better than the con-
trol (Hnatiuk et al. 2016).
In skin wounds the main concern remains to improve angiogenesis along with the
collagen deposition. Although skin healing is good, but in some cases it takes longer
6 Mesenchymal Stem Cell Genetic Engineering and Regenerative Medicine 95
6.4 Conclusion
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Sheep Mesenchymal Stem Cell Basic
Research and Potential Applications 7
M. B. Gugjoo and Amar Pal
Abstract
Mesenchymal stem cells (MSCs) act as reservoir of repair system in an adult tis-
sue. These cells are available in almost all the tissues. The cells have been har-
vested and isolated from numerous tissue sources. Due to their miniscule number
in tissues culture expansion followed by characterization becomes imperative.
Sheep MSCs (sMSCs) too have been obtained from numerous sources including
foetal membranes. These cells have been evaluated in various human transla-
tional models and have variably proven to be useful. Such studies additionally
form basis of the MSCs therapeutic applications in veterinary practice. The pres-
ent chapter throws light on various aspects of sheep mesenchymal stem cells
including their potential therapeutic applications.
7.1 Introduction
Veterinary and human medicine is intricately dependant and linked to each other,
forming important constituents of “One Health” initiative (Gugjoo et al. 2018).
Various ailments among the animals and human have similar pathophysiology and
established therapeutics in one species can form basis of its applications in other
(Gugjoo et al. 2018). Sheep acts as a suitable model animal for various ailments of
epithelium, cardiovascular, skeletal and neurological systems for human (DiVincenti
M. B. Gugjoo
Division of Veterinary Clinical Complex, FVSc & AH, SKUAST-Kashmir,
Jammu and Kashmir, India
A. Pal (*)
Division of Surgery, Indian Veterinary Research Institute, Izatnagar, UP, India
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 99
Springer Nature Singapore Pte Ltd. 2020
M. B. Gugjoo, A. Pal (eds.), Mesenchymal Stem Cell in Veterinary Sciences,
https://doi.org/10.1007/978-981-15-6037-8_7
100 M. B. Gugjoo and A. Pal
et al. 2014; Pelagalli et al. 2018). Sheep being neurologically less developed than
carnivores and equines has an edge in the particular area. Sheep body features like
long bone size and its macrostructure structure are approximate to that of human
(Anderson et al. 1999; Van Der Donk et al. 2001). Due to their comparable bone
biomechanics, biochemistry and histology sheep bone fracture healing turn over
markers may be adopted for human (Sousa et al. 2015). Sheep have sufficient super-
ficial space on their backs that may be utilized to study MSCs regeneration potential
in experimental lesions (Chevrier et al. 2009; Music et al. 2018; Yr et al. 2018). The
current chapter details sheep mesenchymal stem cell (sMSCs) characteristics and
their potential therapeutic applications.
Different tissue sources have variable sMSCs population and proliferation potential.
These cells become senescent after certain passages. Among various sources, sheep
peripheral blood harboured lowest number of MSCs (sPB-MSCs) and had shown
lower ability to proliferate (Lyahyai et al. 2012). MSCs from various sources may
have different proliferation properties. Comparative studies have demonstrated that
sBM-MSCs had higher proliferation potential as compared to cotyledon derived
MSCs (Cot-MSCs) (Ribitsch et al. 2017) and sheep adipose tissue MSCs (sAD-
MSCs) (Heidari et al. 2013). Apart from source based differences, culture expan-
sion may also affect their proliferation. In initial passages, sheep dermis derived
MSCs (sD-MSCs) in comparison to sBM-MSCs may show lower proliferation
potential but that becomes higher with extended passaging (Jahroomishirazi et al.
2014). Likewise sheep liver-MSCs had shorter population doubling time as com-
pared to sAD-MSCs (Heidari et al. 2013). Further incites in this regard are required
to confirm the reason behind such variabilities.
7 Sheep Mesenchymal Stem Cell Basic Research and Potential Applications 101
Table 7.1 Sheep mesenchymal stem cell (MSC) sources and their characteristics (Gugjoo and
Amarpal 2018)
Positive markers Negative markers Differentiation characteristics References
Adipose tissue MSCs (AD-MSCs)
CD29, CD44 – – Grzesiak et al.
(2011)
CD90, CD105, HLA-DR, Adipogenic, chondrogenic, Zorzi et al.
CD73, CD29 CD45,CD34, osteogenic (2015)
STRO-1
CD44, CD90 CD31, CD45 Vahedi et al.
(2016)
CD44 CD31, CD45 Adipogenic, chondrogenic, Feng et al.
osteogenic (2018)
Amniotic fluid MSCs (am-MSCs)
CD13, CD29, CD44, CD45 Adipogenic, chondrogenic, Tian et al.
CD90, CD106, osteogenic (2016)
OCT4
CD44, CD58, CD14, CD31, – Shaw et al.
CD166 CD45 (2011)
Amnion MSCs (A-MSCs)
CD29, CD44, CD31 Adipogenic, osteogenic Klein et al.
CD105, CD90 (2011)
CD166 antigen at CD14, CD31, – Colosimo et al.
low levels and CD29 CD45, CD49f (2013)
and CD58
intermediate levels
Bone marrow MSCs (BM-MSCs)
CD44, CD105, CD45, CD34 Adipogenic, chondrogenic, Mrugala et al.
Vimentin osteogenic (2008)
CD29, CD44, CD14, CD31, Adipogenic, chondrogenic, Mccarty et al.
CD166 CD45 osteogenic (2009)
CD9, CD44, CD45 – Rentsch et al.
CD54,CD73, CD90, (2010)
CD105, CD166
CD44 CD34 Adipogenic, chondrogenic, Czernik et al.
osteogenic (2013)
CD44, CD90 (low CD45, HLA-DR Adipogenic, chondrogenic, Caminal et al.
expression) osteogenic (2014)
CD44, CD58, HLAI, CD31, CD34, – Desantis et al.
CD90 CD45, HLA-DR (2015)
CD29, CD36, CD73, CD45 and CD34 Adipogenic, chondrogenic, Mediano et al.
CD90, CD166, (variable osteogenic, neuronal like cells (2015a, b)
CD29 expression) (downregulation)
CD44, CD90, CD45,CD73 Adipogenic, chondrogenic, Caminal et al.
CD140a, CD105 and osteogenic (2017)
CD166
CD146 CD34, CD45 Adipogenic, chondrogenic, Hindle et al.
osteogenic (2016)
(continued)
102 M. B. Gugjoo and A. Pal
Table 7.1 (continued)
Positive markers Negative markers Differentiation characteristics References
CD73 (96.9 ± 5.9), Negative (CD117 Adipogenic, chondrogenic, Khan et al.
CD90 (99.6% ± 0.3), (0.1%60.1) or low osteogenic (2016)
CD105 (MHCII
(99.16 ± 1.5), (10.5% ± 16.0);
CD271 (97.762.0) CD31
and MHCI (94.0% (14.6% ± 4.2) and
67.2) CD45
(39.4% ± 31.8)
CD90 – Adipogenic, chondrogenic, Landa-Solis
osteogenic et al. (2016)
CD44, CD90, – Adipogenic, chondrogenic, Vivas et al.
CD140a, CD105 and osteogenic (2018)
CD166
Cotyledonary MSCs
CD29, CD33 and CD31, CD45 Adipogenic (weak), Ribitsch et al.
CD166 chondrogenic, osteogenic (2017)
Dermal MSCs (D-MSCs)
𝛽-integrin, CD71, – Adipogenic, chondrogenic, Cui et al. (2014)
CD44 and CD73 osteogenic, neurogenic
CD271 CD45 Adipogenic, chondrogenic, Jahroomishirazi
osteogenic et al. (2014)
Endometrium MSCs (end-MSCs)
CD271 CD49f Adipogenic, chondrogenic, Letouzey et al.
osteogenic, smooth muscle (2015)
Kidney MSCs (K-MSCs)
Oct-4, VIM, CD44, CD34 Adipogenic, chondrogenic, Ji et al. (2016)
FN1, CD73, CD44 hepatocellular
and PAX2
Lung MSCs (L-MSCs)
Oct4, Nanog, CD29, CD34, CD45 Adipogenic, chondrogenic, Ma et al. (2017)
CD44, CD71, CD73 osteogenic, hepatocyte
and CD90
Periodontal ligament MSCs (Pl-MSCs)
CD106, CD44, – – Gronthos et al.
CD166, CBFA-1, (2006)
collagen-I, bone
sialoprotein
Peripheral blood MSCs (PB-MSCs)
CD29, CD73, CD90 CD45, and CD34 Adipogenic, chondrogenic, Lyahyai et al.
and CD105 (low (variable osteogenic, neurogenic (2012)
expression) expression)
CD29, CD36, CD73, CD45 and CD34 Adipogenic, chondrogenic, Mediano et al.
CD90, CD166, (variable osteogenic, neuronal like cells (2015a, b)
CD105 expression) (downregulation)
Stro-1, CD90, CD34, CD45 Adipogenic, Chondrogenic, Hopper et al.
CD106, CD105, Osteogenic, (2015)
CD146, CD166,
CD44
(continued)
7 Sheep Mesenchymal Stem Cell Basic Research and Potential Applications 103
Table 7.1 (continued)
Positive markers Negative markers Differentiation characteristics References
Synovial MSCs (Sy-MSCs)
CD73, CD90, – Adipogenic, chondrogenic, Burk et al.
CD105 osteogenic (2017)
Umbilical cord MSCs (UC-MSCs)
CD44 CD38, CD45, – Fadel et al.
CD41/61. (2011)
CD29, CD44, CD73, CD34 Adipogenic, chondrogenic, Chen et al.
CD90 osteogenic (2018)
Placental MSCs (Pl-MSCs)
CD29, CD44, CD73, CD31, CD45 Early stage Adipogenic, Brown et al.
CD90, CD105, Sox2 gestation chondrogenic, (2016)
CD29, CD44, CD73, CD31, CD45, Late stage osteogenic
CD90, CD105 Sox2 gestation
AD-MSCs adipose tissue MSCs, Am-MSCs amniotic fluid MSCs, A-MSCs amnion MSCs,
BM-MSCs bone marrow MSCs, D-MSCs dermal MSCs, End-MSCs endometrium MSCs, K-MSCs
Kidney MSCs, L-MSCs lung MSCs, Pl-MSCs periodontal ligament MSCs, PB-MSCs peripheral
blood MSCs, Sy-MSCs synovial MSCs, UC-MSCs umbilical cord MSCs, Pl-MSCs placental MSCs
sMSCs express various surface markers and lack haematopoietic markers. Variability
in surface markers of the sMSCs with respect to the tissue source has been demon-
strated (Table 7.1). The variability in marker expression is attributed to the lack of
species specific antibodies, besides differences in the type of tissue sources and
harvesting methods (Gugjoo et al. 2020a, b). Additionally, sMSCs have also been
demonstrated to express various pluripotency markers like Oct4, Nanog and Stro-1
(Hopper et al. 2015; Brown et al. 2016; Ji et al. 2016; Ma et al. 2017).
104 M. B. Gugjoo and A. Pal
sMSCs exhibit variability in their differentiation potential on the basis of their tissue
source. The musculoskeletal differentiation potential of sCot-MSCs and sBM-
MSCs had been akin but sBM-MSCs adipogenic potential was more pronounced
(Ribitsch et al. 2017). Foetal MSCs usually show low PPARγ pathway and as such
may be less demanding for adipogenic activity (Ragni et al. 2013). Among various
musculoskeletal tissues, sheep synovial fluid derived MSCs (sSy-MSCs) had shown
higher chondrogenic expression in comparison to sBM-MSCs (Burk et al. 2017).
However, MSCs from adipose tissue, bone marrow and liver-MSCs had comparable
differentiation activity (Heidari et al. 2013). Such variabilities in differentiation
potential of MSCs with respect to source need further investigation especially on
molecular basis. This may help to better utilize MSCs for therapeutic applications.
Scrapie infected sheep carry MSCs with normal tri-lineage differentiation ability.
However, their proliferation and neurocyte like cell differentiation may be reduced
(Mediano et al. 2015a, b) and as such may offer limited therapeutic benefits in neu-
ral ailments. The cellular characteristics of MSCs may also be influenced by the
factors like feeding standard, age of the animal and disease status of the animal.
Low feeding status of ewe may affect their foetal MSCs characteristics (Pillai et al.
2016). Ageing may or may not decrease MSCs proliferation and clonogenic poten-
tial (Rhodes et al. 2004; Han et al. 2010). In sheep unlike dog, breed variation had
non-significant effect on BM-MSCs proliferation (Rhodes et al. 2004).
MSCs in sheep have been implanted through various routes including parenteral
considering their homing property (Gugjoo and Amarpal 2019). MSCs secretory
properties and differentiation in particular tissue is being determined by the avail-
able local milieu. The cells (including of xenogenic and/or allogeneic sources) have
been well tolerated under in vivo conditions. In a pre-immune sheep foetus, xeno-
genic MSCs (human source) had differentiated into hepatic cells upon intrahepatic/
intraperitoneal implantation (Chamberlain et al. 2007) or secreted insulin upon pan-
creatic transplantation (Ersek et al. 2011). Xenogeneic MSCs too had been demon-
strated to have tissue specific trans-differentiation even in immuno-potent sheep
(Liechty et al. 2000; Mackenzie and Flake 2001; Shaw et al. 2011). Likewise, allo-
geneic applications of these sMSCs had been demonstrated to be safe (Berner et al.
2013; Dooley et al. 2015; McDonald et al. 2015; Zorzi et al. 2015). Apart from
being immuno-compromised, the cells have been demonstrated to have immune-
modulatory properties (Dooley et al. 2015). Considering such immuno-compromised
and immuno-modulatory properties, allogeneic MSCs are increasingly being stud-
ied to make ready-to-use therapy possible.
7 Sheep Mesenchymal Stem Cell Basic Research and Potential Applications 105
In foetal tissues wound healing occurs through regeneration and in adults by repair
(Klein et al. 2011). Sheep has been chosen as wound model as it has sufficient space
on back and limited neurological development in comparison to the canine and
equine (Martinello et al. 2018). In utero implanted xenogeneic or autologous MSCs
have been demonstrated to home to the site of foetal wounds. The implanted cells
have been able to expedite wound healing by increasing the secretion of extracel-
lular matrix (Table 7.2) (Mackenzie and Flake 2001; Klein et al. 2011). Apart from
foetal wounds, adult sheep wounds too tend to heal faster with allogeneic PB-MSCs.
The wound had closed within 15 days and might have been due to the cell induced
inflammation inhibition and enhanced re-epithelialization, neo-vascularization and
contraction. These treated wounds had significantly higher expression of hair-kera-
tine (hKER) and collagen type I (Col1α1) being observed from day 15 up to day 42
(Martinello et al. 2018). It may be concluded that MSCs application are quite prom-
ising for wound management. Further studies are desired to confirm the posology of
the MSCs applications.
An extensive literature on MSCs osteochondral studies exist. The results have been
mostly favourable and without any serious adverse effect as detailed in Table 7.3. A
single study however had failed to demonstrate any obvious improvement with
MSCs as compared to the control (Delling et al. 2015a).
MSCs intra-articular implantation remains safe (Caminal et al. 2014; Abdulmula
et al. 2017). Intra-articularly implanted cells remain viable and attached to the joint
structures (Delling et al. 2015b; Feng et al. 2018; Markides et al. 2019). The avail-
able inflammatory environment however, may reduce their chondrogenic potential,
although without affecting their phenotype (Ando et al. 2012). MSCs possible path-
way of neo-cartilage formation may be ensued via endochondral ossification pro-
cess (Lydon et al. 2019). MSCs tend to undergo hypertrophic chondrocyte formation
and may lead to osteogenesis (Gugjoo et al. 2018).
Intravenous implantation of BM-MSCs had been able to reduce lameness, joint
pain and swelling in a sheep mono-arthritis model. The experimental joints had less
cartilage erosions, limited inflammatory cells and were supplemented with activated
stromal cells and angiogenesis (Abdalmula et al. 2017). In chronic induced osteoar-
thritis models, improved parameters (histological and/micro-CT scores) of healed
Table 7.2 In vivo preclinical experimental mesenchymal stem cell studies on cutaneous wounds
106
Model
defect
Number of size/study Biomaterial Cell dose/
Model type animals period used assembly Evaluation criteria Overall result References
In utero 29 animals 13 months Xenogeneic 5–20 × 106 Polymerase chain PCR confirmed cellular engraftment Mackenzie
tracking of (human) early reaction (PCR), to one or more tissues. Relatively and Flake
cells to MSCs gestation immuno- large engraftment was seen in (2001)
wounds or (65 days histochemistry and wounded areas. Cells persisted in
developing gestation); in situ hybridization foetal tissue up to a year
organs 1 × 108 cells/
kg (mid-
gestation,
85 days
gestation)
Full Six wounds 15 and Allogeneic 1 × 106 Clinical, At 15 days post-lesion, the wounds Martinello
thickness (three wounds 42 days PB-MSCs, (injected in histopathological, treated with MSCs showed a higher et al.
square treated with hyaluronic wound and molecular degree of wound closure, a higher (2018)
wound conventional acid margins), analysis percentage of re-epithelialization,
(4 × 4 cm, topical cream or 1 × 106 proliferation, neo-vascularization and
six wounds) gel, fourth with (loaded with increased contraction in comparison
cold ionized hyaluronic to a control group. Later, the wounds
plasma, fifth acid and treated with MSCs had more mature
with PBS and implanted at and denser cutaneous adnexa as
sixth with wound compared to the control group with
MSCs) centre) absence of inflammation and
expression of CD3+ and CD20+.
Moreover, the mRNA expression of
hair-keratine (hKER) and collagen I
was observed in the MSCs treated
group 15 days
M. B. Gugjoo and A. Pal
Table 7.3 In vivo chondrogenic preclinical experimental mesenchymal stem cell studies in sheep
Model defect size/
Model type Number of animals study period Biomaterial used Cell dose Evaluation criteria Overall result References
Medial 28 (n = 16, cell + 8 mm (diameter) and Autologous 3 × 107 Macroscopic observation, Experimental animal Guo et al.
femoral β-TCP; n = 8 in 4 mm BM-MSCs + beta- histological, immuno- defects were (2004)
condyle β-TCP only and (depth)/24 weeks tricalcium phosphate histochemical, biochemical resurfaced with
defect n = 4 in control) (β-TCP) analysis hyaline-like tissue.
An ideal interface
formed between the
engineered cartilage,
adjacent normal
cartilage and the
underlying bone
Osteonecrosis 8 animals (n = 4 in 10 ml of absolute Sheep BM-MSCs 1 × 106 (each Light microscopy Better bone Feitosa et al.
of femoral control group; n = 2 ethanol induced (transfected) and cell type) regeneration in cell (2010)
head each in sheep MSC human dental stem treated group
group and human cells animals
MSC group treated
after 8 weeks of
induced necrosis)
Chronic 10 animals (n = 40 7 mm/6 months Autologous 4 × 105 MSCs Histopathology Group I had Zscharnack
model of defects; group I: BM-MSCs /collagen mixed with significantly better et al. (2010)
medial chondrogenically I hydrogel constructs collagen I histologic scores
femoral differentiated MSC/ with morphologic
condyles hydrogel constructs; characteristics of
osteochondral group II: hyaline cartilage
lesions Undifferentiated such as
ovine MSC/hydrogel columnarization and
constructs; group III: presence of collagen
7 Sheep Mesenchymal Stem Cell Basic Research and Potential Applications
had significantly
better cartilage than
control
(continued)
109
Table 7.3 (continued)
110
tissue had been observed with the implantation of BM-MSCs (Caminal et al. 2014)
and AD-MSCs (Feng et al. 2018). The variable concentrations of AD-MSCs
implanted along with hyaluronic acid at 3 and 6 weeks post injury were viable for
more than 2 months and had restricted osteoarthritis progression (Feng et al. 2018).
Even xenogenic AD-MSCs (human) along with scaffold (chitosan/collagen) had
significantly improved ICRS-I score in a femoral condyle partial thickness defect as
compared to the scaffold treated and control (Zorzi et al. 2015).
The hyaline tissue formation has not been reported in all the studies and indi-
vidual cases with some demonstrating chondroid metaplasia. The integration of
healed tissue to native cartilage too had been the problem (Caminal et al. 2014). It
may thus, be concluded that MSCs may repair the osteochondral injuries but does
not guarantee the same (Gugjoo et al. 2020a, b).
One of the sheep osteoarthritis model studies had failed to yield improved results
at 12 weeks post autologous MSCs implantation (Delling et al. 2015a). This may
have been due to the weak joint injury failing to induce obvious osteoarthritic
changes or else due to the less concentration of implanted cells (Feng et al. 2018).
Bone tissue engineering is increasing by each passing day to ensure early and better
healing (Chandran et al. 2017). MSCs due to their characteristic properties are being
made subject. Most of the sheep preclinical experimental bone model studies have
shown better and early bone healing upon MSCs implantation as enlisted in table
(Table 7.4). One of the sheep study had shown early osteogenic synthesis with scaf-
fold laden sMSCs, implanted subcutaneously (Boos et al. 2014). Apart from culture
expanded MSCs, bone marrow mononuclear cells (contains MSCs) along with
sheep bone mineral too have favoured improved sinus augmentation at 8 and
16 weeks in comparison to the autogenous graft (Gutwald et al. 2010).
MSCs bone healing potential has been evaluated in different sheep bone models.
Among various bones studied are metatarsal-, tibial- mandible- and cranial-critical
bone defects. Mostly autologous or allogeneic BM-MSCs have been evaluated in
these studies. BM-MSCs along with coral granules (Viateau et al. 2007; Manassero
et al. 2013), cortical allograft (Fernandes et al. 2014), polycaprolactone and
hydroxyapatite (Berner et al. 2015), serum scaffold (Gallego et al. 2015) and cal-
cium alginate (Shang et al. 2001) have been implanted to evaluate healing potential
in critical size bone defects. The implantation of scaffold laden cells had led to an
improved early bone healing with better structural and morphological features
(Fernandes et al. 2014; Gallego et al. 2015). The healed tissue had better quality and
approximated to normal bone tissue (Gallego et al. 2015). The healed tissue strength
could commensurate with clinical standards (Berner et al. 2015). In all these cell
and scaffold treated defects healing was much improved as compared to those of
scaffold treated. Incorporation of growth factor like rhBMP-2 to BM-MSCs and/
autologous serum loaded on to the scaffold and implanted subcutaneously had led
to subcutaneous ossification (Boos et al. 2014).
Cell immunogenicity may or may not affect healing potential of MSCs.
Autologous and allogeneic MSCs had shown comparable tibial bone healing
(Berner et al. 2013) while xenogeneic BM-MSCs had failed to improve sheep tibial
bone healing (Niemeyer et al. 2010). Mononuclear cells (MNCs) may show
improved healing over raw bone marrow concentrate (Ardjomandi et al. 2015).
MSCs may also be effective to repair osteoporotic bone (Chandran et al. 2017).
MSCs along with the tricalcium phosphate scaffold or graft may also be used to
promote interbody spinal fusion and spinal arthrodesis (Table 7.4).
Combination of MSCs and other implants/scaffolds like TiAl6V4 constructs
with a calcium–phosphate coating (Garcıa-Gareta et al. 2015) and polyetherke-
toneketone (Adamzyk et al. 2016) had failed to enhance healing in a sheep trabecu-
lar bone and calvarial defects, respectively. The scaffolds although had favoured in
vitro cell adhesion, growth and osteogenic differentiation of MSCs (Adamzyk et al.
Table 7.4 In vivo preclinical experimental osteogenic mesenchymal stem cell studies in sheep
114
Model defect
size/study
Model type Number of animals period Biomaterial used Cell dose/assembly Evaluation criteria Overall result References
Tibial defects 4 animals (cell-loaded 2-month Autologous 0.5–1.0 × 108 in Indentation assay In cell-loaded implants, Kon et al.
implants, n = 2; BM-MSCs, fibrinogen bone formation occurred (2000)
cell-free HAC cylinders, hydroxyapatite both within the internal
n = 2) ceramic (HAC) macropore space and
around the HAC cylinder
while in control cell-free
implants, bone formation
was limited mostly to the
outer surface and was not
observed in most of the
inner pores. As tested in
an indentation assay, the
stiffness of the complex
HAC-bone material was
found to be higher in
cell-loaded implants as
compared to controls
Cranial bone 8 animals with 16 6 weeks and Autologous – Gross, histological New bone tissues were Shang et al.
defect defects (MSCs and 18 weeks post BM-MSCs and and computerized observed with MSCs/ (2001)
(parietal calcium alginate, n = 8; repair calcium alginate tomography (CT) scan scaffold as early as
bone, 20 mm calcium alginate, n = 4; 6 weeks post-repairing
in diameter, control unrepaired, but not in control groups.
bilateral) n = 4) The engineered bone
tissue became more
mature at 18 weeks
post-repairing. CT scan
revealed an almost
complete repair of the
defect of MSCs/scaffold
at 18 weeks
M. B. Gugjoo and A. Pal
Left 21 animals (group I: 6 months Autologous 138 ± 22 (coral Radiology, histology Non-significant Viateau et al.
metatarsal Empty, n = 5; group II: BM-MSCs pieces), and CT differences in newly (2007)
bone defect Coral scaffold, n = 4; 8.28 ± 1.32 × 106 formed bone in defects
(25 mm) group III: Coral (BM-MScs) were seen in coral/MSCs
stabilized scaffold/MSCs, n = 6; and autograft groups.
with plate autogenous cortico- Significant differences in
and PMMA cancellous graft, n = 6) radiological scores in
space being MSCs/coral and autograft
replaced after groups were seen (21%
6 weeks and 100%, respectively)
Postero- 24 (autograft (AG), 6 months Bone marrow – Micro-CT, manual 33% and 25% of the Gupta et al.
lateral n = 6; cell enriched (selective cell palpation and MSCs and AG groups (2007)
lumbar tricalcium phosphate retention technology), histology had fused vertebrae while
fusion (TCP), n = 6; TCP tricalcium phosphate bone marrow/TCP had
alone, n = 6; TCP/10 ml 8% and TCP alone had
bone marrow, n = 6) 0% fusion
Critical sized 45 animals (n = 15 in 20–24 weeks Autologous 70–100 × 106 Micro-radiography, Radiography revealed Giannoni
tibial defect each group) autologous BM-MSCs, silicon histology and vascular best healing in cell/ et al. (2008)
(stabilized by bone chips of iliac crest stabilized tricalcium density scaffold treated group.
internal graft; ceramic scaffold phosphate Histology showed better
locking only; ceramic vascular density in cell/
compression scaffold + cells scaffold group. Partial
plate and bone deposition occurred
external at periphery of bone
skeletal stumps (scaffold group)
fixator)
(continued)
7 Sheep Mesenchymal Stem Cell Basic Research and Potential Applications
115
Table 7.4 (continued)
116
Model defect
size/study Cell dose/
Model type Number of animals period Biomaterial used assembly Evaluation criteria Overall result References
Anterior 18 animals (gelfoam 3 months Allogeneic MSCs, 1 × 106 Radiology and New bone formation in Goldschlager
cervical sponge, n = 6; Gelfoam sponge, histology gelfoam sponge only and et al. (2010)
discectomy MSCs + gelfoam, n = 6; pentosan polysulphate MSCs/gelfoam group
(C3-C4 and gelfoam sponge + (PPS) was observed in 9/12,
C4-C5) MSCs + pentosan 11/12 animals,
polysulphate, n = 6) respectively. In group
additionally implanted
with PPS had
significantly higher
cartilaginous tissue
within the interbody
cages as compared to
other groups. Only
1/12 in the PPS group
had new bone formation
Critical sized 21 animals (treated 3 months Human BM-MSCs 2 × 107 (oBM- Radiology and Radiology and histology Niemeyer
tibial bone with mineralized (n = 2 each and oBM-MSCs and MSCs or human histology revealed significantly et al. (2010)
defect (3 cm, collagen, n = 7; human group) and mineralized collagen BM-MSCs) better bone formation
stabilized by MSCs, n = 7 and ovine 6 months scaffold (matrices) upon oBM-MSCs seeded
plate) MSCs, n = 7) (n = 5) on matrices implantation
compared to matrices and
human BM-MSCs
seeded matrices
implantation. However,
human BM-MSCs did
not evoke any local or
systemic reaction
M. B. Gugjoo and A. Pal
Critical size 24 animals (control 9 months BM-MSCs 225 × 106 Radiology, CT, The cell treated group Field et al.
tibial scaffold only, n = 12; biomechanical, had significantly higher (2011)
segmental cell + scaffold) histology callus formation and rate
defect (3cmm of bone apposition in the
length and defect
8 mm
diameter)
(stabilized by
interlocking
nail)
Cervical 30 animals (autograft 3 months Allogeneic BM-MSCs 5 × 106 and CT, functional 9/12 of the cell/scaffold Goldschlager
interbody (AG) implantation, + hydroxyapatite/ 10 × 106 radiography, treated animals had et al. (2011)
fusion n = 6; hydroxyapatite/ tricalcium phosphate histomorphological, continuous bony bridging
(C3-C4) tricalcium phosphate (HA/TCP) biomechanical while AG had 1/6 and
(HA/TCP), n = 6; HA/ analysis HA/TCP had 2/6. New
TCP + 5million cells, bone density was 121%
n = 6; HA/TCP + ten higher in cell groups
million cells, n = 6; compared to HA/TCP
control unoperated and 128% as compared
sham, n = 6) to AG groups. Functional
radiography revealed
significantly reduced
macromotion between
C3/C4
Critical sized 24 animals (6 in 12 weeks BM-MSCs + scaffold 35 × 106 Radiology, No significant differences Berner et al.
tibial allogeneic MSCs (polycaprolactone + micro-computed in bone formation in (2013)
segmental treated + scaffold; 6 in β-tricalcium phosphate) tomography allogeneic and
bone defect autologous cell (micro-CT), autologous groups but
treated + scaffold; 6 biomechanical testing, better bone formation
7 Sheep Mesenchymal Stem Cell Basic Research and Potential Applications
(continued)
Table 7.4 (continued)
118
Model defect
size/study Cell dose/
Model type Number of animals period Biomaterial used assembly Evaluation criteria Overall result References
Metatarsal 10 animals (scaffold, 6 months BM-MSCs + acropora 7.5 ± 1.2 × 106 Micro-CT and Two-fold increase in Manassero
bone defect n = 4; MSCs on coral scaffold histology bone formation with et al. (2013)
(25 mm) scaffold, n = 5) cell + scaffold
supported by transplantation as
3.5 mm compared to scaffold
dynamic only at 6 months
compression
plate
Radial and 7 animals (n = 2 tibial 3 and BM-MSCs, PRP and 30–50 × 106 Clinical evaluation, At 6 months significant Casanas
tibial nerve nerve and n = 2 radial 6 months scaffold (neurolacRX (repair of nerve neuro-physiological myelinated nerve fibres et al. (2014)
section- nerves: Treated with biodegradable after 3 weeks) study, morphology and increased conduction
resection scaffold + PRP; n = 4 scaffold) study in MSCs treated with or
(1 cm) tibial nerve and n = 2 without PRP
radial nerves; control
n = 1 tibial nerve and
n = 3 radial nerves:
Scaffold + pooled
human serum)
Postero- 34 animals (lumbar 6 months Osteo-induced 50 × 106 Clinical observation, Lumbar fusion rates were Cuenca-
lateral fusions: Treated with BM-MSCs, scaffolds, radiography, CT, higher for auto and Lopez et al.
lumbar autograft n = 17 on right mineral scaffold and histology, allograft (70%) than (2014)
fusion half and allograft on left hybrid scaffold histomorphometry mineral scaffold (22%)
(L4–L5) half n = 17; and hybrid scaffold
hydroxyapatite n = 17 (35%) based on histology
on left half; and CT. Quantity of bone
hydroxyapatite + cells formation is also higher
(hybrid scaffold) on in autograft and allograft.
right half n = 17) Hybrid scaffold had
higher but statistically
insignificant differences
from hydroxyapatite
M. B. Gugjoo and A. Pal
scaffold based on
histology
Midshaft 8 animals (n = 4 18 weeks Autologous 6 × 107 Radiography, CT and More conspicuous Fernandes
critical sized control; n = 4 treatment BM-MSCs on histology healing and union et al. (2014)
tibial bone group) allogeneic graft between host bone and
defect allograft in cell treated
group. Complete
remodelling at 18 weeks
in cell treated groups.
Histologically marrow
cavity re-established,
intertrabecular spaces
filled with adipose
marrow
Femoral 12 animals (right femur 8 and 16 week BM-MSCs derived 2 × 106 loaded on Radiography, Bone defect was Li et al.
defect treated with scaffold osteoblasts + β-TCP β-TCP histology successfully repaired at (2014)
(10 mm plus cells; left femur 16 week. Sufficient
diameter and with scaffold) volume of bone tissue
3 cm deep) volume formed in cell
groups compared to
scaffold only
Postero- 6 animals (group I: 12 weeks BM-MSCs, 5–6 × 107 Radiography, manual TCP/HA achieved Shamsul
lateral spinal Cell-seeded hydroxyapatite (HA), palpation, histology, superior lumbar et al. (2014)
fusion hydroxyapatite; at L-L2; tricalcium scanning electron inter-transverse fusion
(three-level group II: Cell-seeded phosphate (TCP) microscopy compared to HA but
bilateral HA/tricalcium autograft had best fusion
postero- phosphate at L2–L3;
lateral autograft at L5–L6)
inter-
transverse
fusion,
7 Sheep Mesenchymal Stem Cell Basic Research and Potential Applications
L1–L6)
(continued)
119
Table 7.4 (continued)
120
Model defect
size/study Cell dose/
Model type Number of animals period Biomaterial used assembly Evaluation criteria Overall result References
Sinus 16 animals (ficoll 8 and BM-MSCs, bone – Histological and The approach of using Ardjomandi
augmentation density isolated MSCs, 16 weeks marrow aspirate, histomorphometrical, BBM and MSCs in et al. (2015)
n = 8; bone marrow bovine bone mineral CT combination with fibrin
aspirate, n = 8) adhesive was sufficient
for new bone formation
as the FICOLL
experiment indicated.
However, due to
significantly lower cell
numbers isolated using
the BMAC in sheep, less
new bone was formed in
the test arm
Critical sized 24 animals 12 months Allogeneic 100 × 106 cells Biomechanical testing Improved bone Berner et al.
tibial defect (polycaprolactone- BM-MSCs, PCL/HA and micro-CT, regeneration in cell/ (2015)
(3 cm, plate hydroxyapatite scaffold (4 weeks after defect histology and scaffold group
stabilized) (PCL/HA), n = 8; PCL/ surgery) immune- comparable to autograft
HA + 100x106, n = 8; histochemistry, group
autologous bone defect, scanning electron
n = 6) microscopy
Segmental 15 animals (n = 5 12 and Autologous 25–30 × 106 Computed and Complete bone healing in Gallego
mandibular control, serum scaffold; 32 weeks BM-MSCs and micro-CT treatment group was et al. (2015)
bone defect n = 10 treatment: cell + autologous serum observed at 12 and
scaffold) scaffold 32 weeks but only at
32 week in control
M. B. Gugjoo and A. Pal
Lumbar 36 animals [n = 8 16 weeks Allogeneic 2.5 × 106; Plain radiography, CT, No adverse reaction to Wheeler
interbody cortico-cancellous BM-MSCs, scaffold 6.25 × 106; histopathology, cell implantation. The et al. (2016)
spine fusion autograft; n = 8 in (hydroxyapatite, 12.5 × 106 histomorphometric cell/scaffold implantation
lower concentration tricalcium phosphate and biomechanical achieved similar or better
(2.5 × 106) cell group; and type I collagen) competency fusion as that of autograft
n = 8 in medium after 16 weeks
cell concentration
(6.25 × 106) group;
n = 8 in higher
cell concentration
(12.5 × 106) group]
Tibial (2 10 (n = 8 defects 2 and Autologous 2 × 106 seeded on Macroscopy, Tibial bone is Lovati et al.
holes each in per animals, group I: 4 months osteodifferentiated scaffold radiography, characterized by limited (2016)
right and left) Scaffold without cells; BM-MSCs + micro-CT and repair capability as
and femoral group II: Scaffold hydroxyapatite histology compared to femur in
defects (2 seeded with cells in which cell implantation
holes each in static conditions; group is not crucial. Dynamic
right and left) III: Scaffold seeded cell-loaded implants
(8 mm with cells in dynamic performed better
diameter and conditions; group IV: compared to groups I and
4 mm deep) Control empty defects) II
(continued)
7 Sheep Mesenchymal Stem Cell Basic Research and Potential Applications
121
Table 7.4 (continued)
122
Model defect
size/study Cell dose/
Model type Number of animals period Biomaterial used assembly Evaluation criteria Overall result References
Osteoporotic 8 animals 10 months AD-MSCs, strontium – Histological, cSrHA along with MSCs Chandran
bone model hydroxyapatite histomorphometry, enhanced osteogenic et al. (2017)
(ovariectomy (SrHA) micro-CT and ability with mature
induced, elemental analysis lamellar bone formation
12 mm × 4 mm as compared to bare HA,
defect size) SrHA and tissue
engineered HA implanted
groups.
Histomorphometry data
substantiated improved
osteogenesis on par with
material resorption, as
cSrHA implanted group
exhibited highest
regeneration ratio.
Density histograms from
micro-CT further
signified the enhanced
osteointegrative ability of
cSrHA implants. The
therapeutic potential of
cSrHA in osteoporotic
bone healing and
proposes the use of
allogeneic AD-MSCs for
fabricating “off the shelf
tissue engineered
products”
M. B. Gugjoo and A. Pal
7 Sheep Mesenchymal Stem Cell Basic Research and Potential Applications 123
2016). Thus, ex vivo cell compatible tissue engineered constructs may not necessar-
ily prove useful under in vivo environment.
The tendons and ligaments have limited ability to self-heal due to low cell concen-
tration and associated low matrix turnover. For proper functioning, the healed tissue
should be free of adhesions (Gugjoo et al. 2018a). Healing potential of stem cells
has been studied in various types of sheep tendon models. An improved healing but
variably has been demonstrated (Table 7.5).
MSCs from bone marrow (Crovace et al. 2008; Lacitignola et al. 2014), amniotic
fluid (Colosimo et al. 2013), and peripheral blood (Martinello et al. 2013) has been
demonstrated to improve tendon healing in sheep collagenase-induced Achilles and
digital flexor tendinitis, respectively. Tissue remodelling and improved structural
organization was observed in healed tendons (Martinello et al. 2013). Even mono-
nuclear cells (MNCs) too had improved tendon healing (Crovace et al. 2008).
BM-MSCs implanted locally were demonstrated to survive up to 6 weeks and
engraft efficiently (Colosimo et al. 2013; Lacitignola et al. 2014). MSCs and MNCs
had shown comparable fibre and extracellular matrix restoration in treated tendons
(Table 7.5). However, the CD34 positive cells were highly appreciable in the
BM-MNCs treated cases (Crovace et al. 2008). Similarly, PB-MSCs implantation in
sheep digital flexor tendon (DFT) model had improved the healing of tendon.
MSCs from amnion (Am-MSCs) together with scaffold had improved diaphrag-
matic tendon repair. The repaired tendon tissues had better functional and mechani-
cal parameters in comparison to the tendons treated with acellular grafts (Fuchs
et al. 2004; Turner et al. 2011). Implantation of MSCs along with demineralised
bone matrix in a sheep tendon-bone defect model also had promoted healing. Tissue
repaired with MSCs laden on allogeneic graft had better mechanical and histologi-
cal properties as compared to that of the xenogeneic graft assembly (Thangarajah
et al. 2016).
Model
defect size/ Cell dose/
Model type Number of animals study period Biomaterial used assembly Evaluation criteria Overall result References
Tendinitis of 30 (n = 6 each 8 weeks BM-MSCs, 1.1 × 106 ± 1.4 × 105 Histomorphology, BM-MSCs and Crovace
the Achilles group: BM-MSCs; after BM-MNCs and (MSCs); immuno- BM-MNCs showed et al. (2008)
tendon BM-MNCs; fibrin; treatment fibrin glue after 101 × 106 ± 26 × 106 histochemistry of similar restoration of
(collagenase- saline; sham 2 weeks of (MNCs) collagen type I, III, the architecture of
induced) control) induced cartilage fibres and extra
tendinitis Oligomeric matrix cellular matrix
protein (COMP) (ECM), with a high
and CD34 positive expression of
cells expression collagen type I and
COMP and a very
low expression of
collagen type III in
treated tendons. The
presence of CD34
positive cells was
appreciable in the
BMMNC group
while few cBMSC
showed this cluster
of differentiation,
not expressed in
tendons treated with
fibrin or saline
M. B. Gugjoo and A. Pal
Deep digital 18 animals (groups 30 and Autologous PRP Clinical, Significant Martinello
flexor tendons 1 and 4 received 120 days PB-MSCs, PRP, (882 ± 199 × 103/ ultrasonographic, differences were et al. (2013)
lesions injections of 1 ml of hyaluronic acid ml); MSCs histology, found between
(bilateral) PRP into the (only left limb (10 × 106) immuno- treated groups and
tendon; groups 2 lesions treated histochemical their corresponding
and 5 1 ml of while expression controls (placebo)
PRP + autologous contralateral limb regarding tendon
MSCs; groups 3 and served as control) morphology and
6 received extracellular matrix
autologous (ECM) composition.
PB-MSCs in 1 ml of However, our results
hyaluronic acid) indicate that the
combined use of
PRP and MSCs did
not produce an
additive or
synergistic
regenerative
response and
highlighted the
predominant effect
of MSCs on tendon
healing, enhanced
tissue remodelling
and improved
structural
organization
7 Sheep Mesenchymal Stem Cell Basic Research and Potential Applications
(continued)
125
Table 7.5 (continued)
126
Model
defect size/ Cell dose/
Model type Number of animals study period Biomaterial used assembly Evaluation criteria Overall result References
Tendinitis of 9 animals 3, 4 and Allogeneic 6 × 107 after Evaluation for BM-MSCs into Lacitignola
the Achilles (BM-MSCs/fibrin 6 weeks BM-MSC 2 weeks morphology, tendon lesions had et al. (2014)
tendon glue, n = 9; fibrin (transfected with collagen I positive effects on
(collagenase- glue, n = 9) red fluorescent deposition and the injured tendons.
induced, protein), fibrin presence of The BM-MSCs
bilateral) glue CD34 + cells survived at three,
four and 6 weeks
after treatment.
These cells enhanced
quality healing of
tendons as compared
to the controls,
where no labelled
cells were detected.
Interestingly, we
demonstrated high
expression of
CD34+ cells in
tendons that had
been treated with
BM-MSCs
M. B. Gugjoo and A. Pal
Tendon defect 10 animals 6, 9 and BM-MNCs – Force plate Allograft was Thangarajah
model of (cells + Xenogenic 12 weeks analysis, CT, associated with et al. (2016)
retraction demineralised bone histology significantly higher
matrix, n = 5; weight bearing as
cells + allogeneic compared to
demineralised bone xenograft. Greater
matrix, n = 5) remodelling of
demineralised bone
matrix into tendon
like tissue at defect
area and more direct
type of enthesis
characterized by
significantly more
fibrocartilage. No
failure of tendon-
bone healing was
noted in either group
(continued)
7 Sheep Mesenchymal Stem Cell Basic Research and Potential Applications
127
Table 7.5 (continued)
128
Model
defect size/ Cell dose/
Model type Number of animals study period Biomaterial used assembly Evaluation criteria Overall result References
Lesion of 50 (group 1: 1 day, and Magnetic 5 × 106 MRI, histology Cell treated limbs Khan et al.
lateral branch Sacrificed at 1 day 1–2, 4 12 iron-oxide and flow indicated cellular (2018)
of the ovine (n = 2), treated with and nanoparticles cytometry distribution
deep digital MION-labelled 24 weeks labelled throughout the
flexor tendon MSCs. Group 2: autologous tendon synovial
(lateral Sacrificed BM-MSCs sheath but restricted
border) 1–2 weeks (n = 4), (implanted to the synovial
three sheep treated 2 weeks after tissues, with no
with MION-labelled lesion) MSCs detected in
MSCs and one the tendon or
sheep with surgical lesion. The
PBS. Group 3: lesion was
4 weeks (n = 12), associated with
six sheep treated negligible morbidity
with non-labelled with minimal
MSCs and six sheep inflammation
with PBS. Group 4: post-surgery.
12 weeks (n = 16), Evaluation of both
eight sheep treated cell treated and
with non-labelled control lesions
MSCs and eight showed no evidence
sheep with of healing of the
PBS. Group 5: lesion at 4, 12 and
24 weeks (n = 16), 24 weeks on gross
eight sheep treated and histological
with non-labelled examination
MSCs and eight
sheep with PBS)
M. B. Gugjoo and A. Pal
Table 7.6 In vivo preclinical mesenchymal stem cell studies on cardiac/arterial models in sheep
Number of Model defect Cell dose/
Model type animals size/study period Biomaterial used assembly Evaluation criteria Overall result References
Foetal cardiac 21 animals Analysis done at GFP transducted – Immuno- Cells were detected in 20/21 foetal Airey et al.
development 115–131 days of MSCs from histochemistry hearts. At least 10 areas of cell (2004)
(intraperitoneal gestation (appx human BM, engraftment were reported. Human
implantation of 50–55 days later) foetal brain and areas represented 43.2% areas of
MSCs into sheep liver the total Purkinje fibre areas. No
foetus at difference in engraftment could be
55–62 days of observed based on cell type.
gestation) ~0.01% cardiomyocytes were from
human origin in sheep foetal heart.
In conclusion, stem cells take part
in development of heart
Myocardial 47 animals (group 8 weeks Stro-3 positive 25 × 106; Echocardiography, End diastolic volume increased in Dixon et al.
infarction I: 25 million cells, cells derived 75 × 106; histology, collagen all groups but in group I and II, it (2009)
(coronary n = 7; group II: 75 from BM 225 × 106; content and MMPs was attenuated. ICTP, an index of
ligation) Million cells, 450 × 106 level of collagen degradation was more in
n = 7; group III: (injected at myocardium group I. Matrix metalloproteinases
225 Million cells, border zone (MMPs) were higher in group I as
n = 10; group IV: after 1 h) compared to other groups
450 Million cells, including group V
n = 8; group V:
Free media group)
Transmural 46 animals (group 4 and 8 weeks Stro-3 positive 25 × 106; Echocardiography, No evidence of myocardial Hamamoto
myocardial I: 25 million cells, cells derived 75 × 106; CD31 and α smooth regeneration. Low dose cell groups et al. (2009)
infarction n = 7; group II: 75 from BM 225 × 106; muscle actin had better ejection fraction and
(coronary Million cells, 450 × 106 immune- CD31 and α smooth muscle actin
ligation) n = 7; group III: (injected at histochemistry immune-histochemistry showed
7 Sheep Mesenchymal Stem Cell Basic Research and Potential Applications
(continued)
129
Table 7.6 (continued)
130
(continued)
133
Table 7.6 (continued)
134
The infusion rate may also affect the results. In normal heart, 1.25 million cells
per minute had appeared safer as compared to the 2.5 million cells that had led to
the myocardial necrosis. In an infarcted myocardium, MSCs at the rate of 01 million
cells/min had led to sluggish coronary flow after 25 million cell implantation while
the cell implantation at 0.5 million cells had led to sluggish flow after 40 million
dose (Houtgraaf et al. 2013).
Intraperitoneal implantation of xenogeneic MSCs from BM, foetal liver at
55–62 days of gestation had engrafted into heart and exhibited properties of various
cardiac cells (Airey et al. 2004). These xenogenic MSCs too had enhanced myocar-
dial perfusion although without any significant effect on ejection fraction (Dayan
et al. 2016). To evaluate long term effects of MSCs extended studies are desired.
Bone marrow implantation may not be feasible option to treat myocardial infarction
(Bel et al. 2003).
Even transfection of MSCs with some specific factors may improve their healing
potential. Among notable options are hypoxia-inducible factor 1-α (HIF1-α) and
vascular endothelial growth factor (VEGF). BMSCs transfected with these factors
had significantly reduced infarct size in a sheep acute myocardial infarction (AMI)
model. Improved results might have been due to enhanced angio−/arteriogenesis
through cell paracrine secretion (Locatelli et al. 2015; Hnatiuk et al. 2016).
Apart from myocardial repair, MSCs have been evaluated in skeletal and blood
vessel repair. MSCs had significantly improved rotator muscle cuff repair irrespec-
tive of their source. Experimental muscles had increased contraction force, vascu-
larity, and increased myocyte concentration over adipocytes (Coleman et al. 2011).
For blood vessel repair, MSCs have been employed along with tissue engineered
scaffold patches (Mendelson et al. 2007; Zhao et al. 2010). The cell-seeded scaffold
patches implanted in sheep pulmonary artery had remodelled to a layered and viable
tissue. The tissue had been well integrated into the native arterial wall. The key
remodelling processes included are intimal overgrowth at the luminal surface (pan-
nus formation; neointima) and granulation tissue formation, and fibrosis with for-
eign body reaction (Mendelson et al. 2007). MSCs laden tissue engineered blood
vessels (TEBVs) had also been implanted in case of carotid artery resection model.
The constructs were patent, anti-thrombotic and stable up to 5 months. TEBVs had
smooth muscle, endothelium and collagen and elastin up to 5 months. Non-seeded
grafts contrarily were occluded within 2 weeks (Zhao et al. 2010).
Model
Number of defect size/ Biomaterial Cell dose/
Model type animals study period used assembly Evaluation criteria Overall result References
Radial and tibial 7 animals 3 and BM-MSCs, 30–50 × 106 Histological, At 3 months period Casañas
nerve resection (MSCs + 6 months repairmen with immuno- results were not good. At et al. (2014)
(1 cm) scaffolds) Neurolac™ histochemistry and 6 months results showed
biocamera Morphometry significantly myelined
scaffold nerve fibres with
increased conduction as
compared to control in
both radial and tibial
nerves
In utero repair of 4 animals (1 case 145 days Placenta 5 × 105 Histology and Increased preservation of Brown et al.
Myelomeningocele treated with term of derived MSCs (early motor assessment spinal cord architecture (2016)
(at 75 days of derived placental pregnancy in collagen gel gestation and large neurons in early
pregnancy) MSCs + foetal at delivery MSCs); gestation MSCs treated
membrane (FM) time 1 × 106 compared to others. The
patch; other (term animal treated with early
with early gestation gestation MSCs was
gestation MSCs) capable of normal
placental ambulation. Limited
MSCs + FM distal motor function in
patch; others
1 case with FM
only; 1 case
with skin closure
only)
Myelomeningocele 12 animals (cell At term Human 5 × 105 Motor functional, Significant and clinically Wang et al.
(created at 75 day of treated, n = 6; (145 days) placental MSCs histological and relevant improvement of (2015)
pregnancy) control, n = 6) immune- motor function with
histochemical increased preservation of
M. B. Gugjoo and A. Pal
(continued)
137
Table 7.7 (continued)
138
Model
Number of defect size/ Biomaterial Cell dose/
Model type animals study period used assembly Evaluation criteria Overall result References
Intervertebral disc 8 animals 6, 9 and Allogeneic 1 × 106 Radiography, MRI, Significant improvement Freeman
degeneration (IVD) (MSCs into 12 months BM-MSCs biochemical and in disc height index and et al. (2016)
(Postero-lateral nucleosus 6 months after histology disc height in both cell
annulotomy at 3 pulposus; MSCs defect creation treatment groups at
levels: L3/L4, L4/L5, into annulus 6 months. Mean
L5/L6) fibrosus and Pfirrmann and
phosphate saline histopathological grade
control) improved in cell treated
animals. Spontaneous
repair of antero-lateral
annular lesion in cell
treated groups
Intervertebral disc 45 animals 3 and Allogeneic 1 × 107 Histology and gene MSC treatment prevented Dowdell
degeneration (control n = 10; 6 months BM-MSCs expression increased adipose and et al. (2017)
(antero-lateral early treatment connective tissue
annulus fibrosus group at formation, normally forms
lesion; 20 mm wide 4 weeks after after damage. MSCs
6 mm deep: Lesion at lesion creation, treatment did not prevent
L1–L2, L3–L4 and n = 24; late slow-to-fast muscle fibre
L5–L6) treatment group formation. Alteration in
at 12 weeks, gene expression of
n = 12) pro-inflammatory
cytokines within the
muscle. Increased
interleukin-1 expression
was reduced by early
MSCs treatment. TNF and
TGF-β1 expression was
M. B. Gugjoo and A. Pal
increased at 6 months
Lumbar disc 18 animals 6 months Pentosan 5 × 105 3 T MRI, standard Both the MSCs- and Daly et al.
herniation (repair by (pMSCs, n = 6; polysulphate- radiography, pMSCs-groups exhibited (2018)
microdiscectomy) MSCs, n = 6; primed morphologically, significantly less
control, n = 6) mesenchymal histologically, reduction in disc height
progenitor cells biochemically for and lower Pfirrmann
(pMSCs) and their proteoglycans grades as compared to the
MSCs (PGs), collagen and control, but morphologic
DNA content scores for the pMPC-
injected discs were
significantly lower. The
PG content of the annulus
Fibrosus injury site
region 1 of pMSCs discs
was significantly higher
than MSCs and injury
control AF1. At the AF1
and contralateral AF2
regions, the DNA content
of pMSCs discs was
significantly lower than
injured control discs and
MSCs-injected discs.
Histologic and
birefringent microscopy
revealed increased
structural organization
and reduced degeneration
7 Sheep Mesenchymal Stem Cell Basic Research and Potential Applications
in pMSC discs as
compared to the MSCs
and controls
139
140 M. B. Gugjoo and A. Pal
sBM-MSCs may steadily improve radial and tibial nerve repair. Initially up to
3 months poor nerve regeneration and conduction upon MSCs implantation had
been demonstrated. However, later at 6 months period myelinated nerve fibres had
generated both proximally and distally. Further, nerve action potential had also
improved (Casañas et al. 2014).
Amniotic fluid MSCs (AF-MSCs) may improve motor functions and increase
large neuron preservation in spinal cord (Wang et al. 2015). The early gestation
AF-MSCs in comparison to late stage gestation had better ability to preserve spinal
cord architecture and large neurons in myelomeningocele (MMC). Lambs that
received early gestation cells had normal ambulation while only limited distal motor
function was demonstrated in animals that received late gestation MSCs (Brown
et al. 2016). This questions if MSCs are able to maintain their neural specific prop-
erties with developmental advancement.
MSCs may offer therapeutics in intervertebral disc degeneration (Shamsul et al.
2014). Sheep intervertebral disc disease model treated with BM-MSCs had quantifi-
able improvement at 6 months period. Annulus fibrosis implantation may be better
than nucleus pulposus transplantation (Freeman et al. 2016). Their disc regeneration
potential may be promoted through pentosan polysulphate priming (Daly et al. 2018).
MSCs application in neurological ailments is in infancy and extensive research
studies are desired for any conclusion.
Sheep has been used as an animal model for human respiratory ailments. Acute
respiratory distress syndrome (ARDS) in these animals has been created through
endotoxaemia. BM-MSCs or lung derived MSCs in combination with other support
system had better managed the ARDS. The inflammation along with oedema had
subsided post cell implantation (Ingenito et al. 2012; Asmussen et al. 2014;
Kocyildirim et al. 2017) (Table 7.8).
MSCs have also been studied in acute renal injuries. The cells had ability to
engraft although without any observable beneficial effect (Behr et al. 2007, 2009)
(Table 7.8).
As MSCs are able to trans-differentiate into germinal cells, these may be utilized for
in vivo applications. One available study had demonstrated that transdifferentiated
cells carry ability to home to seminiferous tubules of ram testis. Even these cells had
formed colonies at the area (Ghasemzadeh-Hasankolaei et al. 2015) (Table 7.9).
Initially, it is mandatory to develop differentiation media cocktail that ensures the
typical germ cells properties of transdifferentiated cells. This may be followed by in
vivo studies of these differentiated cells.
Table 7.8 In vivo preclinical experimental mesenchymal stem cell studies on respiratory and renal affections
Model
defect size/ Cell dose/ Evaluation
Model type Number of animals study period Biomaterial used assembly criteria Overall result References
Emphysema 10 animals (control 4 weeks Autologous lung 5–10 × 106 Clinical Cells were well tolerated, Ingenito
scaffold treated n = 5; MSCs + scaffold toxicity, tissue increased perfusion and et al. (2012)
cell treated n = 5) (1% mass (CT), tissue mass as compared
fibrinogen + 20 μg/ lung to control. Histology
ml physiology, confirmed cell retention,
fibronectin + 20 μg/ scintigraphy, increased cellularity and
ml poly-L-lysine) histology extracellular matrix in
cell treated animals
Acute 19 animals (control, 24 h Human BM-MSCs 5 × 106/kg Post-mortem Cells were well tolerated. Asmussen
respiratory PlasmaLyte A, n = 8; (low dose); lung wet-to- PaO2/FiO2 ratio was et al. (2014)
distress low dose MSCs, n = 7; 10 × 106/kg dry weight significantly improved in
syndrome high dose MSCs, n = 4) (high dose) ratio, cell treated groups
(ARDS, broncho- compared to control.
bacterial alveolar Median lung water
pneumonia) lavage, serum content was significantly
biochemical lower in high cell treated
parameters group than control
Acute 11 animals (control, – BM-MSCs 40 × 106 Blood gas Better histopathologic Kocyildirim
respiratory n = 5; extracorporeal results, changes with less et al. (2017)
distress membrane oxygenation radiography, inflammation in combined
syndrome (ECMO), n = 3; MSCs + histopathology cell and ECMO treated
(Escherichia ECMO, n = 3) group
7 Sheep Mesenchymal Stem Cell Basic Research and Potential Applications
coli exdotoxin
induced)
(continued)
141
Table 7.8 (continued)
142
Model
defect size/ Cell dose/ Evaluation
Model type Number of animals study period Biomaterial used assembly criteria Overall result References
Renal ischaemic 24 animals [sham group 3–5 weeks Autologous MSCs 80 × 106 Identification Renal (tubules and Behr et al.
reperfusion (balloon catheterization of engrafted glomeruli) engraftment of (2007)
injury performed but no cell, immune- MSCs. MSCs expressed
(percutaneous ischaemic reperfusion phenotype tubular epithelial cell
transluminal injury (IRI), n = 3; control analysis markers and podocyte
placement of group (IRI induced phenotype. Significant
balloon catheter without any cell treatment, increase in engraftment of
in renal artery) n = 3); cell treated but no MSCs was seen when
IRI induced, n = 3; IRI injected immediately than
induced and cells injected at later period
1 h. later, n = 5; IRI
infused and cells infused
2 weeks afterwards. This
group divided into two,
one evaluated at 3 week,
n = 5 and other at 5 week,
n = 5]
Renal ischaemic Sham group; control 7 days BM-MSCs 50 × 106 Immuno- Transplanted cells were Behr et al.
reperfusion injected culture medium; phenotypic found in glomeruli but not (2009)
injury (bilateral) cell treated group analysis, renal tubules. Cell did not
(balloon function, express glomerular cell
catheter) histology and markers (podocin and von
urine analysis Willebrand factor).
Functional evaluated
revealed no beneficial
effects of implanted cells.
MSCs failed to repair renal
parenchyma and failed to
M. B. Gugjoo and A. Pal
7.8 Conclusion(s)
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Goat Mesenchymal Stem Cell Basic
Research and Potential Applications 8
M. B. Gugjoo, Amar Pal, M. R. Fazili, R. A. Shah, M. S. Mir,
and G. T. Sharma
Abstract
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 153
Springer Nature Singapore Pte Ltd. 2020
M. B. Gugjoo, A. Pal (eds.), Mesenchymal Stem Cell in Veterinary Sciences,
https://doi.org/10.1007/978-981-15-6037-8_8
154 M. B. Gugjoo et al.
8.1 Introduction
There exists a considerable body of literature that has focussed on goat mesenchy-
mal stem cells (gMSCs) properties to be translated to their therapeutic applications.
An understanding of the basic cellular physiological properties, although currently
very limited, may pave the way for their applications in veterinary medicine.
Utilization of goat as translational animal model and application of gMSCs in these
models has been aimed to provide further incites on stem cell applications in human
medicine. The current chapter focuses on the gMSCs culture characteristics and
their in vivo applications.
In comparison to other species, gMSCs have been harvested from a limited number
of tissue/organs. gMSCs from adult tissue organs and foetal membranes are enlisted
in Table 8.1.
gMSCs are characterized as per the standard criteria given for human and as fol-
lowed by other species. gMSCs attach to the plastic surfaces and appear spindle-
shaped fibroblasts. Short or long spindle size at various passages may be seen as has
been reported for goat umbilical cord MSCs (gUC-MSCs). These cells may self-
renew and grow as colonies for certain passages (Martins et al. 2017). The surface
marker expression in general follows pattern as per standard recommendations
(Table 8.1). However, weak expression of markers like CD90, CD166, and CD105
has also been reported in few studies (Knippenberg et al. 2005; Ghaneialvar et al.
2018). In addition to the surface markers, gMSCs express pluripotency markers
(Table 8.1) and have been demonstrated to show plasticity and undergo extended
differentiation into putative germ cells and myocyte-like cells (Tripathi et al.
2010;Li et al. 2017; Zhang et al. 2019).
Donor tissue determines the gMSCs initial concentration and the ability to prolif-
erate and differentiate. Additionally, available culture environment too affects
gMSCs characteristics. Bone tissue (ilium bone) in comparison to bone marrow
may provide a higher yield (Akram et al. 2017). The presence of the periosteal
layer (inner cambium) in bone chip may have led to the higher cell yield. gMSCs
tend to proliferate for certain passages (p 20) beyond which these become
8 Goat Mesenchymal Stem Cell Basic Research and Potential Applications 155
Table 8.1 (continued)
Negative
Positive markers markers Differentiation potential References
CD29, CD44 CD34 – Xu et al. (2017)
CD44 and CD166, CD105, CD34, – Ghaneialvar et al.
CD90 (weak expression) CD45 (2018)
Cord blood MSCs (CB-MSCs)
CD73, CD90 and CD105 CD34 Adipogenic, Somal et al.
Pluripotency markers (Oct4, chondrogenic, and (2016, 2017)
Sox2, Nanog, KLF, cMyc, osteogenic
FoxD3)
Deciduous teeth MSCs (Dt-MSCs)
CD29, CD146 – Osteogenic Zhao et al. (2017)
Endometrium MSCs (End-MSCs)
– – Adipogenic, Tamadon et al.
chondrogenic, and (2017)
osteogenic
Skeletal muscle MSCs (S-MSCs)
– – Osteocytes, adipocytes, Tripathi et al.
and myogenic (2010)
Umbilical cord MSCs (UC-MSCs)
CD73, Thy-1, CD105 CD34 Adipogenic, osteogenic Kumar et al.
(2016)
CD90, CD105, CD44 CD34 Adipogenic, Martins et al.
chondrogenic, and (2017)
osteogenic
CD90, CD105 CD34, Adipogenic,
CD44 chondrogenic, and
osteogenic
Wharton’s jelly MSCs (WJ-MSCs)
CD-73, STRO-1, CD-105 CD34 Adipogenic, Pratheesh et al.
chondrogenic, and (2014)
osteogenic
CD73, CD90 and CD105 CD34 Adipogenic, Somal et al.
Pluripotency markers (Oct4, chondrogenic, and (2016, 2017)
Sox2, Nanog, KLF, cMyc, osteogenic
FoxD3)
AD-MSCs adipose-derived MSCs, AF-MSCs amniotic fluid-derived MSCs, AS-MSCs amniotic
sac-derived MSCs, BM-MSCs bone marrow-derived MSCs, CB-MSCs cord blood-derived MSCs,
Dt-MSCs deciduous teeth-derived MSCs, End-MSCs endometrium-derived MSCs, Skeletal
M-MSCs skeletal muscle-derived MSCs, UC-MSCs umbilical cord-derived MSCs, WJ-MSCs
Wharton’s jelly-derived MSCs
senescent. With each passage, the population doubling time (PDT) tends to
increase (Mohamad-Fauziet al. 2015). Average PDT of gUC-MSCs had increased
from 33.49 h at passage 5 to 34.91 h at passage 12 (Qiuet al. 2012). gMSCs pro-
liferation differences with tissue source also occur. The mean PDT of goat bone
marrow (gBM)-, amniotic fluid (gAF)-, and Wharton’s jelly (gWJ)-derived MSCs
8 Goat Mesenchymal Stem Cell Basic Research and Potential Applications 157
had been 24.94 ± 2.67 h (Eslaminejad et al. 2009); 33.1 h (Pratheesh et al. 2013);
36.06 ± 1.2 h (Pratheesh et al. 2014), respectively, at specific passage. MSCs have
also been harvested from goat foetal adnexa. The cells from particular adnexal
tissues (gWJ-MSCs and goat cord blood-derived MSCs) tend to show faster pro-
liferation as compared to others (gAF-MSCs and amniotic sac-derived MSCs)
(Somal et al. 2016). Additionally, gWJ-MSCs had significantly higher expression
of some of the surface and pluripotency markers as compared to MSCs from other
sources. The expression of few pluripotency markers (Klf and cMyc), however,
had remained comparable between gWJ-MSCs and goat amniotic fluid MSCs
(gAF-MSCs) (Somal et al. 2017).
Self-renewal and differentiation properties of MSCs may be regulated by epigen-
etic modifications. MSCs derived from adipose tissues, bone marrow, and muscle
may not carry identical promoter methylation profiles (Sorensen et al. 2010). The
culture condition sensitivity of gMSCs might change their methylation pattern.
Goat adipose tissue MSCs (gAD-MSCs) tend to undergo higher epigenetic changes
as compared to goat bone marrow (BM)-MSCs (Wang et al. 2017). Likewise gAD-
MSCs may have superior adipogenic but weaker osteogenic differentiation potential
as compared to musculoskeletal sources like gBM-MSCs (Mohamad-Fauzi et al.
2015). The upregulation of osteogenic pathways in these cells had been variable;
wap38 MAPK is upregulated in gBM-MSCs while p44/42 MAPK in gAD-MSCs
(Elkhenany et al. 2016). gBM-MSCs tend to undergo low whole genome methyla-
tion as compared to fibroblasts as bone marrow requires cells that have high prolif-
eration ability (Schopet al. 2009).
Reproductive cyclicity too may affect MSCs proliferation. Anestral and cyclic
endometrium-derived MSCs (End-MSCs) at passage 4 had 40.6 h and 53 h PDT,
respectively (Tamadon et al. 2017). MSCs proliferation in anestrus may be pro-
moted by the available higher concentration of growth factors like basic fibroblast
growth factor (bFGF) and transforming growth factor-β (TGF-β) in endometrial
tissue (Tamada et al. 2000; Tamadon et al. 2017).
Donor age and type of ailment may also affect MSCs characteristics. MSCs har-
vested from an osteoporotic goat tend to proliferate slowly and show reduced osteo-
genic potential. Such a reduced differentiation potential may be enhanced through
specific scaffold (β-TCP) incorporation (Cao et al. 2012). Ageing tends to decrease
MSCs proliferation and differentiation potential. A single gMSCs study, however,
had failed to demonstrate decrease in such properties with ageing (Vertenten
et al. 2009).
158 M. B. Gugjoo et al.
Goat has been utilized as the translational model animal for human. As such gMSCs
have been evaluated in various models of different conditions of bone and cartilage,
among others for their therapeutic effects (Gugjoo et al. 2020). To avoid animal suf-
ferings, substitutes like organ culture system are being developed. One of the stud-
ies had shown comparable results in organ culture system and in vivo conditions
with respect to the gAD-MSCs spatial and temporal behaviour (Peeters et al. 2015).
The current chapter, however, focuses on the in vivo therapeutic applications
of gMSCs.
MSCs local implantation makes them available to the tissue directly in sufficient
concentrations, while distant sources like intravenous routes may be required in
large quantities for effective utilization. Additionally, intravenous application of
gMSCs may activate disseminated intravascular coagulation (DIC) cascade and
lead to hypercoagulable state, but the condition is transient (Liao et al. 2017). This
may be due to the MSCs ability to express pro-coagulant factors, which increases as
the cells are culture expanded (Tatsumi et al. 2013; Liao et al. 2017) and can be
controlled by heparin therapy (Liao et al. 2017). The potential therapeutic applica-
tions of gMSCs are discussed below.
Model defect
Model type Number of animals size/study period Biomaterial used Cell dose Evaluation criteria Overall result References
Full-thickness 32 (menisci repaired 4 months Autologous 20 × 106 Gross, tensile strength, Statistically Port et al.
meniscal with two vertical BM-MSCs, and histology non-significant (1996)
repair sutures, n = 8; fibrin clot differences in
(0.75 mm) 0.5 mL exogenous healing between
fibrin clot, n = 8; suture repaired and
fibrin fibrin clot repaired
clot + BM-MSCs, groups. Addition of
n = 8; left untreated, MSCs did not
n = 8) enhance menisci
repair
Chronic 24 (6 animals cell 12 and 26 weeks BM-MSCs + 10 × 106 Histochemistry Marked Murphy
model of treated/hyaluronan hyaluronan loaded in regeneration of the et al.
excision of and 3 animals control hyaluronan medial meniscus. (2003)
the medial for 12 weeks Degeneration of the
meniscus and evaluation; 9 animals articular cartilage,
resection of cell treated/ osteophytic
the anterior hyaluronan and 6 remodelling, and
cruciate animals for 26 weeks subchondral
ligament evaluation) sclerosis were
reduced in
cell-treated group
compared to control
at 12 weeks.
However, later the
cell treated and
control had severe
osteoarthritis
M. B. Gugjoo et al.
Mandibular 50 (12 in each group) 3 mm NELL-1 3 × 106 cell Macroscopic, Group I showed Zhu et al.
condyle (group I: NELL-1- diameter × 5 mm transfect seeded on histology, and rapid and vigorous (2011)
osteochondral modified BM-MSCs/ depth/6 weeks autologous PLGA immunohistochemistry, healing leading to
defect model PLGA, group II: and 24 weeks BM-MSCs and scaffold micro-CT fibrocartilage
BM-MSCs/PLGA; poly-lactic-co- formation at
group III: PLGA polyglycolic acid 6 weeks. At
alone; group IV: scaffold 24 weeks, complete
control) repair of native
articular cartilage
and subchondral
bone at 24 weeks. In
group II: Repaired
completely filled the
defect with
fibrocartilage at
24 weeks, but the
cartilage was less
well-organized
group I. In groups
III and IV, the
defects were poorly
repaired, and no
cartilage in the
group IV or only
small portion of
cartilage in the
8 Goat Mesenchymal Stem Cell Basic Research and Potential Applications
Model defect
Model type Number of animals size/study period Biomaterial used Cell dose Evaluation criteria Overall result References
Full-thickness 8 (16 knees) 5 mm/6 months Chondron 10% Macroscopic and Combination of Bekkers
chondral defect chondron and cell (chondrocytes in chondron/90% microscopic scoring, BM-MSCs and et al.
in medial treatment vs. own matrix) and BM-MSC biochemical analysis,chondrons leads to (2013)
femoral microfracture BM-MSCs combination and histological and significantly better
condyles at a immunohistochemical microscopic,
concentration analyses macroscopic, and
of 1 × 106 biochemical cartilage
cells/mL regeneration
compared to
microfracture
treatment
Osteochondral 8 (group I: Scaffold 5 mm × 3 mm/1 Stromal vascular 5 × 106 (SVF) Macroscopy, Cell-treated groups Jurgens
defects created seeded with cultured month and fraction (SVF), and 5 × 105 immunohistochemistry, had more extensive et al.
in medial AD-MSCs; group II: 4 months AD-MSCs along (AD-MSCs) biomechanical analysis, collagen type II, (2013)
condyles and Scaffold seeded with with collagen seeded on micro-CT analysis, and hyaline-like cartilage,
trochlear SVF cells; group III: type I/III scaffold biochemistry and higher elastic
grooves Acellular scaffold) scaffold moduli, and their
glycosaminoglycan
content in the
cartilaginous layer
that approached
native tissue values.
In control, lesser
regenerative effect
was seen. No
difference in healing
was seen between
SVF-treated and
AD-MSC-treated
M. B. Gugjoo et al.
animals
Chronic 18 (36 defects) group 5 mm/6 months BM-MSCs 1 × 107 cells Hyaline-like tissue
Macroscopic, histology, Nam et al.
full-thickness I: Bone marrow after biochemical assays with higher (2013)
chondral defect stimulation and 2 weeks (glycosaminoglycans), glycosaminoglycans
in medial BM-MSCs; group II: after bone and gene expressions and chondrogenic
femoral Bone marrow marrow (aggrecan, collagen IIgene expression in
condyles stimulation; group stimulation and Sox9) group I compared to
III: Control for three group II that had
consecutive fibrocartilage.
weeks Lowest healing in
control
Osteochondral n-12 (TEO cultured 12 and 24 weeks Autologous – Histology and Reparative effects Pei et al.
defect in double-chambered BM-MSCs, immunohistochemistry, of TEO cultured in (2014)
(bilateral, 6 mm bioreactor without β-TCP gross morphology bioreactor were
diameter and mechanical better than control,
12 mm depth) stimulation of and mechanical
stirring, n = 4; stimulation could
stimulation of further improve the
stirring, n = 4; no effects
TEO implantation,
control, n = 4)
(continued)
8 Goat Mesenchymal Stem Cell Basic Research and Potential Applications
163
Table 8.3 (continued)
164
Model defect
Model type Number of animals size/study period Biomaterial used Cell dose Evaluation criteria Overall result References
Full-thickness 6 (microfracture and 6.5 mm Human 1 × 106 cells Analysis of No significant Zhang
femoral cell/scaffold groups) diameter/6 and WJ-MSCs seeded on inflammatory response, differences between et al.
condyle 9 months seeded in an ACECM magnetic resonance the two groups in (2018)
cartilage acellular imaging, gross immuno-
defects cartilage morphology, histology, inflammatory
extracellular immunohistochemical parameters. MRI
matrix and immunofluorescent demonstrated
(ACECM)- staining, biomechanical higher-quality
oriented scaffold testing, and biochemical cartilage and
quantitative analyses complete
subchondral bone at
defect sites in the
cell-treated group at
9 months.
Histological
revealed
extracellular
cartilage, cartilage
lacuna, and collagen
type II levels were
higher in cell-
treated group
compared to the
microfracture, while
the cell-treated
group exhibited a
higher elasticity
modulus
M. B. Gugjoo et al.
8 Goat Mesenchymal Stem Cell Basic Research and Potential Applications 165
Various sources like bone marrow- (Murphy et al. 2003; Bekkers et al. 2013;
Nam et al. 2013), adipose tissue- (Jurgens et al. 2013), and Wharton’s Jelly- (Zhang
et al. 2018) derived MSCs have been evaluated in osteochondral defect models. The
cells along with various scaffolds like hyaluronan, collagen I/III, natural cartilage
ingredients, and poly lactic co-glycolic acid (PLGA) have been implanted to pre-
vent loss of cells through adverse effects of local environment. An overall
improvement in osteochondral defect healing have been demonstrated to occur with
MSCs and scaffold implantations (Table 8.3). The improved healing has been char-
acterized by enhanced secretion of cartilage-specific matrices like collagen type II
and glycosaminoglycan. The regenerated hyaline-like cartilage had biomechanical
factors almost comparable to native tissue (Jurgens et al. 2013). These cells tend to
retard degeneration of the articular cartilage and prevent osteophyte formation and
sclerosis of subchondral bone (Murphy et al. 2003). Even mechanical stimulation of
these cells seeded in scaffolds may further enhance their repair potential (Pei
et al. 2014).
The femoral condyle defect-healing potential of MSCs (BM-MSCs or xenogenic
WJ-MSCs) seeded over cartilage extracellular matrix may be better than commonly
used surgical techniques (microfracture and bone marrow stimulation). The healing
had been evaluated utilizing International Cartilage Repair Society (ICRS) and
O’Driscoll scores, in addition to the expression profile of cartilage-specific genes
(Bekkers et al. 2013; Nam et al. 2013; Zhang et al. 2018). However, no differences
in inflammatory response at the defect site had been demonstrated among the cel-
lular implantation- and surgical techniques-treated animals (Zhang et al. 2018). One
of the studies had failed to demonstrate any significant improvement in meniscal
repair with gBM-MSCs and fibrin clot (Port et al. 1996).
Transfected MSCs may further enhance osteochondral defect healing (Zhu et al.
2011; Wei et al. 2019). Among various factors, transfection of gBM-MSCs by
NELL-like molecule-1 (Nell-1) seeded on poly lactic co-glycolic acid (PLGA) had
repaired goat mandibular condyle defects by fibrocartilage at 6 weeks and by
24 weeks with native articular cartilage. Simple BM-MSCs and PLGA assembly
had repaired the defect by fibrocartilage even at 24 weeks (Zhu et al. 2011). Nell-1
is a novel growth factor that specifically target cells committed to osteochondral
lineage. Further studies in the area are desired.
Bone tissue engineering is carried out extensively in humans (Costa-Pinto et al.
2011). Goat is increasingly being used as human orthopaedic research animal model
(Proffen et al. 2012). Goat and human long bones have approximate size and mac-
rostructure, in addition to the biomechanics and biochemistry (Anderson et al. 1999;
Van Der Donk et al. 2001; Sousa et al. 2015). Furthermore, goat osteoporosis effec-
tively mimics human condition (Cao et al. 2012).
Various goat bone defect models have been created to study MSCs therapeutic
potential (Table 8.4). Variable critical size defects have been created that varied
Table 8.4 In vivo preclinical experimental mesenchymal stem cell studies on osteogenesis in goat
166
Model defect
size/study
Model type Number of animals period Biomaterial used Cell dose Evaluation criteria Overall result References
Tibial bone defects 26 (BCB + transfected 8 and Biphasic calcined 2 × 108 Histology, 5/8 defects were completely Dai et al.
(2.6 cm) cells, n = 9; β-gal- 26 weeks bone(BCB), biomechanical healed, while 3/8 had partial (2005)
transfected cells + BCB, BMP-2-transfected examination healed in BCB + transfected
n = 6; BCB + simple BM-MSCs, simple cell-treated group. In other
cells, n = 8, BCB, BM-MSCs experimental groups, slight or no
n = 3) healing was reported
Cranial suture 10 (MSCs, n = 5; PBS, 4 weeks MSCs 5 × 109 SEM and histology The new bone formation at the Shen et al.
distraction n = 5) edge of suture was superior in (2006)
(coronal) (0.4 mm/ cell-treated group than control
day for 8 days)
Bilateral early stage 23 (right and left 16 weeks Bone 1 × 107 Radiography, bone The femoral heads in BMP-2- Tang et al.
osteonecrosis of femoral defect-treated morphogenetic mineral density, transfected cell-treated group had (2007)
femoral head BMP-2- and protein-2 histology, and normal density and surface, while
(ligation of lateral β-gal-transfected cells, (BMP-2)- or biomechanical testing those in β-gal-treated cell group
medial circumflex respectively, n = 20; β-galactosidase had low density and irregular
arteries and control, n = 3) (β-gal)-transfected surface. Histologically, lamellar
delivery of liquid BM-MSCs, bone was formed in BMP-2
nitrogen to femoral β-tricalcium cell-treated group, while in β-gal
head, 6-mm defect phosphate (β-TCP) cell-treated group residual fibrous
filled with tissue was formed. The new bone
biomaterials) formed in BMP-2 group had
significantly higher bone as
compared to β-gal. The maximum
compressive strength and Young’s
modulus of repaired tissue in
BMP-2 treated cell group were
similar to normal bone and
significantly higher as compared
to β-gal cell-treated group. In
non-treated group, femoral heads
collapsed at 16 weeks
M. B. Gugjoo et al.
Segmental femoral 6 (HASi, n = 4; 4 months BM-MSCs, 1 × 105/ Radiography, Good osteointegration and Nair et al.
defect (2 cm, nail tissue-engineered HASi, triphasic cm2 of micro-CT, histology, osteoconduction were observed in (2009)
stabilized) n = 2) ceramic-coated HASi histomorphometry, TE HASi and HASi. Tissue-
hydroxyapatite scanning electron engineered HASi showed better
(HASi) microscopy, and and faster performance evident by
inductive couple lamellar bone organization of
plasma spectrometry newly formed bone with the
degradation of material. In HASi
only group, immature interwoven
bony bridges still intermingled
with scattered small remnants of
material
Bilateral segmental 6 (β-TCP seeded 24 weeks Autologous 2 × 106/ Radiography, histology Significant osteogenic capacity of Wang
tibial defect BM-MSCs cultured in BM-MSCs mL and micro-CT experimental group compared to et al.
(30 mm) dynamic perfusion control. Segmental bone defect (2010)
reactor implanted in left repair by dynamic perfusion
tibia; β-TCP seeded reactor culture MSCs outweigh
BM-MSCs cultured static mode
static implanted in right
tibia)
Tibial unicortical 8 (4 defects in each 2, 4, 8, and BM-MSCs, β-TCP, – Radiography, Cell survival and proliferation in Vertenten
defect (6 mm) (four tibia; PLA, PLA + cell; 12 weeks methacrylate- histology, polymer-substituted bone et al.
defects per tibia) PLA + cells + β-TCP; endcapped histomorphology, and defects was demonstrated. The (2009)
control) poly(D,L-lactide- immunohistochemistry incorporation of β-TCP was
Co-έ-caprolactone) associated with less expansion and
growth of MSCs
Non-critical-sized 8 (autologous 2 and Autologous 4 × 105 Clinical, radiology, Faster bone repair in defects filled Lippens
unicortical tibial BM-MSCs, Pluronic® 4 weeks, 6 BM-MSCs, cultured and histology with hydrogel plus cultiSpher-S et al.
defect model F127 hydrogel; tubular and 8 weeks Pluronic® F127 on carriers in comparison to control. (2010)
8 Goat Mesenchymal Stem Cell Basic Research and Potential Applications
(bilateral, 4 holes, sintered hydroxyapatite) hydrogel, tubular carriers No new bone formation was
6 mm) sintered originating from hydroxyapatite
hydroxyapatite carriers. However, the hydrogels
stimulated the natural repair
process
(continued)
167
Table 8.4 (continued)
168
Model defect
size/study
Model type Number of animals period Biomaterial used Cell dose Evaluation criteria Overall result References
Segmental tibial 32 (nHACP/CF + cells, 8 weeks Autologous 2 × 106 Radiography, Cell along with scaffold and Liu et al.
defect (25 mm) n = 8; autograft, n = 8; BM-MSCs, histology, and autograft groups had defect (2010)
nHACP/CF, n = 8; nano- biomechanics repaired by 8 weeks
control, n = 8) hydroxyapatite/
collagen/poly
(L-lactic acid),
PLLA/chitin fibres
(nHACP/CF)
Medial femoral 16 (sham, n = 6; β-TCP 16 weeks Autologous – Radiography, CT, MSCs/ β-TCP treated animals had Cao et al.
condyle defect of treated, n = 10; after defect BM-MSCs micro-CT and successful repair of bone defects (2012)
osteoporotic defect BM-MSCs/β-TCP, creation (oestrogen deficient histomorphometry
(oestrogen n = 16) donor), β-TCP
deficiency induced,
0.8-cm diameter
and 8-mm deep
cylindrical defects)
Maxillary sinus 9 (cells/CPC, n = 6; 3 months BM-MSCs, calcium 2 × 107 Micro-CT, histology Group treated with cells/CPC Zou et al.
augmentation CPC alone, n = 6; phosphate and histomorphometry promoted early bone formation (2012)
(bilateral) autogenous graft, n = 6) cement(CPC) and mineralization and maximally
maintain volume and height of
augmented maxillary sinus as
compared to other groups.
Osteointegration was significantly
higher in cells/CPC treated group
as compared to others
M. B. Gugjoo et al.
Critical sized femur 12 (right defects treated 8 and Bone marrow – Radiography, CT and Volume of new bone formed in Li et al.
defects (bilateral, with SAP/DBM/BMA, 16 weeks aspirate (BMA), 3D image processing SAP/DBM/BMA assembly (2015)
20 mm) n = 12; left with DBM/ nano-scale techniques implanted bone defects was
BMA, n = 12) self-assembly significantly higher as compared
peptide (SAP) to DBM/MSCs
modified
demineralized bone
matrix (DBM)
Iliac crest defect (4 10 (one defect received 3–12 weeks Allogeneic 2 × 106 Histomorphometry, Fluorochrome incorporation Loozen
defects bilaterally) either autologous bone BM-MSCs, fluorescent evaluated at 3, 6 and 9 weeks and et al.
graft or was left empty bicalcium microscopy, histomorphometry at 12 weeks (2015)
(n = 5). In the other 3 phosphate (BCP) immunohistochemistry after implantation revealed clear
locations, the differences between the groups,
experimental groups with pBMP-2 combined with
(n = 10) were tested: (1) MSCs being the most effective.
biphasic calcium The BMP-2 was demonstrated in a
phosphate (BCP) variety of bone-residing cells
control, (2) BCP with through immunohistochemistry.
alginate, pBMP-2 and Further analysis indicated that
allogeneic MSCs and multinucleated giant cells might
(3) BCP with alginate have an important role in
and pBMP-2 transgene expression. The model
appeared robust, showed no
neighbouring effects and
demonstrated effectivity of
combined cell and gene therapy
Distraction 12 (MSCs, n = 6; 30 days BM-MSCs 1.5 × 106 Energy dispersive An increased calcification and El Hadidi
osteogenesis control, n = 6) (two X-ray, scanning significant trabecular bone size et al.
(mandibular) injections electron microscopy and limited osteoid formation in (2016)
8 Goat Mesenchymal Stem Cell Basic Research and Potential Applications
Model defect
size/study
Model type Number of animals period Biomaterial used Cell dose Evaluation criteria Overall result References
Maxillary sinus 18 (n = 6 each group); 3 months Deciduous teeth Computed tomography At 3 months, SGDs-CPC Zhao et al.
floor elevation group A (SGDs-CPC MSCs (DT-MSCs), (CT), histology, and compound had promoted bone (2017)
compound), group B calcium phosphate histomorphometric formation and mineralization. The
(CPC alone), and group cement (CPC) analysis areas of new bone formation in
C (autogenous bone elevated sinuses were DT-MSCs/
obtained from an iliac CPC group, which was
crest) significantly higher than the
CPC-alone group or in the
autogenous bone group.
Immunohistochemical staining
revealed that GFP and OCN were
both expressed in the new bone
tissue, which suggested that the
implanted DT-MSCs might have
contributed to new bone formation
on the elevated sinus floor.
DT-MSCs can promote new bone
formation and maturation in the
goat maxillary sinus, and the
tissue-engineered bone composite
of SGDs and CPC might be a
potential substitute for existing
maxillary sinus floor elevation
methods
M. B. Gugjoo et al.
8 Goat Mesenchymal Stem Cell Basic Research and Potential Applications 171
among studies even on the same bone type. Critical size defect for ilium had been
17 mm (Anderson et al. 1999), femoral defect 20 mm (Li et al. 2015), and tibial
defects 25 mm (Liu et al. 2010) and 30 mm (Wang et al. 2010). Various bone tissue-
engineered constructs along with MSCs have been employed in goats for critical
size defects of mandible, femur, and tibia (Table 8.4). Mostly BM-MSCs have been
utilized along with various kinds of scaffolds (Table 8.4). The cellular scaffolds had
led to the early and improved bone repair both in quality and quantity as compared
to the acellular scaffolds. The lamellar bone organization and its osteo-integration
in the cell-treated defects had been better than those treated without cells. In vitro
cell-scaffold compatibility does not guarantee in vivo regenerative effects (Vertenten
et al. 2009). Thus, it is important to confirm in vivo applicability of MSCs and/or
scaffolds.
gBM-MSCs genetic engineering with human bone morphogenetic protein
(hBMP) gene (Tang et al. 2007; Loozen et al. 2015) and beta-galactosidase (gal)-
gene (Tang et al. 2007) have been demonstrated to further promote healing of bone.
The healed bone tissue formed had higher volume with better mechanical strength
as compared to that with normal MSCs. BMP-2 transduced gBM-MSCs, however,
may be more pro-healing as compared to BM-MSCs transfected with beta-galacto-
sidase (Tang et al. 2007). Bone remains under stress that may determine the healing
outcome. On that principle, dynamically cultured MSCs and/scaffolds and distrac-
tion osteogenesis had favoured bone healing (Shen et al. 2006; Cao et al. 2012;
Gardel et al. 2013).
Bone tissue-engineered cellular scaffolds have favoured early and better bone
regeneration. To achieve evidence-based medicine and for definitive applications of
these constructs, more uniform and extensive studies are desired.
Goats carry larger size of lumbar intervertebral discs (~5 mm disc height) and have
disc mechanical properties and biochemical composition resembling to that of
human. This makes goats more suitable animal model for human (Table 8.5)
(Beckstein et al. 2008). Goat preliminary ex vivo studies on MSCs and/scaffold
have shown favourable role in intervertebral disc diseases (Gullbrand et al. 2017).
Few in vivo studies conducted have been pro to the utilization of MSCs along with
scaffolds. The healing may be observed as early as 3 weeks of time (Xu et al. 2017).
Among various factors, basement membrane molecules too determine nucleus
pulposus chondrogenesis and cartilage regeneration (Toh et al. 2013).
The cellular grafting of periodontal defects may further promote periodontal tissue
regeneration against the normal grafting. Among various tissue-regenerated struc-
tures are cementum, bone, and periodontal ligament (Table 8.5) (Marei et al. 2009).
The preliminary reports demand in-depth studies for any definitive outcome.
Table 8.5 In vivo preclinical experimental mesenchymal stem cell studies on different conditions in goat
172
Goat has also been used to study the effect of MSCs in cardiovascular affections
relevant to the humans. Autologous gBM-MSCs and/or scaffold in coronary artery
ligation model has been evaluated. The assembly could prevent left ventricular
chamber dilatation and had improved myocardial perfusion and contraction (Liao
et al. 2006). It is a preliminary study that demands further evaluation.
Lung cancer remains one of the leading causes of death (Wakelee et al. 2007). Surgical
resection in limited lung disease is preferable option but tends to develop broncho-
pleural fistula (BPF) causing higher mortality (Sonobe et al. 2000). In an induced
bronchopleural fistula, implantation of the gBM-MSCs had led to the healing of fis-
tula in about a month (Table 8.5) (Petrella et al. 2014), demanding further studies.
8.3.8 Reproduction
Nuclear Transfer (NT) technology is used to develop clones. The current efficiency
remains low, and one of the important limiting factors is the nucleus of donor cell
type (Kwong et al. 2014). gMSCs as donor karyoplast appear promising to improve
such efficiency as these cells can be passaged for extended periods (Ren et al. 2014).
Ex vivo studies have demonstrated gBM-MSCs as donor karyoplast to produce
cloned caprine embryos. These cells had better developed up to the hatched blasto-
cyst stage than that with ear fibroblasts. Compared to somatic cells, better prolifera-
tion rate, growth capacity, transfection efficiency and convergence, and cleavage
had been demonstrated with MSCs (Kwong et al. 2014; Ren et al. 2014). Contrarily,
one of the studies had failed to show any difference in fusion and cleavage rates
between gBM-MSCs and somatic cells (Kwong et al. 2014).
Apart from the NT, MSCs have been evaluated for their effect on the growth and
activation of pre-antral follicles. A significant reduction in the percentage of mor-
phologically normal follicles cultured along with BM-MSCs had been demonstrated
as compared to the fresh control. However, a significantly higher rate of morpho-
logically normal pre-antral follicles had been reported when cultured in BM-MSCs
as compared to those cultured normally. The follicular diameter, however, had
remained comparable in all the follicles irrespective of the culture conditions. In
conclusion, BM-MSCs had no influence on follicular or oocyte development but
had promoted survival of pre-antral follicles (Arrivabene Neves et al. 2019).
8.3.9 Mastitis
Stem cells have been harnessed from mammary gland and as such are considered as
potential targets to improve milk production (Martignani et al. 2009; Capuco et al.
174 M. B. Gugjoo et al.
2012; Martignani et al. 2014). MSCs due to their characteristic features have poten-
tial to regenerate mammary tissue. A single MSCs study on goat chronic mastitis
had demonstrated their potential to reduce fibrosis and in maintaining normal tissue
architecture and ability to secrete milk (Costa et al. 2019).
Goats may act as a model for stress urinary incontinence disorder of human
(Burdzinska et al. 2017) and as such applicability of gMSCs in such ailments may
be evaluated.
8.4 Conclusions
gMSCs have been harvested from various sources including foetal membranes.
MSCs characteristic properties like proliferation and differentiation potential have
been evaluated in relation to tissue sources. The cells have been characterized as per
the standard protocols. Bone marrow, adipose tissue, and foetal adnexa-derived
MSCs have been mainly evaluated under experimental studies. A wide array of
gMSCs research mainly focussed on human translational studies has been con-
ducted. Preliminary therapeutic literature on gMSCs has variably favoured their
in vivo applications. Different biomaterials like cellularized scaffolds and geneti-
cally engineered cells together with growth factors have also been evaluated.
Currently, the studies remain inconclusive.
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Abstract
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 181
Springer Nature Singapore Pte Ltd. 2020
M. B. Gugjoo, A. Pal (eds.), Mesenchymal Stem Cell in Veterinary Sciences,
https://doi.org/10.1007/978-981-15-6037-8_9
182 M. B. Gugjoo et al.
9.1 Introduction
Among various domestic animals, cattle/buffalo has a crucial position in the eco-
nomics of the livestock industry, worldwide. These animals mainly contribute
through meat and milk production, in addition to hides and other biological materi-
als (Jean and Anderson 2014; Aghamohammadi et al. 2018; Das et al. 2018).
Medical conditions like endometritis, mastitis, lameness, fracture, and others can
badly affect their production and reproductive efficiency (Izquierdo et al. 2017).
With the biotechnological advancement, cattle- or buffalo-MSCs (c-/buf-MSCs) are
being studied for improved production, reproduction, and/or genetic improvement
(Shibiru et al. 2017). Additionally, bovines can offer suitable translational experi-
mental animal model for human medicine (Bosnakovski et al. 2004). c-/buf-MSCs
potential applications are being evaluated mostly in relation to reproduction and/ or
production. The current chapter throws light on the c-/buf-MSCs characteristics and
their potential applications.
Various sources have been utilized to harvest c-/buf-MSCs. These cells are being
evaluated and characterized as per the standard criteria laid down for humans.
Cattle- and buffalo-MSCs characterization markers including differentiation poten-
tial have been detailed in Tables 9.1 and 9.2, respectively (Gugjoo et al. 2018).
Plastic adherent c-/buf-MSCs exhibit fibroblastic morphology. All these studies
have not demonstrated all the laid down characterization principles (Donofrio et al.
2008). Even some of the studies have failed to report typical surface marker expres-
sion of these MSCs as shown in Table 9.1. In addition to the trilineage, the cells
have been studied for their extended differentiation potential including germ cell-,
hepatocyte-, islet cell-, myocyte-, and neurocyte-like cells (Cardoso et al. 2012a; Lu
et al. 2014a, b, c; Xiong et al. 2014; Peng et al. 2017; Deng et al. 2018).
Plastic adherent c-/buf-MSCs show variable morphologies. These cells may exhibit
variable morphologies in early passages followed by mostly fibroblastic in extended
passages as has been reported for cattle amniotic fluid MSCs (cAF-MSCs). The
early passage morphology includes fibroblastic, and flat and circular type (Corradetti
et al. 2013) or fusiform (Yang et al. 2016). These fibroblasts tend to show higher
mean population doubling potential and are better able to migrate and home to dis-
tant tissues. The higher migration potential and homing was confirmed by their
ability to show greater CD44 expression (Rossi et al. 2014). Buffalo amniotic mem-
brane MSCs (bufAm-MSCs) too had shown mixed cell morphologies in early pas-
sages, which had changed to mostly fibroblastic subsequently (Dev et al. 2012). The
time of cell plastic adherence may also determine their ability to appear fibroblasts.
9 Cattle/Buffalo Mesenchymal Stem Cell Basic Research and Potential Applications 183
Table 9.1 Cattle mesenchymal stem cell sources and their characterization
Positive markers Negative markers Differentiation References
Adipose tissue MSCs (AD-MSCs)
Vimentin, CD49d, CD34 Adipogenic Ren et al.
CD13 (2010)
β-Integrin, CD44, – Adipogenic, osteogenic Lu et al.
and CD73 (2014c)
CD29, CD73, CD34, CD45 Adipogenic, chondrogenic, da Silva et al.
CD90, CD105 osteogenic (2016a)
CD70, CD90 CD34, CD45 Adipogenic Cebo (2017)
CD29, CD44, CD34, MHC-II Adipogenic, chondrogenic, Queiroz et al.
Vimentin osteogenic (n.d.)
CD73, Oct4 and CD34 – Cahuascanco
Nanog et al. (2019)
Amniotic fluid MSCs (AF-MSCs)
CD29, CD44, CD14, CD34, CD45, Adipogenic, chondrogenic, Corradetti et al.
CD73, CD166, CD90, MHC-II neurogenic (2013)
MHC-I, Oct-4,
C-Myc
CD44, CD73, CD34, CD45 Adipogenic, osteogenic, Gao et al.
CD166, Oct-4 neurogenic (2014)
CD90, CD105, CD14, CD73, CD34, Adipogenic, osteogenic, Rossi et al.
CD34 Oct-4, Sox2, chondrogenic (2014)
Cytokeratin,
E-Cadherin
CD29, CD73, CD34, CD44, CD45 Adipogenic, chondrogenic, da Silva et al.
CD90, CD105 osteogenic (2016a)
β-Integrin, CD44, CD34, CD45 Adipogenic, osteogenic, Chang et al.
CD73, CD166, chondrogenic (2018)
Oct-4
Amniotic membrane MSCs (Am-MSCs)
CD29, CD44, CD14, CD34, CD45, Adipogenic, chondrogenic, Corradetti et al.
CD73, CD105, MHC-II neurogenic (2013)
CD166, MHC-I,
Oct-4, C-Myc
Nanog, Oct-4, Sox-2 – Osteogenic Mann et al.
(2013)
CD44, Nanog, Oct-4 MHC-II Adipogenic, chondrogenic, Campos et al.
osteogenic (2017)
Bone marrow MSCs (BM-MSCs)
CD29, CD44, and CD14, CD31, CD34, Adipogenic, osteogenic, Kato et al.
CD166 CD45, CD90, CD105, neuroectodermal (2004)
CD140a, and CD117
CD73, CD90, CD34, CD45 Adipogenic, chondrogenic, Cortes et al.
CD105 osteogenic (2013)
CD29, CD73, Oct-4, CD34, CD45 Hepatogenic, neurogenic Dueñas et al.
Nanog (2014)
(continued)
184 M. B. Gugjoo et al.
Table 9.1 (continued)
Positive markers Negative markers Differentiation References
CD29, CD44, CD73 CD31, CD34, CD45 Osteoblasts, adipocytes, Lu et al.
hepatic, and islet-like cells (2014a)
CD29, CD90, CD45 Adipogenic, chondrogenic, Lee et al.
CD44, CD105, osteogenic (2015)
Oct-4, Sox2 Nanog
CD73, Oct4 and CD34 – Cahuascanco
Nanog et al. (2019)
Dermal MSCs (D-MSCs)
CD29, CD44, CD34, CD106, CD166, Adipogenic, osteogenic, Sun et al.
CD73, CD90 Oct-4 neurogenic (2014)
Endometrium MSCs (End-MSCs)
CD44, CD117, Nanog Chondrogenic, osteogenic Cabezas et al.
STAT-3, Oct-4, (2014)
Sox-2
CD44, CD29, CD34 (low Adipogenic, osteogenic de Moraes
Vimentin expression), MHC-II et al. (2016)
OCT4, SOX2, NANOG Adipogenic, osteogenic, Lara et al.
CD44, c-KIT chondrogenic (2017a, b)
Liver MSCs (Li-MSCs)
CD29, CD44, CD34, CD45, Adipogenic, osteogenic, Lu et al.
CD73, CD90, BLA-DR chondrogenic (2014b)
CD106, CD166
Lung MSCs (L-MSCs)
CD29, CD44, CD34, CD45, Osteogenic, neural lineages Hu et al. (2015)
CD73, CD166 BOLA-DRα
Placental MSCs (Pl-MSCs)
CD166, CD73, CD45 Islet like cells (insulin, Peng et al.
β-integrin, Oct-4 glucagon, pax-4, Nkx6.1, (2017)
pax-6, Fox)
Tendon MSCs (T-MSCs)
CD44, Tenascin, Collagen II Adipogenic, chondrogenic, Yang et al.
Collagen I, Collagen osteogenic (2016)
III
Umbilical cord MSCs (UC-MSCs)
Oct-4, CD73 – Adipogenic, chondrogenic, Raoufi et al.
osteogenic (2011)
CD29, CD44, CD34, CD45, Adipogenic, osteogenic, Xiong et al.
CD73, CD90, BLA-DR hepatocyte-, Islet cell-, (2014)
CD166 neurocyte-like cells,
CD44, Nanog, Oct-4 MHC-II Adipogenic, chondrogenic, Campos et al.
osteogenic (2017)
Wharton’s jelly MSCs (WJ-MSCs)
CD29, CD73, CD34, CD45 Adipogenic, chondrogenic, Cardoso et al.
CD90, CD105, osteogenic, neural-like (2012b)
POUF51, ITSN1 cells
(continued)
9 Cattle/Buffalo Mesenchymal Stem Cell Basic Research and Potential Applications 185
Table 9.1 (continued)
Positive markers Negative markers Differentiation References
CD105, CD29, – Adipogenic, chondrogenic, Cardoso et al.
CD73, CD90 osteogenic, astrocyte (2017)
CD29, CD73, CD34, CD44, CD45 Adipogenic, chondrogenic, da Silva et al.
CD90, CD105 osteogenic (2016a)
Yolk sac MSCs (YS-MSCs)
CD90, CD105, and CD45, CD44, and Adipogenic, chondrogenic, Mançanares
CD79 CD79 osteogenic et al. (2015)
Table 9.2 Buffalo mesenchymal stem cell sources and their characterization
Negative
Positive markers markers Differentiation References
Adipose tissue MSCs
Oct-4, CD44 – Adipogenic, chondrogenic, Hepsibha
osteogenic et al. (2011)
CD105, CD90, CD73 CD34, Adipogenic, osteogenic, Sampaio
CD45, chondrogenic et al. (2015)
CD79A
Amniotic membrane MSCs
Oct-4, Sox-2, Nanog – Neurogenic Dev et al.
(2012)
CD29, CD44, CD105, OCT4, CD34 Adipogenic, chondrogenic, Ghosh et al.
SOX2, NANOG, TERT osteogenic (2015)
CD29, CD44, Nanog, Oct-4, CD34 Adipogenic, chondrogenic, Sadeesh
Sox-2 osteogenic et al. (2016)
CD44, CD90, CD105, CD73, CD34, Adipogenic, osteogenic, Deng et al.
CD29, CD166, OCT4, SOX2, CD45 chondrogenic, and neural (2018)
NANOG, REX-1, SSEA-1, lineages (FABP4, Ost, ACAN,
SSEA-4 and TRA-1-81, COL2A1, Nestin, and β
Cytokeratin 18 (epithelial) III-tubulin)
Bone marrow MSCs
CD73, CD90, CD105, Nanog, CD31, Adipogenic, chondrogenic, Gade et al.
Oct-4, STRO-1, Sox2 CD34 osteogenic (2013)
Umbilical cord MSCs
OCT4, NANOG and SOX2 – Adipogenic, osteogenic Singh et al.
(2013)
Wharton’s jelly MSCs
CD-73, CD-105 and CD-90, CD34 Adipogenic, chondrogenic, Sreekumar
Stro-1, Oct-4, Nanog, Sox-2 osteogenic et al. (2014)
AD-MSCs adipose tissue MSCs, BM-MSCs bone marrow MSCs, d-MSCs dermis MSCs, Am-MSCs
amniotic membrane MSCs, UC-MSCs umbilical cord MSCs, WJ-MSCs Wharton’s jelly MSCs
186 M. B. Gugjoo et al.
Earlier the cells attach to plastic, earlier the fibroblastic cells appear as hasd
been demonstrated for early gestation-derived bufAm-MSCs (Deng et al. 2018)
Cattle and buffalo MSCs tend to have similar culture characteristics. Only differ-
ence may remain in their ability to survive for longer periods. Buffalo adipose tissue
(AD)-MSCs had been proliferated for more passages in comparison to cattle (cAD)-
MSCs, yielding higher cell concentration in the former sources (Sampaio et al.
2015). Such differences may arise with respect to tissue sources, their physiological
state, and age of donor.
MSCs proliferation potential tends to decrease with age. cBM-MSCs from young
donor had demonstrated vigorous proliferation in comparison to the adult source
(Jiwu et al. 2008). Health status also affects the MSCs culture characteristics as
infections like endometritis had reduced proliferation potential of cEnd-MSCs
(Lara et al. 2017a, b), while tumor growth (adenomyotic uterus) had enhanced their
pluripotency expression profile (Nanog and Oct-4) (Łupicka et al. 2015).
the ovary had induced estrus in bilateral ovarian dystrophic cattle. Fifty percent of
animals (4 out of 8 animals) had come into heat. Out of those four animals, 50% (2
out of 4 animals) had ovulated, formed corpus luteum (CL), and attained pregnancy.
It could be inferred that cAF-MSCs injection may bring animals into heat and sub-
sequently CL formation can occur but may also be affected by other factors (Chang
et al. 2018). Endometritis reduce cEnd-MSCs proliferation potential (Lara et al.
2017a, b), and as such extrinsic implantation of MSCs may be effective way to con-
trol endometritis due to their immunomodulatory or anti-inflammatory properties.
The cellular implantation may be made through per vaginal route where cells may
migrate and home to endometrium as has been demonstrated in horse (Mambelli
et al. 2013; Falomo et al. 2015).
MSCs may be utilized to improve and/ or maintain elite bull germplasm. In vitro
cMSCs differentiated into germ cell-like cells may be implanted into the testicles. It
has been demonstrated that ram testis had supported germ cell-like cells growth and
survival. The cells had even migrated and homed to the basement membrane of
seminiferous tubules. These cells had formed spermatogonia-like cell colonies sim-
ilar to testicular native spermatogonia (Ghasemzadeh-Hasankolaei et al. 2015). In
lieu cBM-MSCs trans-differentiated to germ cell-like cells may be evaluated for
their utility.
In livestock sector, elite animals are preferred for their higher production potential,
which creates an environment of stress (Jóźwik et al. 2012). Stress makes these tis-
sue cells more amenable to early aging. MSCs exhibit dynamic antioxidant action
through endogenous production of hemeoxygenase-1 (HO-1) and glutathione and
restricting cellular production of reactive oxygen species (Vanella et al. 2012; Calió
et al. 2014; Ayatollahi et al. 2014). These features also attribute to MSCs tolerance
toward the oxidative stress (Brandl et al. 2011). Due to these features, MSCs may
prevent telomere shortening in cells preventing their senescence (Estrada et al. 2013).
Mastitis that causes decrease in milk production has lot more associated problems
as it makes human health amenable to diseases through bad quality milk and through
antibiotic resistance. Currently, no ideal treatment is available to combat this prob-
lem in productive cattle. Mammary stem cells are being isolated and characterized
from mammary gland of bovines in addition to other species (Shackleton et al.
2006; Martignani et al. 2009; Martignani et al. 2014). Isolation of different stem cell
types from mammary gland is currently under process (Thomas et al. 2011; Rauner
and Barash 2012; Martignani et al. 2014).
MSCs can prove useful in two ways: on one way the cells may secrete specific pro-
teins and on other way their antimicrobial effects may prevent mastitis. Co-culture of
190 M. B. Gugjoo et al.
mammary epithelial cells and cUC-MSCs had enhanced quantity of β-casein and κ
casein, in addition to the expression of other proteins in mammary epithelial cells. The
possible pathway followed in the mammary epithelial cells might have been JAK/
STAT as IGF-1 had inhibited their expression profile (Zhao et al. 2017).
Fetal cBM-MSCs and cAD-MSCs conditioned medium carries antibacterial
activity. The conditioned medium had decreased about 30% of the growth of
Staphylococcus aureus under in vitro conditions (Cahuascanco et al. 2019).
Transfection of c-/buf-MSCs with bovine lactoferricin (LFcinB) gene may endow
them with the properties to express antibacterial peptide LfcinB (Sharma et al.
2017). Such LFcinB-cloned MSCs if injected into the mammary gland may prove
fruitful through their higher antibacterial properties especially against Staphylococcus
aureus and Escherichia coli (Sharma et al. 2017). Thus, extensive studies are desired
to confirm the feasibility and efficacy of using these transfected MSCs in clinical
settings.
Cattle/ buffalo teat fistula and fibrosis have huge economic impact through loss
of milk and potential to develop mastitis. MSCs applications in such conditions
are desired. In our own unpublished study, allogeneic MSCs applications in
post-surgical repair of teat fistula promote early healing of wounds. Thus,
MSCs applications appear promising in such cases. The possible beneficial
effects may be through MSCs differentiation into various types of epithelial
cells (Wu et al. 2007; Borena et al. 2009) and/or through vasculogenesis (Borena
et al. 2009).
9.3.6 B
ovine Spongiform Encephalopathy (BSE)
and Nerve Injuries
the spleen, lymph nodes and brain assays (Sohn et al. 2009). Thus, there is need to
confirm if the said potential is also reduced in BSE and whether these cells can be
used to treat infected animals.
Stem cells may trans-differentiate into the neural cell-like cells or secrete trophic
factors (Hokari et al. 2008; Saito et al. 2008; Cardoso et al. 2012a; Xiong et al.
2014), though the direct neuronal differentiation is questionable (Hokari et al. 2008;
Wu et al. 2003; Dobkin et al. 2006). Commonly affected peripheral nerves like
radial, obturator, and sciatic nerves make large ruminants lame and/or recumbent.
Thus, MSCs studies may be undertaken to evaluate their therapeutic potential in
these peripheral nerve injuries.
MSCs are extensively studied for their therapeutic effects in osteoarthritis. Cattle
chronic osteoarthritis (OA) or degenerative joint disease (DJD) tends to cause male
infertility (impotentia coeundi) in bulls (Heinola et al. 2013; Wolfe 2018). MSCs
may be effective in OA; however, currently the therapy has not been able to provide
desired results and may be due to the inhibitory effects of locally available inflam-
matory mediators. The inflammatory environment tends to enhance expression of
adhesion molecule preventing cell migration and decrease their secretion of matrix
(Zayed et al. 2016; Reesink et al. 2017).
Cattle and horse laminitis carry almost similar pathogenesis (Nilsson 1982) and
as MSCs have been demonstrated to be play effective role in equine acute laminitis
(Dryden et al. 2013; Morrison et al. 2014), studies in cattle may also be undertaken.
9.4 Conclusions
Cattle and buffalo MSCs have been harvested from numerous sources and are well
characterized. MSCs properties may vary with respect to the source and may be
affected with respect to the age and health status. Literature on cattle/ buffalo MSCs
applications mainly focuses on their production and reproduction-related traits. The
results of these studies however are currently inconclusive. MSCs potential applica-
tions go beyond these reproductive and/ production-related traits and may be stud-
ied in such areas as well.
192 M. B. Gugjoo et al.
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Cat Mesenchymal Stem Cell
Characteristics and Potential 10
Applications
M. B. Gugjoo and Amar Pal
Abstract
Cat mesenchymal stem cells (fMSCs) have been culture expanded and character-
ized from various tissue sources, though mainly from adipose tissues. The cells
have been evaluated for their therapeutic effects in various experimental models
and clinical trials relevant to veterinary and human practice. The studies have
potentially favored MSCs applications; however, limited available studies cur-
rently fail to provide any conclusive result(s). The current chapter discusses
fMSCs characteristics and their potential applications.
10.1 Introduction
Cat is increasingly being kept as a companion animal, and as such their clinical
practice is increasing by each passing day. Regenerative medicine involving MSCs
is gradually being employed in feline practice. Cat and human genome are highly
linked, and as such cat MSCs experimental/clinical studies may provide proof of
therapeutics for human medicine (Munoz et al. 2012), in addition to the therapeutics
in veterinary practice.
M. B. Gugjoo (*)
Division of Veterinary Clinical Complex, FVSc & AH, SKUAST-Kashmir,
Jammu and Kashmir, India
A. Pal
Division of Surgery, Indian Veterinary Research Institute, Izatnagar, UP, India
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 197
Springer Nature Singapore Pte Ltd. 2020
M. B. Gugjoo, A. Pal (eds.), Mesenchymal Stem Cell in Veterinary Sciences,
https://doi.org/10.1007/978-981-15-6037-8_10
198 M. B. Gugjoo and A. Pal
An initial report on feline mesenchymal stem cells was reported by Martin and co-
workers in 2002 that had isolated and characterized the cells from bone marrow
(Martin et al. 2002). Since then many reports have been published on their isolation
and culture expansion. The cells have been isolated from various although limited
tissue types as detailed in Table 10.1. Most of the studies have focused on the adi-
pose tissue derived MSCs. Cat MSCs (fMSCs) are being characterized based on the
standard criteria laid down by the International Society for Cellular Therapy (ISCT).
fMSCs are plastic adherent with spindle-shaped fibroblast-like appearance (Quimby
et al. 2011; Arzi et al. 2015; Sato et al. 2016; Quimby and Borjesson 2018). These
cells variably express various surface markers and lack hematopoietic markers.
Among various sources, some of the studies had shown MSCs expression of the
hematopoietic (CD34) and other markers like MHC-II, while others had reported
MSCs failure to express CD90 (Iacono et al. 2012; Mumaw et al. 2015; Vidane et al.
2016). The cells show trilineage differentiation (adipogenic, chondrogenic, and
osteogenic lineages) (Table 10.1).
The cells have been culture expanded in growth medium containing the fetal
bovine serum (FBS). A study that utilized combination of FBS (10%) and autolo-
gous plasma had shown similar fPB-MSCs characteristics as that of cells being
cultured under FBS. Currently, studies on FBS alternatives to culture expand fMSCs
is lacking. fMSCs tend to undergo early senescence with extensive passaging. Cat
peripheral blood (fPB)-MSCs had steadily expanded up to 5–6 passages, but almost
all the cells had stopped proliferation at passages 7–9. Among 15 cultures, only 01
culture had proliferated up to passage 10. The cellular proliferation, however, had
remained unaffected by age, sex, and breed (Sato et al. 2016). The population dou-
bling levels of cat adipose tissue (fAD)-MSCs demonstrated at different passages
were 2.08 ± 0.02 (P1), 3.95 ± 0.07 (P2), 5.88 ± 0.15 (P3), 7.14 ± 0.15 (P4),
8.00 ± 0.16 (P5), and 8.78 ± 0.19 (P6). From P6 onward, minimal increase in dou-
bling levels had occurred. As such the population doubling time had significantly
increased to an average of 95.7 h. at P4 and 141.3 h. at P5 from 62.1 h. at P3 (Kim
et al. 2017). Similarly, another study had shown that fAD-MSCs population dou-
bling time at passage 5 had increased significantly. The decreased expression of
pluripotency markers and of surface markers was also seen after extended culture,
significantly at P5. The musculoskeletal (especially osteogenic and chondrogenic)
differentiation potential too had reduced with progressive culturing in these cells
(Lee et al. 2018). fAD-MSCs at P2 had been characterized by their normal meta-
phase and karyotype (Villatoro et al. 2018).
To improve cellular proliferation and self-renewal, culture modifications are
being worked out. Culture addition of recombinant acid ceramidase (rAC) had
improved growth of cat bone marrow (fBM)-MSCs by twofold at 1 week. Even
addition of rAC to chondrogenic medium had improved cellular chondrogenic
potential, synergistic with TGF-β1 (Simonaro et al. 2013). rAC might have reduced
the cell stress that arises with the harvesting procedure and during their ex vivo
expansion. The possible beneficial effect of the rAC treatment in chondrogenic
10 Cat Mesenchymal Stem Cell Characteristics and Potential Applications 199
Table 10.1 (continued)
Positive markers Negative markers Differentiation References
Bone marrow MSCs (BM-MSCs)
CD44, CD90, CD105 CD4, MHC-II Adipogenic, Quimby et al.
chondrogenic, and (2011)
osteogenic
CD44, CD90, CD105 CD4, MHC-II (low Adipogenic, Webb et al.
expression) chondrogenic, and (2011)
osteogenic
CD29, CD44, CD105, CD14, CD34, Adipogenic, osteogenic, Munoz et al.
Stro-1 CD45, MHC-II and neurogenic (2012)
CD29, CD44, CD105, CD45 CD73, CD90, Adipogenic, Mumaw et al.
(30%), MHC-II (39%) CD79α, and CD34, chondrogenic, and (2015)
osteogenic
Peripheral blood MSCs (PB-MSCs)
CD44, CD90 CD4, MHC-II Adipogenic, Sato et al.
chondrogenic, and (2016)
osteogenic
AD-MSCs adipose tissue MSCs, Am-MSCs amniotic membrane MSCs, BM-MSCs bone marrow
MSCs, PB-MSCs peripheral blood MSCs
differentiation process may occur through better TGF-β pathway signaling, in addi-
tion to the effects of sphingolipid changes on chondrogenic differentiation. Further,
stress induced with serum-free chondro-differentiation of MSCs may also be con-
trolled (Simonaro et al. 2013).
The cells isolated from various tissues and at variable locations may have vari-
able culture characteristics. fBM-MSCs and fAD-MSCs had similar surface mark-
ers expression, but fAD-MSCs had proliferated significantly faster as compared to
BM-MSCs. Thus, fBM-MSC and fAD-MSC were phenotypically similar, but
higher intrinsic proliferation rate had made fAD-MSC culture proliferation easier
(Webb et al. 2011). In relation to variable donor tissue locations, abdominal fAD-
MSCs harbored higher percentage of cells expressing positive surface markers
(CD90 and CD105) than those of subcutaneous fAD-MSCs. Although comparable
pluripotency marker (Oct-4 and Klf4) expression had been reported, except for
Nanog that had been highly expressed in subcutaneous fAD-MSCs. The cells had
shown an equivalent in vitro multilineage differentiation including the neuron-like
cells (Gómez et al. 2015).
fAD-MSCs can contract latent Feline Foamy Virus (FFV, retrovirus) infection that
induces formation of syncytial cell and subsequent apoptosis in them. FFV in cul-
tured cells had led to their morphologic and proliferative alterations. FFV-infected
fAD-MSC lines cultured in growth media had formed multinucleated giant cells
and had topped proliferation at passages 3–5. Incorporation of antiviral tenofovir
10 Cat Mesenchymal Stem Cell Characteristics and Potential Applications 201
MSCs culture expansion is aimed to achieve enough concentration for their in vivo
applications. MSCs have been applied either through intravenous and intraperito-
neal or through local routes. All these routes have been found to be safe and without
any potential adverse effects. For better concentration of MSCs in cerebrospinal
fluid (CSF), intraventricular route of implantation was demonstrated to be superior
as compared to the subdural and brain parenchyma route (Glage et al. 2011). The
mild reactions in the form of local lymph node swelling and transient lethargy and
inactivity had been encountered (Webb and Webb 2015; Trzil et al. 2016; Parys
et al. 2016). The intravenous applications of MSCs have usually been followed by
coagulation cascade being prevented by heparin. Below are the in vivo fMSCs
experimental (Table 10.2) and clinical (Table 10.3) studies.
Kidney ailments are one of the common clinical conditions with life-threatening
potential in domestic cats (Lawson et al. 2015). Currently, an effective and specific
treatment to restore the affected kidney to normalcy is unavailable (Vidane
et al. 2016).
Table 10.2 In vivo preclinical experimental mesenchymal stem cell studies in cats
202
10.3.7 Cardiomyopathy
10.4 Conclusions
fMSCs have been harvested from various sources mainly from adipose tissues. The
cells have been culture expanded and characterized as per the ISCT. The cells
exhibit limited self-renewal properties in currently available media as their viability
decreases and become more senescent at early culture passages (p6). fAD-MSCs
are more prone to FFV that leads to their morphologic and proliferative alterations.
The cellular applications have been made in various experimental models and or
clinical conditions. The results of these studies have variably been pro-therapeutic.
In case of kidney failure, however, little to no success with fMSCs has been demon-
strated. These studies are very limited with lack of sufficient data to be considered
sufficient for any conclusion(s).
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Dog Mesenchymal Stem Cell Basic
Research and Potential Applications 11
M. B. Gugjoo, Amar Pal, and G. T. Sharma
Abstract
Mesenchymal stem cells (MSCs) being available in numerous adult tissues and
foetal membranes have been harvested from almost all these sources in dog. Due
to the presence of miniscule number of MSCs in these tissues, culture expansion
followed by their characterization becomes imperative. These characterized cells
have been made subject in preclinical experimental and clinical settings in dogs.
The studies have variably but undeniably supported their therapeutic effects,
although currently inconclusive. Number of dog clinical ailments compeer to
that of human. Therapeutic benefits of dMSCs in these ailments may provide
proof-of-therapeutic principle for human medicine. The current chapter details
dMSCs culture characteristics and potential applications.
11.1 Introduction
The population of pets like dog is increasing in the world. These pets are considered
as family members and owners spent many dollars to keep them healthy. Dog, Canis
familiaris, suffers from numerous ailments that lack standard treatment. Many of
M. B. Gugjoo (*)
Division of Veterinary Clinical Complex, FVSc & AH, SKUAST-Kashmir,
Jammu and Kashmir, India
A. Pal
Division of Surgery, Indian Veterinary Research Institute, Izatnagar, UP, India
G. T. Sharma
Division of Physiology and Climatology, Indian Veterinary Research Institute,
Izatnagar, UP, India
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 213
Springer Nature Singapore Pte Ltd. 2020
M. B. Gugjoo, A. Pal (eds.), Mesenchymal Stem Cell in Veterinary Sciences,
https://doi.org/10.1007/978-981-15-6037-8_11
214 M. B. Gugjoo et al.
A diverse set of tissues have been evaluated for stem cell reserve in dogs. This
includes adult issues and the foetal membranes (Table 11.1) (Gugjoo et al. 2019a).
These cells have also been harvested from induced pluripotent stem cells (Chow
et al. 2017). The current tissue harvesting procedures are invasive; efforts are being
made to develop advanced harvesting procedures that reduce animal sufferings. For
muscle tissue harvesting, minimally invasive technique of microbiopsy (leads to
insignificant muscle damage) had been demonstrated as reasonably safe way to har-
vest MSCs (Ceusters et al. 2017). Foetal membrane tissues are limited in dogs as
pets undergo elective castration in juvenile age (Sultana et al. 2018). dMSCs in
compensation, may be harvested from ovaries and/or uterus of spayed animals
(Sultana et al. 2018).
Table 11.1 Dog Mesenchymal stem cell (MSC) sources and their characterization
Positive markers Negative markers Differentiation References
Adipose MSCs (AD-MSCs)
CD44, CD90, CD14 and CD45 Osteogenic Kang et al.
MHC-I, CD34 (2008)
(partially)
OCT4, NANOG, Not available Osteogenic, Neupane et al.
SOX2 Chondrogenic and (2008)
limited Adipogenic
CD44, CD90, CD14, CD34, CD45 Osteogenic, Ryu et al.
CD105 Adipogenic, (2009)
neurogenic
CD44, CD29, CD14, CD34, CD45, CD117, Osteogenic, Vieira et al.
CD90 CD13, CD105, CD73 Adipogenic, (2010)
Chondrogenic,
myogenic
CD90, CD44, CD34, CD45 Osteogenic, Martinello
CD140a, CD117 Adipogenic, myogenic et al. (2011)
CD29, CD44, thy CD31, CD34, CD73, CD105 Osteogenic, Oh et al.
1.1 Adipogenic, (2011)
Chondrogenic,
myogenic, neurogenic
OCT4, NANOG, – Osteogenic, Guercio et al.
SOX2 Adipogenic, (2012)
chondrogenic
CD44, CD90 CD34, CD45, CD146 Osteogenic, Kisiel et al.
SOX2, OCT4, Adipogenic (2012)
NANOG
CD29, CD44, CD CD34, CD45, SSEA-3, Osteogenic, Takemitsu
90, SSEA-1 (low), SSEA-4, TRA-1-60 and Adipogenic et al. (2012)
OCT3/4, SOX2, TRA-1-81
NANOG
CD44, CD73, CD14, CD34, CD45 Neurogenic Ryu et al.
CD90, CD105 (2012)
CD29, CD90 and CD34, CD45 and MHC-II Osteogenic, Villatoro et al.
STRO-1 Adipogenic, (2015)
Chondrogenic
CD90, CD44, CD34, CD45 Primordial germ Wei et al.
CD166, Oct4, Sox2 cell-like cells and male (2016)
germ cells
CD 90, CD 105 CD 14, CD 45 Osteogenic, Blecker et al.
Adipogenic, (2017)
Chondrogenic,
neurogenic
CD9, CD44, CD90, CD34, CD45 and STRO-1 Osteogenic, Bearden et al.
and CD105, Adipogenic, (2017)
NANOG, OCT4, Chondrogenic
and SOX2
(continued)
216 M. B. Gugjoo et al.
Table 11.1 (continued)
Positive markers Negative markers Differentiation References
CD44, CD90 CD34, CD45 Osteogenic, Kriston-Pál
Adipogenic, et al. (2017)
neurogenic
CD90, CD44, CD34, CD45 Osteogenic, Trindade et al.
NANOG, TERT, Chondrogenic, (2017)
SOX2, OCT4 Adipogenic,
neurogenic, endoderm,
primordial germ cells
CD90, CD44, Sox2 CD45 Osteogenic, Ayala-Cuellar
and Nanog Chondrogenic et al. (2019)
CD73, CD90, CD45, CD14, HLA-DR and Osteogenic, Bçrziòð et al.
CD44 CD34. Chondrogenic, (2018)
Adipogenic
CD90, OCT4, CD34, CD45, MCH-II – Enciso et al.
SOX9, RUNX2, (2018)
PPARG
CD90, CD44 CD45, CD11b Osteogenic, Sasaki et al.
Chondrogenic, (2018)
Adipogenic
CD44, CD90 CD 34 and CD45 Osteogenic, Shah et al.
Chondrogenic (2018)
CD29, CD44, CD11/18, CD34, CD45, Osteogenic, Villatoro et al.
CD73, CD90 and MHC-II Adipogenic, (2018)
STRO-1 Chondrogenic
Amniotic fluid MSCs
Oct-4, CD44, CD34, CD45 Osteogenic, Uranio et al.
DLA-DRA1 and Chondrogenic, (2011)
DLA-79 Adipogenic,
neurogenic
Amniotic membrane MSCs
CD90, CD105 CD3, CD11c, CD28, CD38, Osteogenic, Park et al.
CD62L, CD34, CD45, Chondrogenic, (2012a)
CD41a Adipogenic,
neurogenic
Oct-4, CD44, CD34, CD45 Osteogenic, Uranio et al.
CD184 and CD29 Chondrogenic, (2011)
Adipogenic,
neurogenic
Bone marrow MSCs
CD90, MHC-I CD34, CD45, MHC-II Neurogenic Kamishina
et al. (2006)
CD105, CD90 CD45, CD34 Osteogenic, Csaki et al.
Chondrogenic, (2007)
Adipogenic
(continued)
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications 217
Table 11.1 (continued)
Positive markers Negative markers Differentiation References
CD10, CD13, CD3, CD14, CD34, CD45 – Lee et al.
CD29, CD44, (2011b)
CD73/SH-3, CD90/
Thy-1 and CD106/
VCAM-1
CD 44 and CD 90 CD34 Osteogenic Tharasanit
Chondrogenic, et al. (2011)
Adipogenic,
neurogenic
CD44, STRO-1 CD34, CD45 Chondrogenic Hodgkiss-
Geere et al.
(2012)
CD44, CD90. CD34, CD45, CD146 Osteogenic, Kisiel et al.
SOX2, OCT4, Adipogenic (2012)
NANOG
CD44, CD73, CD14, CD34, CD45 Neurogenic Ryu et al.
CD90, CD105 (2012)
CD29, CD44, CD CD34, CD45, SSEA-3, Osteogenic, Takemitsu
90, OCT3/4, SOX2, SSEA-4, TRA-1-60, Adipogenic et al. (2012)
NANOG TRA-1-81
CD44, CD105 CD34, CD45 – Kazemi et al.
(2017)
CD29, CD44, CD45 and CD11b Osteogenic, Matsuda et al.
CD90 Adipogenic (2017)
CD90, OCT4, CD34, CD45, MCH-II – Enciso et al.
SOX9, RUNX2, (2018)
PPARG
CD 166, CD 29, CD45 and CD 34 Osteogenic, Li et al.
CD 90, CD 105 and Chondrogenic (2018)
CD 44
CD90, CD44 CD45, CD11b Osteogenic, Sasaki et al.
Chondrogenic, (2018)
Adipogenic
CD9, CD44, CD90, CD34, CD45 and STRO-1 Osteogenic, Bearden et al.
and CD105, Adipogenic, (2017)
NANOG, OCT4, Chondrogenic
and SOX2
Corneal MSCs
CD90, CD73, CD34 Osteogenic, Kafarnik et al.
CD105, N-cadherin, Chondrogenic, (2020)
Pax6 Adipogenic
Dental pulp MSCs
CD90, CD44, – Osteogenic, Khorsand
CD146, SSEA-4 Chondrogenic, et al. (2013)
(low) Adipogenic
(continued)
218 M. B. Gugjoo et al.
Table 11.1 (continued)
Positive markers Negative markers Differentiation References
Endometrium MSCs
CD44, CD29, CD45, CD34, CD31 (low to Osteogenic, De Cesaris
CD90, CD13, nil) Chondrogenic, et al. (2017)
CD133, CD73, Adipogenic
CD105, Oct4
Limbal epithelium MSCs
CD90, CD44, CD45, CD11b\c Osteogenic, Sancak et al.
CD49 Chondrogenic, (2014)
Adipogenic,
keratinocyte
Muscle MSCs
CD44, CD90, CD34, CD45 Osteogenic, Kisiel et al.
SOX2, OCT4, Adipogenic (2012)
NANOG
Olfactory epithelium MSCs
CD73, CD90, CD34, CD45, CD79 Osteogenic, Pinheiro et al.
CD105 Chondrogenic, (2016)
Adipogenic,
neurogenic
Omental MSCs
CD90, CD44 CD45, CD11b Osteogenic, Bahamondes
Chondrogenic, et al. (2017)
Adipogenic
Ovarian MSCs
CD90, CD44, CD34, CD45 Osteogenic, Trindade et al.
NANOG, TERT, Chondrogenic, (2017)
SOX2, OCT4 Adipogenic,
neurogenic, endoderm,
primordial germ cells
Periodontal ligament MSCs
– – Adipogenic, Sedigh et al.
Osteogenic (2010)
Periosteal MSCs
CD44, CD90, CD34, CD45 Osteogenic, Kisiel et al.
SOX2, OCT4, Adipogenic (2012)
NANOG
Synovial membrane MSCs
CD9, CD44, CD90, CD34, CD45 and STRO-1 Osteogenic, Bearden et al.
and CD105, Adipogenic, (2017)
NANOG, OCT4, Chondrogenic
and SOX2.
CD90, CD44 CD45, CD11b Osteogenic, Sasaki et al.
Chondrogenic, (2018)
Adipogenic
Peripheral blood MSCs
CD34(low) CD34 Osteocalcin Huss et al.
(2000)
(continued)
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications 219
Table 11.1 (continued)
Positive markers Negative markers Differentiation References
Placental MSCs
CD90, CD29, CD45, CD34 and MHC-II, Osteogenic, Saulnier et al.
CD44, CD105, NANOG, OCT4 Chondrogenic, (2016)
CD73, SOX2 (low) Adipogenic
CD44, CD29, MHC-II, CD45, CD34 Osteogenic, Cabon et al.
CD90 Adipogenic, (2019)
Chondrogenic
Umbilical cord MSCs
CD29, CD33, CD4, CD8a, CD10, CD14, Osteogenic, Seo et al.
CD44, CD105, CD20, CD24, CD31, CD34, Chondrogenic, (2009)
CD184, Oct4 CD38, CD41a, CD45, neurogenic
CD49b, CD41/61, CD62p,
CD73, CD90, CD133,
HLA-DR
Umbilical cord tissue MSCs
CD44, CD54, CD4, CD8A, CD25, CD33, Chondrogenic Lee et al.
CD61, CD80, CD34 and CD117 (2013)
CD90, CD105,
Flk1, HMGA2,
Sox2, Nanog,
Oct3/4
Oct-4, CD44, CD34, CD45 Osteogenic, Uranio et al.
CD184 and CD29 Chondrogenic, (2011)
Adipogenic,
neurogenic
Umbilical cord vein MSCs
CD44, CD29, CD14, CD34, CD45, CD117 Osteogenic, Zucconi et al.
CD90 Adipogenic, (2010)
Chondrogenic
CD44, CD73, CD14, CD34, CD45 Neurogenic Ryu et al.
CD90, CD105 (2012)
Wharton’s jelly MSCs
CD90, CD105 CD3, CD11c, CD28, CD38, Osteogenic, Seo et al.
CD62L, CD34, CD45,CD41a Chondrogenic, (2012)
neurogenic
CD44, CD73, CD14, CD34, CD45 Neurogenic Ryu et al.
CD90, CD105 (2012)
AD Adipose tissue, AF Amniotic fluid, AM Amniotic membrane, BM Bone marrow, DP Dental
pulp, End Endometrium, LE Limbal epithelium, Li liver, MSC Mesenchymal stem cell, Mu Muscle,
OE Olfactory epithelium, Om Omentum, Ov Ovary, PL Periodontal ligament, Pe Periosteum, PB
Peripheral blood, Pl Placenta, Sy synovium, UCB Umbilical cord blood, UCT Umbilical cord tis-
sue, UCV Umbilical cord vein, WJ Wharton’s jelly
elongated to cuboidal shapes too have been demonstrated (Huss et al. 2000; Csaki
et al. 2007; de Bakker et al. 2013).
potential of dSy-MSCs than dBM-MSCs and MSCs derived from inguinal and
infrapatellar fat pad (Sasaki et al. 2018). Between dBM-MSCs and dLi-MSCs,
chondrogenic potential had been higher in former while liver specific genes were
better expressed in the latter (Malagola et al. 2016). Among various sources like
adipose tissue, bone marrow, umbilical cord and Wharton’s Jelly, dAD-MSCs had
higher potential to proliferate. Vascular endothelial growth factor expression
(VEGF) had been higher in dBM-MSCs. At early phase dAD-MSCs and dog umbil-
ical cord blood- (dUCB-) MSCs had greater ability of osteogenesis, however, com-
parable osteogenesis was demonstrated in all these cell lines (Kang et al. 2012).
From all these studies it can be inferred that tissue source plays an important role in
MSCs proliferation and differentiation properties. Musculoskeletal tissue (synovium
and bone marrow) derived MSCs may be preferable for musculoskeletal tissue
regeneration.
Location of the particular tissue may also affect MSCs properties. Omentum and
subcutaneous adipose tissue derived AD-MSCs had comparable trophic, vasculo-
genic and immuno-modulatory properties. Omentum, however, had significantly
higher cell yield as compared to others (Bahamondes et al. 2017). dMSCs from
subcutaneous fat had shown multipotent differentiation up to passage 8 while such
a potential of MSCs from visceral (periovaric) fat had been only up to passage 4.
The former cell type additionally had higher osteogenic expression (Yaneselli et al.
2018). Among various BM-MSCs sources ilium derived cells had been most prolif-
erative (Volk et al. 2012). Mandible MSCs may carry higher cell proliferation in
comparison to the femur (Bugueño et al. 2017). However, considering factors like
collection technique and cell characteristics, humerus had been considered to
be better source (Laura et al. 2008).
Ageing may reduce dMSCs proliferation and differentiation potential. MSCs from
aged dog bone marrow had a significant (40%) reduction in proliferation potential,
in addition to the osteogenic potential as compared to young ones (Volk et al. 2012).
Other similar study had demonstrated young donor AD-MSCs carried higher popu-
lation doubling potential as compared to those of aged dog MSCs. These cells had
also expressed higher surface and pluripotency markers (Lee et al. 2017). Other
study though had reported comparable osteogenic gene and fibroblast growth fac-
tor-10 expression in young and aged dMSCs but tumour necrosis factor and inter-
feron gamma expression had increased in aged dAD-MSCs (Taguchi et al. 2019).
Thus, available environment may affect dMSCs expression potential.
dMSCs may be affected by steroids (Marycz et al. 2014; Guo et al. 2015; Jo et al.
2017) and vitamin C (Guo et al. 2015). Low dose steroids may improve dMSCs
222 M. B. Gugjoo et al.
activity while higher doses might be cytotoxic (Marycz et al. 2014). Vitamin C may
also improve dMSCs proliferation (Guo et al. 2015).
Dog breeds may vary in their genetics and are expected to show some cellular dif-
ferences including of MSCs. Most of the breeds including German shepherd,
Labrador, Golden retriever, Border collie and Malinois had comparable initial
BM-MSCs population except one (Hovawart) that had much lower cell population.
MSCs from these breeds (German shepherd, Labrador, Golden retriever and Border
collie), however, had undergone early senescence. The cellular differentiation
potential in these breeds may also vary (Bertolo et al. 2015).
MSCs are increasingly being utilized in dog clinical practice. dMSCs have been
studied in various types of musculoskeletal and non-musculoskeletal tissues (Tables
11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 11.10, 11.12, and 11.13) (Gugjoo et al.
2019a). The in vivo studies have largely supported dMSCs therapeutic potential
(Hall et al. 2010; Sousa et al. 2011; Kim et al. 2011; Cuervo et al. 2014; Muir et al.,
2016). Many of these studies have failed to report any major adverse effect (Cuervo
et al. 2014; Bçrziòð et al. 2018; Villatoro et al. 2018) while some have reported
adverse reactions like self-controllable localized inflammation and pulmonary
parenchymal oedema and haemorrhage (Mokbel et al. 2011; Park et al. 2012a, b;
Kang and Park 2014).
In vivo MSCs viability remains important for their effectiveness. MSCs in vivo
implantation exposes them to noxious environment. As such the scaffold seeded
cells are being implanted. dAD-MSCs encapsulated in alginate beads had improved
retention and the cells were viable up to 7 days (Koh et al. 2017). The studies
described in this chapter range from preclinical experimental models to the clinical
trials. The clinical studies described are mostly uncontrolled and have been con-
ducted as case report, case series and/or comparative studies.
11.3.1.1 Osteoarthritis
Osteoarthrosis (OA) affects larger dog population (20%) (Johnston, 1997).
Currently, no successful clinical treatment is available (Gugjoo et al. 2016; Gugjoo
et al. 2017; Li et al. 2018; Gugjoo et al. 2019b). As such stem cell applications in
dog are being evaluated under experimental settings (Table 11.2) and in also clinical
trials (Table 11.3).
Table 11.2 In vivo chondrogenic preclinical experimental mesenchymal stem cell studies in dogs
Model defect
Number of size/Study Cell
Model type animals period Biomaterial used dose Evaluation criteria Overall result References
Partial 32 (8 3 mm diameter Autologous 1.4– Morphological, Recovery was significant Mokbel
thickness control; 12 and 1 mm BM-MSCs 1.6 × 106 histological and both clinically and et al.
chondral group II; 12 depth/8 weeks fluorescence analysis histologically in the two (2011)
defects of group III) cell treated groups (group
lateral femoral II and III) as compared to
condyle the control (group I). In
the meantime, group II
showed better results at
8 weeks than group
III. Homing was
confirmed by the
incorporation of injected
GFP-labelled MSCs
within the newly formed
cartilage
Osteochondral Group I: 4.2 mm Chondrogenically- – Gross morphology and Statistically significant Yang et al.
defects of Cell + diameter and induced BMSCs by histological, improvement in gross and (2011)
medial femoral scaffold; 6 mm and scaffold biochemical, histology and cartilage
condyles group II: depth/3 months biomechanical and stiffness in cell/scaffold
Control and 6 months micro-CT analyses treated animals as
compared to control.
Comparable micro-CT
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications
analysis of the
subchondral bone in two
groups. Better results in
later period than at early
period
(continued)
223
Table 11.2 (continued)
224
Model defect
Number of size/Study Cell
Model type animals period Biomaterial used dose Evaluation criteria Overall result References
Osteonecrosis 24 (54 hip 2 mm diameter VEGF 165 2 × 107 Radiography, single- Better results in group I as Hang et al.
of the femoral joints) group and 2 mm transgenic bone photon emission, compared to group II and (2012)
head I: Transgenic width/12 weeks marrow computed tomography, group III. A regular
BM-MSCs; mesenchymal stem histopathology, arrangement of trabeculae
group II: cells or simple histomorphometric and obvious bone
BM-MSCs BM-MSCs analysis and regeneration with
group III: immunofluorescent significantly increased
Control staining for von capillaries in group I
Willebrand factor animals
Osteochondral 12 (24 6 mm diameter Autologous 1 × 106 Macroscopic and Group I had statistically Kazemi
defects (defects) and depth of BM-MSCs + histopathology significant improvement et al.
group I: Cell 5 mm/4– platelet rich fibrin in gross and histological (2017)
+ PRF 24 weeks (PRF) parameters as compared
treated; to control at 16 weeks.
group II: Compared to cell treated
Control group gross parameters
were deteriorated with
time in control group
animals
M. B. Gugjoo et al.
Chondral 24 (48 4 mm/28 weeks BM-MSCs + 1 × 107 Macroscopy, magnetic Group I had regenerated Li et al.
defects of stifle defects) hyaluronic acid resonance imaging more cartilage-like tissue (2018)
joint group I: Cell (HA) (MRI), histopathology, than did HA alone or
+ HA immunohistochemistry saline. Macroscopic
treated; for type II collagen evaluation and
group II histological assessment
hyaluronic score were significantly
acid; group improved in cartilage
III: Control defects of group I as
compared to those in the
other groups. MRI
demonstrated cartilage-
like signal at defect sites,
and the surface of
cartilage was relatively
smooth. No obvious
defect was found and
cartilage-like tissue with
the same thickness of the
surrounding normal tissue
was formed in group I. for
group II, cartilage-like
signal was also observed,
but the thicknesses of
tissue at the defect sites
were thinner than those at
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications
Number of
Clinical condition/ animals Study Evaluation
ailment included period Study type Cell source Cell dose criteria Overall result References
Chronic 21 90 days Case series Autologous 5 × 106 (20 Orthopaedic Statistically significant Black et al.
osteoarthritis of (randomized, AD-MSCs dogs) and examination improvement in scores (2007)
Coxo-femoral joint double 4.5 × 106 (1 scores, for lameness and the
blinded, dog) lameness and compiled scores for
placebo- composite lameness, pain and range
controlled scores and size of motion as compared
trial) effect to control dogs
Chronic 14 180 days Case series Autologous 3–5 × 106 Orthopaedic Statistically significant Black et al.
osteoarthritis of (randomized, AD-MSCs examination improvement in (2008)
humeroradial joint double score and size lameness, pain on
blinded, effect manipulation, range of
non-placebo- motion and functional
controlled disability in cell treated
trial) animals
Chronic 4 30 days Case series Autologous 3–5 × 106 Clinical tests Improvement with time Guercio
osteoarthritis of (uncontrolled AD-MSCs (laden in like trot pain on as per owner although et al. (2012)
humeroradial joint study) platelet rich palpation and without any statistical
plasma or functional testing
hyaluronic improvement in
acid) disability
Chronic arthritis of 8 180 Autologous 15 × 106 Force platform Significant improvement Vilar et al.
the hip joint AD-MSCs analysis in clinical parameters in (2013)
cell treated cases
M. B. Gugjoo et al.
Hip osteoarthritis 39 180 days Randomized Autologous 30 × 106 Visual analog MSCs and PRGF Cuervo et al.
(autologous comparative AD-MSCs scale (VAS), implantation was safe (2014)
AD-MSCs clinical trial and PRGF bioearth scale and effective in the
vs PRGF) assessment, functional analysis at 1,
range of motion 3 and 6 months; provide
(ROM), a significant
radiography improvement, reducing
dog’s pain, and
improving physical
function. With respect to
basal levels for every
parameter in patients
with hip OA, MSCs
showed better results at
6 months
Hip dysplasia 9 (group I: 30 days Comparative Allogeneic 0.2–0.8 × 106 Physical and Positive results were Marx et al.
SVF, n = 4; study AD-MSCs at orthopaedic more clearly seen in the (2014)
group II: acupoints or examinations SVF-treated group. All
AD-MSCs; stromal dogs had shown
n = 5) vascular improvement in range of
fraction motion, lameness at trot
(SVF) and pain on
manipulation of the
joints, except for one
ASC-treated patient.
These results had shown
that autologous SVF or
allogeneic ASCs can be
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications
pathology
(continued)
Table 11.3 (continued)
228
Number of
Clinical condition/ animals Study Evaluation
ailment included period Study type Cell source Cell dose criteria Overall result References
Chronic arthritis of 15 (group I: 180 days Case control Autologous 15 × 106 X-ray and Mean values of PVF and Vilar et al.
the hip joint MSCs (blinded AD-MSCs platform gait VI were significantly (2014)
treated, control study) analysis: Mean improved within the first
n = 10; values of peak 3 months cell post-
sound dogs, vertical force treatment in the OA
n = 5) (PVF) and group, increasing 9%
vertical impulse and 2.5% body weight,
(VI) respectively, at day 30.
After this, the effect
seems to decrease
reaching initial values
Osteoarthritis of hip, 82 (bilateral 60 days Case series Allogeneic 12 × 106 Owner Success in the primary Harman
elbow, stifle, and or joints: study AD-MSCs (cryopreserved client-specific outcome was variable. et al. (2016)
shoulder joints Treated, (prospective, with 85.1% outcome CSOM was statistically
n = 35; randomized, viability) measurement improved in the treated
placebo masked and (CSOM) and dogs compared to the
control, placebo- secondary placebo dogs. The
n = 32) and controlled) measures veterinary pain on
(single included manipulation score and
joint: veterinary pain the veterinary global
Treated: on score were both
n = 8; manipulation, statistically improved in
placebo veterinary treated dogs as
control, global score, compared to placebo
n = 7) and owner
global score
M. B. Gugjoo et al.
Elbow dysplasia and 30 dogs (39 1 year Case series Allogeneic 12 × 106 ± Owner and A highly significant Kriston-Pál
elbow osteoarthritis elbow (uncontrolled AD-MSCs + 3.2 × 106cells veterinarian improvement was et al. (2017)
joints) study) hyaluronic examination, achieved after cell
acid (0.5%) arthroscopy implantation without any
and medication as
histopathology demonstrated by the
degree of lameness
during the follow-up
period. Control
arthroscopy of 1
transplanted dog
indicated that the
cartilage had
regenerated. Histological
analysis of the cartilage
biopsy confirmed that
the regenerated cartilage
was of hyaline type
Hip, knee, 10 patients 30–90 days/4 Case reports Autologous 3 × 107 Physical Functional outcomes for Dražilov
radiocarpal (n = 5 years (5 AD-MSCs examination all analysed et al. (2018)
intercarpal, elbow evaluated animals) and assessment characteristics improved
and ulnar for 90 days for lameness, significantly at the end
osteoarthritis and n = 5 pain on of this evaluation
for manipulation, compared with the
1–4 years) range of motion baseline. Long-lasting
of the joint and positive effects on two
functional out of five analysed
disability characteristics
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications
(continued)
229
Table 11.3 (continued)
230
Number of
Clinical condition/ animals Study Evaluation
ailment included period Study type Cell source Cell dose criteria Overall result References
Degenerative 203 10 weeks Controlled Allogeneic 128 received a Quality of life Ninety percent of young Shah et al.
arthritis (divided clinical study AD-MSCs single dose of score (QoL), dogs (<9 years) had (2018)
into groups intra-articular lameness and shown excellent
as per age, injection in the pain score improvement in pain and
weight) affected joint. mobility and were able
65 dogs to run and resume
received a normal activity. Sixty
single percent of older dogs
intravenous showed good
injection, and improvement. However,
10 dogs 12% of dogs did not
received both exhibit any change in
IA and IV symptoms; one dog
injections showed worsening of the
symptoms
Osteoarthritis (hip, 22 6 months Non- Allogeneic 10 × 106 Clinical Veterinary clinical Cabon et al.
knee, elbow and and 2 years randomized, MSCs (repeated evaluation, flow evaluation showed a (2019)
tarsal joints) open and injection after Cytometric significant and durable
monocentric 6 months in 8 Crossmatch clinical improvement (up
study dogs or 11 analysis of to 6 months) following
joints) Humoral MSC administration.
response Eight dogs (11 joints)
against cellular were re-injected
product 6 months apart,
sustaining clinical
benefits up to 1 year.
Owner’s global
satisfaction reached 75%
at 2 years post-treatment
M. B. Gugjoo et al.
Elbow osteoarthritis 6 6 weeks Case control Xenogeneic 1 × 106 cells Orthopaedic There was no significant Daems et al.
chondrogenic examination, difference in the (2019)
induced synovial fluid orthopaedic examination
PB-MSCs examination, parameters, the
(equine radiography radiographic
source) examination, synovial
fluid sampling and
pressure plate analysis
between placebo and
MSC treatment. A single
intra-articular
administration of MSCs
proved to be a
well-tolerated treatment,
which reduced lameness
and pain according to
the owner’s evaluations
as compared to a placebo
treatment
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications
231
232 M. B. Gugjoo et al.
In these studies a single dose of dMSCs has been injected locally into joints
except for a study that had implanted cells more than once (Cabon et al. 2019).
One study had utilized acupoint as site of implantation (Marx et al. 2014). Either
only cells have been utilized (Marx et al. 2014; Vilar et al. 2014) or along with
platelet rich plasma/fibrin (Vilar et al. 2013; Kazemi et al. 2017), biphasic scaf-
fold (Yang et al. 2011) or hyaluronic acid (Guercio et al. 2012; Kriston-Pál et al.
2017; Li et al. 2018). Even chondrogenically differentiated (Yang et al. 2011) or
vascular endothelial growth factor transduced dMSCs have also been evaluated
(Hang et al. 2012). In general, these studies have variably supported therapeutic
applications of dMSCs.
Variable evaluation parameters have been undertaken in these studies. Variable
follow-up period ranging from as low as 1 month (Guercio et al. 2012; Marx et al.
2014) to a maximum period of 5 years (Yoon et al. 2012) had been conducted.
Therapeutic evaluation of dMSCs has been made on the basis of clinical evalua-
tion parameters like Pain scales, and MRI, CT scan and histological examination.
Quantitative Force Platform Gait Analysis tends to have greater accuracy and con-
cordance for assessment of AO lameness as compared to pain assessment scales
(Vilar et al. 2014). Improvement on the basis of clinical parameters, arthroscopic
evaluation and histology has been demonstrated (Kriston-Pál et al. 2017). The
histological scores of healed tissues in MSCs-treated groups were higher as com-
pared to control but complete hyaline tissue was lacking. The healed tissues had
been mixed fibrocartilage and/hyaline lacking complete integration to the native
cartilage (Yang et al. 2011; Kazemi et al. 2017). Some of the studies, however,
had failed to demonstrate significant improvement (Daems et al. 2019) and even
some failed to demonstrate long-lasting improvement with MSCs applications
(Vilar et al. 2014).
Several comparative studies have also been conducted. dAD-MSCs had been
compared to platelet rich growth factors (PRGF) (Cuervo et al. 2014), stromal vas-
cular fraction (SVF) (Marx et al. 2014), in addition to the VEGF transgenic dBM-
MSCs against simple BM-MSCs. MSCs though had provided better outcome as
compared to PRGF but not in comparison to SVF. SVF also contain growth factors
and as such might have improved osteochondral defect healing (Kazemi et al. 2017).
VEGF transgenic dBM-MSCs had better osteochondral healing potential as com-
pared to simple MSCs (Hang et al. 2012). There is need for studies that compares
dMSCs to the currently available surgical techniques.
(continued)
Table 11.4 (continued)
234
cells.
Table 11.5 In vivo clinical osteogenic mesenchymal stem cell studies
236
Number
of
animals Study Evaluation
Clinical case included period Study type Cell source Cell dose Route criteria Overall result References
Radial 1 90 days Case report Autologous 3.2 × 107 Local Radiography The composition Lee et al.
fracture with AD-MSCs + implantation + and lameness scaffold of HA (2009a)
sclerotic hydroxyapatite compression containing ADSCs had
ends and chitosan plate induced new bone
formation
Radial 1 8 weeks Case report Umbilical cord 1 × 107 Local Radiography Fracture healing was Jang et al.
non-union MSCs + repeated implantation identified by 6 weeks (2008)
fracture cortical after after cell injection.
allograft 1 month Increased opacity
between radius and
allograft bone, and a
decreased radiolucent
gap was checked. With
time, the radiolucent
site gradually
decreased and the
radiolucent fracture
line disappeared
Oligotrophic 1 dog 9 weeks Case report Bone 1 × 104 Local Lameness Completely healed Song et al.
non-union morphogenetic cells/cm2 implantation + and fracture (2017)
left radial protein-7 locking plate radiography
fracture AD-MSC
sheet
Tibial 9 15, 30, Double-blind MSCs 1.5 × 106 Local Radiography, Cell treated group had dos Santos
tuberosity (control, 60 and experimental application image significant ossification et al.
osteotomy n = 5; cell 120 days study analysis mean 36.45% greater (2018)
treated, software than the control at
M. B. Gugjoo et al.
n = 4) 30 days
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications 237
L-lactide-co-ε-caprolactone (Jang et al. 2008), coral scaffold (Cui et al. 2017) and
porous ceramic cylinder (Bruder et al. 1998). All these studies have demonstrated
improved bone density with uniform growth of regenerated tissue upon dMSCs
implantation. Concentration of the cells and of the growth factor (rBMP) used too
may have effect on bone healing (Itoi et al. 2016). Contrary to ex vivo reports,
dMSCs from varied sources had provided comparable in vivo osteogenesis (Kang
et al. 2012).
Apart from experimental studies, clinical application of MSCs too has supported
their role in enhancement of healing. BMP-7 transgenic dAD-MSC sheets implanted
in a clinical oligotrophic non-union radial fracture and autologous AD-MSCs in
combination with hydroxyapatite and chitosan implanted in a radial fracture with
sclerotic ends had complete healing in about 9 weeks (Song et al. 2017) and
12 weeks (Lee et al. 2009a), respectively. All these studies have utilized MSCs
along with variable scaffolds and/growth factors. There is need to perform compara-
tive uniform studies incorporating various scaffolds and growth factors. Besides,
these studies have to be compared against the current surgical treatment options for
their feasibility and effectiveness.
carrier vs + carrier
sponge sponge
treated vs
control)
(continued)
239
Table 11.6 (continued)
240
Number of
animals Study
Clinical case included period Study type Cell source Cell dose Route Evaluation criteria Overall result References
Chronic Chagas 5 6 months Case series Autologous 100 × 106 Right and left Electrocardiography Significant Sousa et al.
cardiomyopathy BM-MSC coronary artery and improvement in (2011)
implantation echocardiography cardiac function
(peak velocity of
aortic flow) after
cell implantation
Semitendinosus 2 19 weeks Case report Autologous 3.7 × 106 Local Ultrasonography, AD-MSCs Brown
myopathy (case 1) AD-MSCs implantation muscle biopsy, therapy enhanced et al.
and visual assessment of muscle healing (2012)
22 weeks dog’s improvement and prevented
(case 2) in affected tasks and fibrosis in these
surgeon assessments clinical cases,
similar to the
reported literature
in laboratory
animals. The
dogs returned to
their previous
training and
occupations with
a functional gait
and no lameness.
Grade IV 1 5 years Case report Autologous 1 × 106 + Local Lameness and Normal walking Yoon et al.
patellar AD-MSC hyaluronic acid implantation radiography and reduced (2012)
luxation, osteophytes and
arthritis osteochondral
lesions had been
reported after cell
implantation.
M. B. Gugjoo et al.
Gastrocnemius 1 631 days Case report Autologous >20 × 106 Local Serial orthopaedic Lameness Case et al.
tendon injury BM-MSC (cryopreservation) implantation examinations, subjectively (2013)
ultrasonography resolved and peak
and long-term vertical force
force-plate gait increased from
analysis 43% to 92% of
the contralateral
pelvic limb.
Serial
ultrasonographic
examinations had
revealed
improved but
incomplete
restoration of
normal linear
fibre pattern of
the gastrocnemius
tendon.
(continued)
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications
243
Table 11.7 (continued)
244
Number of
animals Study
Clinical case included period Study type Cell source Cell dose Route Evaluation criteria Overall result References
Partial cranial 36 (n = 19 for 90 days Retrospective Bone 2–4 mL Local joint Objective gait Promising Canapp Jr.
cruciate BMC + PRP) comparative marrow (BMC + PRP) implantation analysis, diagnostic healing potential et al.
ligament tear and (n = 17 for study concentrate and 1–2 mL arthroscopy, and of these (2016)
treated with AD-PCs + PRP) + PRP vs (AD-PCs + PRP) validated treatments was
TPLO AD-MSCs + functional demonstrated.
PRP questionnaire Arthroscopic
findings of 13
cases out of 36
cases at 90 days
interval were
available. Nine
dogs had fully
healed CCL
represented by
marked
neo-
vascularization
and a normal
fibre pattern in
the disrupted
areas. Out of
other four cases,
one had improved
healing and was
subjected to
another injection
while rest three
underwent tibial
plateau levelling
osteotomy
M. B. Gugjoo et al.
Cranial 12 8 weeks Case series Autologous 2 × 106 and Intravenous Radiography, No adverse Muir et al.
cruciate (case BM-MSCs 5 × 106 and local arthroscopy, serum events. With (2016)
ligament controlled intra-articular and synovial lower circulating
rupture clinical study) cytokines and CD8+ T
C-reactive protein lymphocytes after
cell treatment
were detected.
Serum C-reactive
protein (CRP)
was decreased at
4 weeks and
serum CXCL8
was increased at
8 weeks.
Synovial CRP in
the complete CR
stifle was
decreased at
8 weeks.
Synovial IFNγ
was also lower in
both stifles after
cell injection.
Systemic and
intra-articular
injection of
autologous
BM-MSCs in
dogs with partial
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications
CR suppresses
systemic and
stifle joint
inflammation,
including CRP
concentrations.
(continued)
245
Table 11.7 (continued)
246
Number of
animals Study
Clinical case included period Study type Cell source Cell dose Route Evaluation criteria Overall result References
Semitendinosus 9 dogs (11 cases) Short-term Uncontrolled AD-MSCs – Local Ultrasonography, At short-term Gibson
myopathy (6 months) retrospective implantation visual assessment follow-up et al.
and comparative score (VAS), ultrasound and (2017)
long-term clinical trial gait analysis
(1 year) showed a mean
follow-up reduction in the
overall
intramuscular
lesion size and
reduction in the
visual assessment
score (VAS). At
long-term
follow-up, in 8
cases had a
normal gait and
in 3 cases the
dogs had an
improved gait as
compared with
initial
examination. All
8 dogs had
returned to active
police work.
Cranial 14 (9 MSCs and 180 days Comparative Allogeneic 10 × 106 Local Clinical score and Better healing in Taroni
cruciate 5 NSAIDs) study foetal implantation gait and bone MSC treated et al.
ligament (blinded) adnexa healing animal at 1 month (2017)
rupture MSCs with comparable
gait and clinical
score at 6 months
M. B. Gugjoo et al.
Muscular 16 (group 5 and Case series Allogeneic 6.3 × 107 to Intrafemoral Immunohisto Persistent clinical Lorant
dystrophy received cells 9 months muscle 1.2 × 108 per kg injections chemistry, improvement of et al.
with transient derived histomorphometry, the GRMDMU/ (2018)
immunosup MSCs + mononuclear cell tr-IS dogs was
pression immunosup proliferation, observed.
(GRMDMU/ pression western blotting GRMDMU/no-IS
tr-IS), n = 4; dogs exhibited no
group received benefit.
cells without Histologically,
immunosup only 9-month-old
pression GRMDMU/tr-IS
(GRMDMU/ dogs showed an
no-IS), n = 4; increased muscle
group received no regenerative
cells without activity. A mixed
immunosup cell reaction with
pression, n = 3; the host
group received no peripheral blood
cells with mononucleated
transient cells (PBMCs)
immunosup and
pression, n = 5) corresponding
donor cells
revealed
undetectable to
weak lymphocyte
proliferation in
GRMDMU/tr-IS
dogs compared
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications
with a significant
proliferation in
GRMDMU/no-IS
dogs
247
248 M. B. Gugjoo et al.
desired involving genetically modified cells to confirm their role in cardiac ail-
ments, if any (Lu et al. 2013).
Dog has been used as myocardial ischemic model animal for human though the
condition has little relevance in dog clinical practice. An increased vascularity and
cardiac functional improvement (Memon et al. 2005; Silva et al. 2005; Bartunek
et al. 2007; Perin et al. 2008) has been reported with MSCs implantation. As stem
cells have self-renewal and multipotent differentiation properties, infarcted myocar-
dium may have been repaired (Linke et al. 2005).
11.3.2.1 Wounds/Ulcers
Numerous MSCs studies are being conducted to evaluate the healing potential of
MSCs in wound and ulcer. In dog MSCs have been employed in management of
wounds (Tables 11.8 and 11.9), and ulcers (Table 11.8). Even dAD-MSCs may suf-
fice as canine dermal papillae substitutes (Bae et al. 2015). Local implantation of
the MSCs like BM-MSCs appears more effective in induced oral ulcers as com-
pared to the systemic implantation (Aly et al. 2014).
An extensive study on full thickness wound model had shown that implantation
of allogeneic BM-MSCs of various concentrations (1 × 104, 1 × 105, 1 × 106 and
1 × 107) had led to wound epithelial regeneration by day 7. Although, collagen was
higher in healed wounds implanted with highest cell concentration but was statisti-
cally insignificant. Further, pro-healing factor like bovine fibroblast growth factor
(bFGF) was highly expressed while inflammatory mediators like IL-2 and INF-γ
were decreased at day 7 in comparison to control confirming their anti-inflammatory
and anti-fibrotic role in skin wounds (Kim et al. 2013). MSCs extracellular vesicles
(microvesicles) may also improve healing of wounds in dogs (El-Tookhy et al.
2017). In chronic clinical cases previously unresponsive to conventional therapy,
MSCs had promoted healing (Madhu et al. 2014; Zubin et al. 2015). In our own
experience allogeneic BM-MSCs had improved and led to faster healing of an
extensive wound in a dog (Madhu et al. 2014).
MSCs tend to decrease inflammation (Yang et al. 2018), suppress collagenous
matrix degradation (Jeon et al. 2010), favour angiogenesis (Kim et al. 2012) and
accelerate epithelialization of wound (Rodriguez-Menocal et al. 2015).
Model
Number defect
of size/study Biomaterial Evaluation
Model type animals period Study type used Cell dose criteria Overall result References
Full thickness 10 (24 7 days Experimental Allogeneic 1 × 104, Histopathology, Wound epithelial regeneration Kim et al.
skin wound wounds) study BM-MSCs 1 × 105, immunohisto was seen with epithelial gap (2013)
1 × 106 and chemistry and significantly smaller in cell
1 × 107 cells PCR treated groups as compared to
/300 μL of untreated. Compared to control
PBS. 200 μL significantly higher
(wound bed proliferating cell nuclear
and 100 μL antigen (PCNA), angiogenesis
in wound and collagen synthesis was
edges) seen in cell treated groups.
Although, collagen was higher
in 1x107 cell treated groups but
was statistically non-
significant. Bovine fibroblast
growth factor (bFGF) was
higher and IL-2 and INF-γ
were lower at day 7
Induced Oral 18 (6 in 15 days Comparative BM-MSCs 2 × 107 Clinical and The treatment resulted in Aly et al.
ulcers each experimental histopathological dramatic wound edge (2014)
group) study activation and resurfacing of
(intravenous oral mucosa wound. Local
Vs local application hastens such
implantation healing compared to systemic
Vs control) application
Laryngotracheal 7 6 weeks Experimental BM-MSCs 2 x 106 and Histology Prominent healing effect of Iravani et al.
stenosis study (cell/ conditioned the cells or conditioned media (2017)
conditioned media in wounds though statistically
M. B. Gugjoo et al.
Number
of animals Study Evaluation
Clinical case included period Study type Cell source Cell dose Route criteria Overall Result References
Inflammatory 11 42 Case series Allogeneic 2 × 106/kg b. Intravascular Clinical Significantly Pérez-
bowel disease (Unblinded, AD-MSCs wt inflammatory decreased CIBDAI Merino
non- bowel disease and CCECAI but et al.
comparator activity index no effect on CRP (2015a, b)
study) (CIBDAI) and
canine chronic
Enteropathy
clinical
activity index
(CCECAI),
and C-reactive
protein (CRP)
Keratoconjunctivitis 15 (24 12 months Case series Allogeneic 1 × 106 Lacrimal Schirmer tear Statistically Bittencourt
sicca eyes) AD-MSCs glands test, ocular significant et al.
(dorsal and surface improvement in (2016)
third evaluation Schirmer tear test
eyelid) from 28 days
Keratoconjunctivitis 12 (24 9 months Case series Allogeneic 5 × 106 & Around Schirmer tear Statistically Bittencourt
sicca eyes) AD-MSCs 3 × 106 lacrimal test, ocular significant et al.
glands and surface improvement in (2016)
third eyelid evaluation Schirmer tear test
and ocular surface
M. B. Gugjoo et al.
Recurrent corneal 1 69 days Case report Allogeneic 3 × 106 Topical Clinical Improved clinical Arantes-
ulcer after first AD-MSCs (two doses application testing parameters like Tsuzuki
treatment at 48 days blepharospasm, et al.
later) conjunctival (2019)
hyperaemia,
mucopurulent
ocular discharge,
photophobia,
corneal opacity,
chemosis,
pigmentation,
neo-
vascularization
and pain. The
fluorescein test
was negative by
day 69
Perianal fistula 6 6 months Case series Human Commercial Local Clinical Completely healed Ferrer
embryonic preparation implantation evaluation fistula at 3 months et al.
stem cell while 2 cases had (2016)
derived relapse at
MSCs + 6 months
cyclosporine
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications
255
256 M. B. Gugjoo et al.
effects
(continued)
257
Table 11.11 (continued)
258
AD-MSCs and
AD-MSCs + MPSS
groups
(continued)
259
Table 11.11 (continued)
260
of dogs
Sciatic nerve 20 (negative 9 months Human umbilical cord 1 × 106, Local Electrophysiological, The LOCC and Cui et al.
resection (a 35 mm control, MSCs longitudinally implantation electron microscopy, hUC-MSCs (2017)
long) LOCC, oriented collagen histological analysis synergistically
hUC-MSC in conduit (LOCC) promoted regeneration
combination and improved the
with LOCC functional recovery in a
(LOCC/ dog model of sciatic
hUC-MSC), nerve injury
and autograft
(positive
control)
groups
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications
261
Table 11.12 In vivo clinical mesenchymal stem cell studies on various neurological affections
262
Number of
animals Study Evaluation
Clinical case included period Study type Cell source Cell dose Route criteria Overall result References
Chronic spinal cord injury 23 (10 6 months Case series Autologous 1.4 × 106–5.6 Subarachnoid TSCIS system Improvement Nishida
BM-MSCs to (uncontrolled, BM-MSCs × 106 space (involves in locomotor function et al. (2011)
and 13 35 months non-blinded implantation evaluation of without nociception in 6
control) clinical study) between L5 and each pelvic cell treated dogs
L6 limb separately
and has 3
components:
Gait,
proprioceptive
positioning and
nociception)
Spinal cord injury 1 180 days Case report Autologous 20 × 106 & 4.16 Local post at T12 Neurological Functional recovery William
bone marrow × 106 BMMNCs left examination, demonstrated with cell et al. (2011)
mononuclear + hemilaminectomy Olby scores implantation
cells thermoreversible and intravenous
(BMMNCs) gelatin polymer implantation
Acute spinal cord injury 85 (34 6 months Case series Autologous 5 × 106 cells Intralesional Gait analysis, Recipients of olfactory Granger
randomized (controlled, olfactory somatosensory- mucosal stem cell et al. (2012)
subjects out randomized mucosal stem evoked potential, transplants gained
of which 23 double- cells Transcranial significantly better
cell treated blinded trial) magnetic forehand coordination than
and 11 no motor-evoked those dogs that did not
cell potentials, received cells. Intraspinal
treatment) Urodynamics olfactory mucosal cell
transplantation improves
communication across the
damaged region of the
injured spinal cord
M. B. Gugjoo et al.
Spinal cord compression 4 18 months Case reports Autologous 1 × 106 + collagen Local lesion Clinically, Post MSCs implantation, Penha et al.
due to herniated (uncontrolled BM-MSCs gelfoam scaffold implantation radiographically, a progressive recovery of (2014)
intervertebral disks (T12 to clinical study) post-surgical and by nuclear the panniculus reflex and
L5 vertebrae) decompression magnetic diminished superficial
resonance and deep pain response,
imaging (MRI) although there were still
low proprioceptive
reflexes in addition to a
hyperreflexia in the
ataxic hind limb
movement responses was
demonstrated at day 10.
Each dog demonstrated
an improvement in these
gains over time.
Conscious reflex
recovery occurred
simultaneously with
moderate improvement
in intestine and urinary
bladder functions in two
of the four dogs. By the
18th month of clinical
monitoring, a remarkable
clinical amelioration
accompanied by
improved movement, in
three of the four dogs
was reported. However,
no clinical gain was
associated with
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications
alterations in magnetic
resonance imaging.
(continued)
263
Table 11.12 (continued)
264
Number of
animals Study Evaluation
Clinical case included period Study type Cell source Cell dose Route criteria Overall result References
Chronic spinal cord injury 7 90 days Case series Allogeneic 1 × 106 Intralesional and MRI and Olby All dogs improved Sarmento
(uncontrolled BM-MSCs caudal to lesion scale locomotor and sensory et al. (2014)
pilot study) implantation function, tail tone was
observed in seven dogs,
pain reflexes and
defecation return were
observed in five dogs.
Paraplegia 13 12 months Case series Autologous 5.0 × 106 (2 doses Percutaneous Texas spinal Improvement in gait, Besalti
(uncontrolled neurogenically- after 21 days of transplantation cord injury scale nociception, et al. (2015)
clinical study) induced interval post (TSCIS), proprioception, SEP and
BM-MSCs hemilaminectomy/ somatosensory- MEP results (2 cases),
laminectomy evoked only gait score
potentials (SEP) improvement (6 cases)
and motor- and no improvement was
evoked recorded in (5 cases)
potentials
(MEP)
Chronic spinal injury/IVDD 6 16 weeks Case series Allogeneic 1 × 107 Intralesional Functional Only single dog regained Escalhão
(pilot clinical AD-MSCs implantation evaluation of ability to walk et al. (2017)
test) Locomotor
activity
Meningo-encephalomyelitis 8 2 years Clinical series Autologous 2 × 106, 4 × 106 Intrathecal (IT)/ MRI, All the dogs had Zeira et al.
of unknown origin (MUO) BM-MSC (3 dogs) and right Intracarotid neurological presented early (2015)
2 × 106, 0.5 × 106 (IC) & intrathecal examination improvement in their
(4 dogs) and intravenous and general and neurological
histopathology conditions, with
particular effect on
cervical pain. The group
of dogs treated by IT+IA
administration showed a
shorter time of reaction
to therapy compared to
the group treated by
M. B. Gugjoo et al.
IT+IC administration
Canine distemper 8 (n = each 15 days Case series Foetal olfactory 1.0 × 106 /kg b. Intravenous RT-PCR and MSC therapy resulted in Pinheiro
group; group (double-blind epithelium wt. laboratory no significant et al. (2016)
I: Cell + randomized stem cells investigations improvement when
standard trial) administered during the
medicine; acute phase of canine
group II: distemper disease, and a
Standard prevalence of animals
medicine) with high mortality rate
was found in both groups
due to the severity of
symptoms
Canine distemper 4 1 year Case series AD-MSCs 1.0 × 107 (3 Intravenous Neurological During the first year after Pinheiro
doses at monthly examination cellular therapy, the et al. (2019)
interval) animals did not
demonstrate significant
changes in their
locomotive abilities.
However, the intense
(grade V) myoclonus in
three animals was
reduced to a moderate
(grade IV) level. At
1 year after the
mesenchymal stem cell
infusions, three animals
regained functional
ambulation (grade I), and
all four dogs started to
move independently
(grades I and II). In two
animals, the myoclonic
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications
Miniscule number of MSCs studies related to various other ailments like diabetes
(Zhu et al. 2011; Gautam et al. 2016), liver fibrosis (Matsuda et al. 2017) and acute
kidney injury (Lee et al. 2017b) have also been undertaken as enlisted in Table 11.13.
In a dog hepatocutaneous syndrome, multiple intrahepatic implantations of
Table 11.13 Miscellaneous in vivo mesenchymal stem cell studies in dog
Number of Model defect Biomaterial
Model type animals size/study period Study type used Cell dose Evaluation criteria Overall result References
Diabetic model 30 dogs 16 weeks Comparative Autologous 2.53106 / Analysis of C peptide, Group 1 had insulin Zhu et al.
(alloxan and (6 in each experimental BM-MSCs mL (total immunohistochemistry secretion through (2011)
streptozotocin group) study (group retrovirus volume and implanted cells;
induced) 1: MSCs carrying 20 mL immunocytochemistry, animals also
transfected insulin and injected glucose tolerance test, responded to glucose
with EGFP gene measurement of blood challenge however
retrovirus glucose, insulin β-cells were
carrying detection undetectable as no C
insulin and peptide secretion was
EGFP gene) recorded. Rest of the
Vs group 2: groups had no control
MSCs on glucose challenge.
transfected Glucose level was
with statistically controlled
retrovirus in group 1 compared
carrying to group 4
EGFP gene
Vs group 3:
MSCs
without virus
infection) Vs
group 4:
Cell-free
11 Dog Mesenchymal Stem Cell Basic Research and Potential Applications
group 5:
Normal dogs)
(continued)
267
Table 11.13 (continued)
268
allogeneic AD-MSCs had been made along with other managemental practices. An
improvement in the clinical signs like hair regrowth and resolution of skin lesions
was demonstrated. The dog, however, had died 32 months after diagnosis. The nec-
ropsy had revealed liver fibrosis and superficial necrolytic dermatitis (Nam et al.
2017). MSCs may also act drug (paclitaxel) carriers and can be used to target
tumours like glioma and glioblastoma (Bonomi et al. 2017). Combined application
of MSCs, recombinant human BMP-2 and cisplatin may prove useful in canine
osteosarcoma (Rici et al. 2018). However, MSCs soluble factors may promote
tumour cell proliferation and invasion (Teshima et al. 2018) and thus, studies are
desired to confirm their safety, feasibility and actual utility.
11.4 Conclusion(S)
Numerous dog tissue sources harbour MSCs although concentration and properties
may be variable. The cellular features also vary with age and health status of the
donor. dMSCs have been evaluated for their therapeutic benefits on musculoskeletal
and non-musculoskeletal tissue ailments. MSCs studies in dog tend to provide
proof-of-therapeutic potential in human medicine. Due to the lack of understanding
in their basic cellular properties, an initial step should be deliberated upon these
properties. Further, case controlled uniform clinical trials need to be conducted to
provide evidence based medicine for clinical conditions. Several issues like MSCs
source, cell concentration, mode of delivery, cell survival on transplantation and
frequency of stem cells need further deliberation and standardization.
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Equine Mesenchymal Stem Cell Basic
Research and Potential Applications 12
M. B. Gugjoo, Amar Pal, D. M. Makhdoomi,
and G. T. Sharma
Abstract
M. B. Gugjoo (*)
Division of Veterinary Clinical Complex, FVSc & AH, SKUAST-Kashmir,
Jammu and Kashmir, India
A. Pal
Division of Surgery, Indian Veterinary Research Institute, Izatnagar, UP, India
D. M. Makhdoomi
Division of Surgery and Radiology, FVSc & AH, SKUAST-K,
Srinagar, Jammu and Kashmir, India
G. T. Sharma
Division of Physiology and Climatology, Indian Veterinary Research Institute,
Izatnagar, UP, India
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 283
Springer Nature Singapore Pte Ltd. 2020
M. B. Gugjoo, A. Pal (eds.), Mesenchymal Stem Cell in Veterinary Sciences,
https://doi.org/10.1007/978-981-15-6037-8_12
284 M. B. Gugjoo et al.
12.1 Introduction
Equine industry is one of the biggest industries be it in the form of racing, polo, or
for recreational and transportation purposes. In racehorses, musculoskeletal disor-
ders reduce their performance and even destroy their carrier (Perkins et al. 2005).
Among soft tissue ailments, skin wounds, eye affections, and reproductive disorders
pose a significant problem to clinicians. Various horse and human developmental
and autoimmune ailments have comparable pathophysiology (Gershwin 2007).
Horse and human cartilage thickness and physiological features approximate to
each other (Aigner et al. 2010). The cartilage defects associated with intense physi-
cal activity too resemble (Desancé et al. 2018). Thus, horse can make an excellent
model animal to provide proof-of-therapeutic principle of mesenchymal stem cells
(MSCs) in human medicine (McIlwraith et al. 2011; De Schauwer et al. 2013;
Gugjoo et al. 2019b). Tissue engineering employing MSCs is being studied exten-
sively. MSCs meager population in adult tissues/organs makes their culture expan-
sion necessary for effective utilization. Innumerable in vitro studies have piled upon
equine MSCs (eMSCs). These studies have been conducted on their culture charac-
teristics, characterization, and their therapeutic properties. The in vivo studies have
supported the therapeutic potentials of MSCs, although variably (Carrade et al.
2011a, b; Spaas et al. 2012; Burk et al. 2013) with mechanism(s) behind the healing
largely being unrevealed. The current chapter deliberates on the eMSCs ex vivo
properties and their potential therapeutic applications.
Horse MSCs (eMSCs) have diverse sources ranging from adult tissues to fetal mem-
branes as detailed in Table12.1. Donor tissues usually harbor small fraction of
MSCs and are isolated along with the other cell types including progenitor cells
(Chong et al. 2011). The characterization of eMSCs has been made as per the stan-
dard protocol of the International Society for Cell Therapy (ISCT) (Dominici et al.
2006). eMSCs and human MSCs tend to have comparable marker expression and
differentiation potential (Hillmann et al. 2016). eMSCs invariably follow the plastic
adherence and are multipotent (Guest et al. 2008; Ranera et al. 2011; De Schauwer
et al. 2012). Their surface marker expression, however, remains controversial as
these cells variably express different surface markers and lack expression of certain
markers. This discrepancy in marker expression has mainly been observed in rela-
tion to the tissue source as shown in Table12.1. eMSCs also express pluripotency
markers like Oct4, Sox-2, and Nanog and show extended differentiation like neural
cell-like cells, myogenic cell-like cells, tenocyte-like cells, and hepatocyte-like cells
(Violini et al. 2009; Coli et al. 2011; Gomiero et al. 2016; Pennington et al. 2016;
Cabezas et al. 2018).
12 Equine Mesenchymal Stem Cell Basic Research and Potential Applications 285
Table 12.1 Horse mesenchymal stem cell sources and their characterization
Positive markers Negative markers Differentiation References
Adipose tissue MSCs (AD-MSCs)
CD29, CD44, CD105, CD45 Osteogenic and Marycz et al.
CD90 (limited adipogenic (2012)
expression)
CD29, CD44, and CD90 MHC-II, CD34 Adipogenic, Alipour et al.
chondrogenic, and (2015)
osteogenic
CD29, CD44, CD105 – Adipogenic, Xie et al.
(87%) chondrogenic, and (2013)
osteogenic
CD73, CD90, CD105 Adipogenic, González-
chondrogenic, and Fernández
osteogenic et al. (2016)
CD105, CD90, CD49, CD43, CD73 Adipogenic, Pall et al.
CD44 chondrogenic, and (2016)
osteogenic
CD90, CD105, CD44, CD45,CD34, CD31, – Angelone
CD73, CD13, CD133, SDF-1 receptor CXCR4 et al. (2017)
CD29,OCT-4
CD44, CD90, CD105 CD45 Adipogenic, Nawrocka
chondrogenic, and et al. (2017)
osteogenic
CD29, CD44, CD90, CD34, CD45, and Chondrogenic and Bundgaard
CD105, CD166 CD79a osteogenic et al. (2018)
CD29, CD44, CD90, MHC-II, CD45, OCT4, Adipogenic, Cabezas et al.
CD-105, MHC-I Sox2 chondrogenic, and (2018)
osteogenic
CD90, CD105, Oct4 Osteogenic Elashry et al.
(2018)
Amniotic membrane MSCs (am-MSCs)
CD90, CD73, CD34, MHC-I, MHC-II, Adipogenic, Iacono et al.
Oct4 (weak), IL-6, IL-β1, CD45, Sox2, Nanog, chondrogenic, and (2017)
IL-8 (weak) INF-γ, TNF-α, IL-4 osteogenic
CD29, CD44, CD106, CD34, MHC-II Adipogenic, Perrini et al.
CD105 chondrogenic, (2016)
osteogenic, and
neurogenic
Bone marrow MSCs (BM-MSCs)
CD29, CD44, CD172a CD34, CD45 – Fortier et al.
(2010)
CD29, CD44, CD105 – Adipogenic, Xie et al.
(64%) chondrogenic, and (2013)
osteogenic
CD11a/18, CD34, CD44, Adipogenic, Lombana et al.
CD90 chondrogenic, and (2015)
osteogenic
(continued)
286 M. B. Gugjoo et al.
Table 12.1 (continued)
Positive markers Negative markers Differentiation References
CD90, CD29 MHC-II, CD44, Adipogenic, Mitchell et al.
CD45RB chondrogenic, and (2015)
osteogenic
CD90, CD105, CD44, CD34, CD45 Adipogenic, Barrachina
CD73 chondrogenic, and et al. (2016)
osteogenic
CD90, CD44, CD29 MHC-II, CD86 – Clark et al.
(2016)
CD73, CD90, CD105 Adipogenic, González-
chondrogenic, and Fernández
osteogenic et al. (2016)
CD44, CD29, CD90, CD34, MHC-II, CD105 Adipogenic, Branly et al.
CD73 (at p3 and p4 only), (P4) chondrogenic, and (2017)
CD105 (at P3 25% osteogenic
expression)
CD29, CD44, CD90, CD34, CD105, Adipogenic, Zahedi et al.
MHC-I, Sox2 MHC-II, Nanog, Oct4, chondrogenic, and (2018)
SSEA-1, −3,-4 osteogenic
CD29, CD44, CD90, CD34, CD45, and Chondrogenic and Bundgaard
CD105, CD166 CD79a osteogenic et al. (2018)
CD29, CD90 CD34, CD45, CD14, Adipogenic, Korchunjit
CD73, and CD105, chondrogenic, et al. (2019)
CD79a, DRB, NANOG, osteogenic, and
and SOX2 tenogenic
CD44, CD90, MHC-I, CD11a/CD18, MHC-II Chondrogenic and Schrock et al.
CD105 (33.9%) osteogenic (2018)
CD29, CD44, CD90, CD45 and CD79a Chondrogenic Gale et al.
CD105, and MHCI, (2019)
MHC-II
Dental pulp MSCs (DP-MSCs)
CD11a/CD18, CD44, CD34, CD45 Adipogenic, Ishikawa et al.
CD90, CD105, MHC-I, chondrogenic, and (2017)
MHC-II osteogenic
Dermal MSCs
CD90, CD105 (48%), CD34, CD45, CD79α, – Michler et al.
CD29, CD44 CD14, MHC-II, CD73 (2018)
Endometrium MSCs (end-MSCs)
CD29, CD44, CD105, CD34, CD45, MHC-II Adipogenic, Rink et al.
CD90, Pericyte (NG2, chondrogenic, (2017)
CD146) osteogenic, and
smooth muscle
CD29, CD44, CD90, MHC-II, CD45, OCT4, Adipogenic, Cabezas et al.
CD-105, MHC-I Sox2 chondrogenic, and (2018)
osteogenic
(continued)
12 Equine Mesenchymal Stem Cell Basic Research and Potential Applications 287
Table 12.1 (continued)
Positive markers Negative markers Differentiation References
Gingiva MSCs
CD90, CD105, Vimentin CD31 Adipogenic, Mensing et al.
chondrogenic, and (2011)
osteogenic
Hoof MSCs (H-MSCs)
CD44, CD29, CD105, – Adipogenic, Yang and
Oct4, Sox2, keratin osteogenic, and Lopez (2019)
(K)14, K15, K19 neurogenic
Peripheral blood MSCs (PB-MSCs)
CD90, CD105 (40.5%), CD34, CD45, MHC-II Adipogenic, Ishikawa et al.
CD44, MHC-1 (60.2%), chondrogenic, and (2017)
CD11a/CD18 (42.1%) osteogenic
Periodontal ligament (PL-MSCs)
CD90, CD105, Vimentin CD31 Adipogenic, Mensing et al.
chondrogenic, and (2011)
osteogenic
CD44, CD29, MHC-II Beerts et al.
(2017)
Synovial fluid MSCs (SF-MSCs)
CD45, CD105, CD90, – – Prado et al.
CD34, CD117, CD133, (2015)
TRAI-1-81, VEGF,
LY6A, OCT3/4, Nanog
CD90, CD44, PCNA, – Adipogenic, Fülber et al.
Vimentin, PGP-9.5, chondrogenic (weak), (2016)
lysozyme and osteogenic
Synovial membrane MSCs (Sy-MSCs)
CD90, CD44, PCNA, Adipogenic, Fülber et al.
Vimentin, PGP-9.5, chondrogenic (weak), (2016)
lysozyme and osteogenic
CD44 and CD90, MHC-I, CD34, CD45. Chondrogenic, Yamasaki
CD11a/18, CD105, osteogenic, and et al. (2018)
MHC-II (weak), Sox2 tenogenic
Umbilical cord MSCs (UC-MSCs)
CD44, CD90 (weak) MHC-II, CD34, Oct4 Osteogenic and Maia et al.
adipogenic (2017)
Wharton’s jelly MSCs (WJ-MSCs)
CD90, CD73, CD34, MHC-I, MHC-II, Adipogenic, Iacono et al.
Oct4, IL-6, IL-β1, IL-8 CD45, Sox2, Nanog, chondrogenic, and (2016)
INF-γ, TNF-α, IL-4 osteogenic
AD-MSCs adipose tissue MSCs, Am-MSCs amniotic membrane MSCs, BM-MSCs bone marrow
MSCs, DP-MSCs dental pulp MSCs, End-MSCs endometrium MSCs, H-MSCs hoof MSCs,
PB-MSCs peripheral blood MSCs, PL-MSCs periodontal ligament, SF-MSCs synovial fluid MSCs,
Sy-MSCs synovial membrane MSCs, UC-MSCs umbilical cord MSCs, WJ-MSCs Wharton’s
jelly MSCs
288 M. B. Gugjoo et al.
Individual health status affects cell viability and as such may affect clinical results.
AD-MSCs harvested from metabolic syndrome (insulin resistance disease) horses
may be more senescent with an insubstantial differentiation potential. Oxidative
stress that promotes accumulation of toxic compounds in the mitochondria might
lead to the cell senescence. Autophagic switch to counter oxidative stress might
decrease MSCs differentiation potential (Marycz et al. 2016a, b). In vitro applica-
tion of Spirulina platensis had restored senescent eAD-MSCs morphology and
functionality and could have been possible through the reduction in oxidative stress
12 Equine Mesenchymal Stem Cell Basic Research and Potential Applications 289
Anesthetics and/drugs affect MSCs viability based on their class (Edmonds et al.
2016; Schrock et al. 2018). Romifidine and mepivacaine had nonsignificant effect,
while detomidine and butorphanol had significantly reduced MSCs viability.
Xylazine had also decreased MSCs viability but insignificantly. Commonly used
steroids like methylprednisolone and triamcinolone may compromise eBM-MSCs
viability (Edmonds et al. 2016). Therapeutic concentrations of antibiotics like ami-
noglycosides (gentamicin and amikacin) had deleteriously affected eBM-MSCs,
while hyaluronic acid had proven beneficial for their proliferation (Bohannon
et al. 2013).
Stem cell therapy in horse medicine mostly remains in experimental phase due to
lack of the literature incorporating uniform case controlled studies (Imamura and
Tanaka 2009; Clegg and Pinchbeck 2011; Volk and Theoret 2013; Dehghan et al.
2015; Ojeh et al. 2015). In horses, the therapeutic applications of stem cells had
been mainly aimed to address the issues of musculoskeletal system and first of its
kind had been conducted on superficial digital flexor tendonitis (Smith et al. 2003).
As the potential goes beyond, MSCs potential applications are now being evaluated
290 M. B. Gugjoo et al.
Horse wounds are predisposed to issues like proud flesh and/infections. The current
standard therapeutic options heal only about 24% of wounds by primary healing
(Wilmink et al. 2002). The limited availability of donor skin and more failures in
“graft take” under grafting makes it a least opted therapeutic solution (Theoret
2009). Potential applications of stem cells in wound healing had come to the fore
when a higher number of the cells exhibiting similar phenotype to that of BM-MSCs
were observed in blood of burn patients (Mansilla et al. 2006; Fox et al. 2008).
Since then stem cell therapies are being evaluated.
Ex vivo horse study had demonstrated that encapsulated MSCs and their condi-
tioned media might promote the repair of skin wounds. The repair could occur
through equine dermal fibroblast mobilization and increased pro-wound healing
gene expression (Bussche et al. 2015). In vivo implanted eBM-MSCs tend to remain
well oriented and integrated under the epithelium of repaired soft palate (Carstanjen
et al. 2006). The locally injected allogeneic equine umbilical cord blood (eUCB)-
MSCs had improved wound healing of hind limb wounds. The cell-injected wounds
had exhibited better tissue regeneration with little inflammatory cells. The expres-
sion of healing factors (transforming growth factor-β1 and cyclooxygenase 2) was
significantly higher in cell-treated wounds as compared to the control. MSCs heal-
ing potential was unaffected by their culture technique (normoxia vs. hypoxia)
(Table12.2) (Textor et al. 2017).
In clinical studies (foal deep sore wounds and decubital ulcers), local implanta-
tion of allogeneic amnion-derived MSCs (Am-MSCs) along with carboxymethyl-
cellulose (Iacono et al. 2016) or platelet-rich plasma (Table12.3) (Iacono et al.
2012) had led to earlier and improved wound healing as compared to wounds treated
without the cells. The cells had been implanted multiple times and along with the
other biomaterials (Iacono et al. 2016). The combined application (involving local
and parenteral injection) of PB-MSCs had improved wound healing of 4 chronic
dermal wounds of adult horse metatarsals. Granulation tissue had formed signifi-
cantly much earlier, while scar formation was delayed in comparison to the control
(Spaas et al. 2013). Thus, MSCs may be employed to repair equine wounds.
Improved healing upon the MSCs transplantation might arise through regeneration
of epidermal cells and/increased angiogenesis (Krause et al. 2001; Wu et al. 2007;
Borena et al. 2009). However, all these studies have mostly been uncontrolled, and
as such evidence-based studies involving large significant numbers of cases are
desired.
Table 12.2 In vivo preclinical experimental mesenchymal stem cell studies on cutaneous wounds in horse
Model
defect
size/ Cell
study Biomaterial dose/ Evaluation
Model type Number of animals period used assembly criteria Overall result References
Bilateral distal 6 horses (each wound in 6 weeks Umbilical 15– Histology, Cell treated irrespective of prior Textor
forelimb full horse treated with cord blood 20 × 106 thermography, preconditioning improved et al.
thickness wounds hypoxic- and normoxia- MSCs per gene expression, histological scoring and wound (2017)
(3 wounds; each preconditioned cells. wound and wound area. No significant advantage of
2.5 × 2.5 cm2 Other wounds were surface area hypoxic conditioning. An
spaced at 2.5 cm treated with above cell improved transforming growth
vertically aligned) types laden in autologous factor beta and cyclooxygenase-2
fibrin gel. Control with gene expression at 1 week.
one saline and other Histology showed wounds
fibrin gel-treated wounds treated with MSCs more of
pro-healing than
pro-inflammatory
12 Equine Mesenchymal Stem Cell Basic Research and Potential Applications
291
Table 12.3 In vivo clinical mesenchymal stem cell studies on cutaneous wounds/eye affection in horse
292
Laterally placed large eyes make horse conducive to their injuries (Wada et al. 2010;
Clode and Matthews 2011). Corneal wounds, especially of ulcerative keratitis,
account 68.5% (Andrew and Willis 2005) to 90.5% (Wada et al. 2010) of the ocular
problems, among which infectious keratitis accounts for 35–39% of all the cases
(Strubbe et al. 2000; Olivier et al. 2003). To address these issues, a study had dem-
onstrated that a significant corneal epithelial restoration and reduction of inflamma-
tory condition occur with conjunctival and/ophthalmic artery injection of PB-MSCs
(Table12.3) (Spaas et al. 2011; Marfe et al. 2012).
Even in an old (2 months) case of equine bilateral retinal detachment, improve-
ment in nerve function and visual acuity had been demonstrated in left eye with
intravenous injection of PB-MSCs. However, right eye had inconsiderable improve-
ment (Marfe et al. 2012). The variable pathophysiology involved and/or insufficient
cellular concentration achieved in the right eye could have been the reasons for
unfavorable results in right eye. Thus, more extensive studies are desired as per the
pathophysiology with MSCs dose titration.
Tendonitis tends to lead to equine lameness (Barreira et al. 2008). This condition is
managed by rest and rehabilitation, although recurrent tendonitis in 43% cases has
been reported (Dyson 1997; Violini et al. 2009). Lack of basic understanding of
tendon cell characteristics and their precursors in tendon healing currently chal-
lenges effective tendon healing (Bi et al. 2007). To gain insights and provide early
healing of tendon, MSCs are being evaluated.
Numerous MSCs studies (experimental or clinical) on tendon or ligament inju-
ries have been conducted as enlisted in Table 12.4 and 12.5. The models have been
created either surgically (Geburek et al. 2017) or by enzymatic action (Nixon et al.
2008; Carvalho et al. 2013a). Apart from experimental models, MSCs therapeutic
applications have also been evaluated in clinical tendinopathies and/desmitis (Renzi
et al. 2013; Van Loon et al. 2014; Beerts et al. 2017). Most of the studies have evalu-
ated BM-MSCs and AD-MSCs with two studies demonstrating umbilical cord
(UC)-MSCs (Van Loon et al. 2014) and PB-MSCs (Beerts et al. 2017). The cells
have been injected intralesionally after some delay. Some studies have additionally
incorporated scaffolds like platelet-rich plasma (Renzi et al. 2013), serum (Pacini
et al. 2007; Geburek et al. 2017), fibrinogen (Crovace et al. 2010), and plasma lysate
(Del Bue et al. 2008). Growth factors like insulin-like growth factor (IGF-1) have
also been added (Smith et al. 2003; Schnabel et al. 2009). In all these studies, a
single dose of the variable cell concentration has been made barring two studies that
had injected the cells twice (Van Loon et al. 2014; Vandenberghe et al. 2015).
Most of the studies have shown positive role of MSCs in tendon healing. One of
the studies, however, had failed to demonstrate significant therapeutic effects of the
MSCs (Caniglia et al. 2012). BM-MSCs suspended in fibrinogen (Crovace et al.
Table 12.4 In vivo preclinical experimental stem cell studies on tendinitis in horse
Model
Number of defect size/ Biomaterial Cell dose/
Model type animals study period used assembly Evaluation criteria Overall result References
Superficial 8 (4 cell 6 weeks AD-nucleated 13.83 ± 3.41 × 106 Biochemical and Ultrasonography revealed no Nixon
digital flexor treated and cell fraction cells (3 doses at molecular and difference in rate or quality of et al.
tendinitis 4 control) 40–53 h range) ultrasonography were repair between groups. (2008)
(collagenase- performed Histologic evaluation revealed a
induced) significant improvement in
tendon fiber architecture;
reductions in vascularity;
inflammatory cell infiltrate;
collagen type III formation; and
improvements in tendon fiber
density and alignment in
ADNC-treated tendons. Repair
sites did not differ in DNA,
proteoglycan, or total collagen
content. Gene expression of
collagen type I and type III in
treated and control tendons was
similar. Gene expression of
COMP was significantly
increased in ADNC-injected
tendons
(continued)
12 Equine Mesenchymal Stem Cell Basic Research and Potential Applications
297
Table 12.4 (continued)
Model
298
(continued)
Table 12.4 (continued)
300
Model
Number of defect size/ Biomaterial Cell dose/
Model type animals study period used assembly Evaluation criteria Overall result References
Bilateral 9 horses 22 weeks AD-MSCs 1 × 107 injected Ultrasonography and Color Doppler signal at 2 weeks Conze
forelimb (2 in each intralesionally histology was significantly more extensive. et al.
superficial horse) after 2 weeks Histology showed significantly (2014)
digital flexor more neovascularization after
tendons 22 weeks
(SDFT)
Bilateral 9 horses 24 weeks AD-MSCs in Ultrasound-guided Ultrasonography, Statistically nonsignificant Geburek
forelimb (2 in each inactivated 10 × 106 cells clinical, biochemical, differences in two treated groups. et al.
superficial horse) serum and were injected and biomechanical Mature collagen crosslink (2017)
digital flexor inactivated intrialesionally evaluation hydroxylysylpyridinoline was
tendons serum only comparable to normal tendon
(SDFT) collagen in AD-MSCs treated
group, while it was lower in the
other group. Stress at failure and
modulus of elasticity in
AD-MSCs group were
significantly lower than the
mature normal tendon
M. B. Gugjoo et al.
Table 12.5 In vivo clinical mesenchymal stem cell studies on tendinitis in horse
Number of Biomaterial Cell dose/ Evaluation
Clinical case animals Study period used assembly criteria Overall result References
Superficial 26 (11 cell Uncontrolled Autologous 9.5 × 106 Ultrasound and Nine MSC-treated animals Pacini et al.
digital flexor treated; 15 study BM-MSCs ability to return recovered from their clinical (2007)
tendinitis control) to race conditions had an excellent
ultrasound image of tendons after
a period ranging from 3 to
6 months and returned to racing
with good or even optimal results
in the previous category of
competition in 9–12 months
without any reinjuring event. All
of them are still active more than
2 years from diagnosis. One of the
2 remaining horses received less
than 1 × 106 of MSCs, and its
tendon did not heal, relapsing after
rehabilitation; the other was lost to
follow-up. Small number of MSCs
may be sufficient to repair
damaged tendons without the use
of scaffold support. Ultrasound
scanning showed that fibers were
correctly oriented in cell-treated
tendons. Further, no ectopic bone
deposition occurred
12 Equine Mesenchymal Stem Cell Basic Research and Potential Applications
(continued)
301
Table 12.5 (continued)
302
was included
Superficial 12 (6 6 months BM-MSCs, 1 × 107 Morphology, Cell-treated tendons had Smith et al.
digital flexor animals cell growth autologous biomechanical, significantly lower structural (2013)
tendon treated; 6 factors, BM-MSCs ultrasonography, stiffness although there is no
control) natural suspended in histology significant difference in calculated
mechanical 2 mL of marrow modulus of elasticity. Lower
stimulus supernatant (improved) histological scoring of
organization and crimp pattern,
lower cellularity, DNA content,
vascularity, water content, GAG
content, and MMP-13 activity
were seen in cell-treated tendons
Tendinitis of 52 animals 6 months Umbilical 2–10 × 106 Clinical and 40/52 (77%) animals returned to Van Loon
superficial (young: cord (single dose) ultrasonography the normal activity. 20/23 SDFT et al. (2014)
flexor tendons 5–9 years; blood- 20 × 106 (double cases had positive result. 15/22
(SDFT) and medium derived dose in 5 horses cases of SL had positive result. 4/6
deep digital aged: MSCs at 4–36 weeks). DDFT and ICL had positive result.
flexor tendon 10–14 years; 4/5 double- Control cases (SDFT: 1/3; SL:
(DDFT), and >15 years) treated animals 2/3) reached to normal or higher
desmitis of were from SL performance levels after 6 months
superficial group or more following proper
ligament (SL) rehabilitation programme. No
and inferior significant difference in results
check with respect to the age was
ligament recorded
(ICL) (first
time injured)
12 Equine Mesenchymal Stem Cell Basic Research and Potential Applications
(continued)
303
Table 12.5 (continued)
304
2010), autologous serum (Pacini et al. 2007), and plasma lysate (Del Bue et al.
2008; Renzi et al. 2013; Vandenberghe et al. 2015) had improved tendon repair
especially in relation to the functional recovery.
The intralesional injected MSCs had remained viable, distributed, and integrated
within the injured tissue. The cell numbers had decreased with the time, and small
numbers had been identified in the peripheral blood up to 24 h and in the contralat-
eral tendon lesions up to 24 weeks (Burk et al. 2016). Even up to 34 days, the cells
had been located in the injected lesions (Guest et al. 2008). AD-MSCs tend to
decrease inflammation markedly and improve alignment of collagen fibrils and ten-
don fibers (Nixon et al. 2008). The cell concentration, however, appears to have a
role to play. One million cells had been insufficient for effective tendon healing
(Smith 2008; Caniglia et al. 2012), while higher cell concentrations like ten million
(Smith et al. 2009) and 20 million (Van Loon et al. 2014) had significantly improved
tissue matrix approximating to the normal tendon tissue.
Delayed implantation (day 30) of AD-MSCs in superficial digital flexor tendon-
itis too had improved healing as better histological and immunohistochemical
scores were demonstrated. However, an insignificant difference in sonographic and
clinical signs had been demonstrated between the cell-treated or nontreated cases
(Carvalho et al. 2011). Combined application of BM-MSCs and IGF-1 had improved
healing of the superficial digital flexor tendonitis. Tendons treated with cells and
IGF-1 were histologically better in comparison to those treated with BM-MSCs
only. The cell-treated tendons had better histological scores as compared to the con-
trol (Schnabel et al. 2009).
Even clinical improvement with the application of MSCs together with the rou-
tine managemental procedures has been demonstrated. The clinical studies on liga-
ment and tendon injuries had demonstrated that AD-MSCs and BM-MSCs had
improved most of the cases (85%) and these animals had returned back to competi-
tion (Ferris et al. 2009; Leppanen et al. 2009). Animals of diverse age, gender, and
breed had similar outcome (Ferris et al. 2009). Single case report of superficial digi-
tal flexor tendonitis locally treated with BM-MSCs and growth factors along with
natural stimulus had improved tendon healing. The stimulus might have triggered
MSCs differentiation into tendon fibroblasts (Smith et al. 2003). Apart from growth
factors, platelet-rich plasma (PRP) scaffold laden allogeneic AD-MSCs (Del Bue
et al. 2008) or PB-MSCs (Beerts et al. 2017) had improved tendonitis and tendon/
ligament injuries, respectively. Most of these animals had returned back to the
normal activity. Combined application of allogeneic PB-MSCs and platelet-rich
plasma (PRP) had moderately improved suspensory ligament (SL) and superficial
digital flexor tendon (SDFT) sonographic features at 6-week period that had fur-
ther improved at 24 months. The reinjury rate has markedly reduced in these cell-
treated cases (18%) in comparison to conventionally treated horses (44%) (Beerts
et al. 2017). Similar biomaterial combination used at 4 weeks after diagnosis and
repeated at 32-week period in a proximal suspensory ligament desmitis had com-
pletely restored its fiber alignment being observed under ultrasonography. These
animals had improved performance almost similar to the initial levels
(Vandenberghe et al. 2015). An equine tendonitis study had failed to demonstrate
306 M. B. Gugjoo et al.
Bone heals naturally with healed tissue approximating to natural native tissue
(Taylor et al. 2007). The healing of a fractured bone in horse is challenging due to
its uncontrolled movement. In case of extensive fractures or tumors, the ability to
heal becomes even more challenging. To improve healing in such conditions, tissue
Table 12.6 In vivo chondrogenic preclinical experimental mesenchymal stem cell studies in horse
308
(continued)
309
Table 12.6 (continued)
310
(continued)
Table 12.7 (continued)
314
12.3.7 Laminitis
Table 12.8 In vivo preclinical experimental mesenchymal stem cell studies on osteogenesis in horse
Number of Model defect Cell dose/
Model type animals size/study period Biomaterial used assembly Evaluation criteria Overall result References
4th 10 (20 metacarpal, 1 cm/12 weeks Periosteum- 20 × 106 Radiography and MSCs along with fibrin McDuffee
metacarpal 10 control, and 10 derived histology glue-treated animals failed et al.
ostectomy experimental) osteoprogenitor to show improved (2012)
cells (osteogenic radiographic and
lineage histological features in
differentiated) comparison to the fibrin
glue alone
Pastern joint 6 (12; 6 control Locking BM-MSC, 1 × 107 Radiography, An improved bone Seo et al.
arthrodesis and 6 compression gelatin/β-- histology, formation and subsequent (2014)
experimental) plate stabilization tricalcium computed arthrodesis in comparison
phosphate and tomography to the use of locking
BMP-2 compression plate
application had occurred
Splint bones 6 animals (group 1 cm/16 weeks BM-MSCs, Autologous Radiography, CT, Group A had statistically Tsuzuki
defect (4 A: BM-MSCs + BMP-2, and BM-MSCs and histology significant radiographic et al.
defects each BMP-2 in sponge; gelatin and and histological scores (2014)
in second group B: β-tricalcium BMP-2 in compared to control, while
metacarpal BM-MSCs in phosphate gelatine CT scores were
and sponge; group C: sponges sponge statistically significant than
metatarsal BMP-2 in sponge; other groups
bone of right group D: Saline)
limbs and
4thmetacarpal
and
metatarsal in
left limbs)
M. B. Gugjoo et al.
Table 12.9 In vivo clinical mesenchymal stem cell studies on osteogenesis in horse
Number of Evaluation
Clinical condition/ailment animals Study period Biomaterial used Cell dose criteria Overall result References
Phalanx digitalis distalis 1 12 weeks Orthopedic screw 6 × 106 in Clinical After 3 months, Marycz et al.
fracture after surgery wrapped with plasma plasma evaluation and hoof was (2012)
protein adhesive/ protein radiography significantly
MSCs biofilm adhesive regenerated with
biofilm bone completely
regenerated
12 Equine Mesenchymal Stem Cell Basic Research and Potential Applications
317
Table 12.10 In vivo mesenchymal stem cell applications in horse laminitis
318
Clinical
condition/ Study Biomaterial Evaluation
ailment Number of animals period used Cell dose criteria Overall result References
Chronic 12 14 months Allogeneic 20–25 × 106 – 10/12 horses (83%) responded Morrison
laminitis UC-MSCs (3–4 well. However, long-term et al. (2014)
treatments) success rates still need to be
(retrograde determined. The first
venous digital “successful” case is only
perfusion) 14 months out from initial
treatment
Chronic 30 (onset of laminitis 4 months Allogeneic 20–30 × 106 (4 Radiographically Of the 30 cases, 21 patients Dryden et al.
laminitis < 30 days [10 cases], UC-MSCs or treatments at (70%) had a successful outcome. (2013)
30–60 days [4 cases], BM-MSCs monthly In the cases receiving stem cells
60–90 days [2 cases], interval) <30 days after the onset of
and > 90 days [14 (retrograde laminitis, the success rate was
cases]) venous digital 100%. The success rate in the
perfusion, using cases receiving stem cells
the palmar/ >90 days after the onset of
plantar digital laminitis was only 50%.
vein) Statistical analysis is pending
Chronic 9 1 year Allogeneic 15 × 106 in Clinical Progressive amelioration of Angelone
laminitis AD-MSCs 15 mL PRP observations and vascularization of foot. An et al. (2017)
(III, IV (first dose) (implanted venography improvement in structure and
grade) and thrice at function of hoof. At 1 year, 7/9
autologous 1 month horses were in activity with 2
AD-MSCs interval) developing recurrent laminitis.
(two doses) After 1 year, only two animals
could be seen in activity and rest
either deceased due to colic or
were euthanized (recurrent
laminitis cases). Two cases were
M. B. Gugjoo et al.
variable retention of MSCs and poor distribution within the hoof. Coronary band
injection had failed to show any migration of MSCs within the hoof (Spriet
et al. 2013).
MSCs may prove beneficial within 30 days of third phalanx displacement after
which lamellar wedge develops (Dryden et al. 2013; Morrison et al. 2014). Post
lamellar wedge development, little observable improvement with MSCs has been
reported. Autologous or allogeneic MSCs from adipose tissue and umbilical cord
have been used. These cells have been injected more than once along with other
biomaterials like platelet-rich plasma. An overall improvement in the hoof anatomy
(based on quality and shape) and laminitic foot weight bearing and mobility had
been demonstrated (Angelone et al. 2017).
Neuron is a highly differentiated cell with almost no regenerative power. Stem cells
though are present in the nervous tissue, but their role is yet to be understood.
Various studies including that of horse have variably supported neural or mesenchy-
mal stem cell differentiation into the neuron-like cell (Hoynowski et al. 2007;
Jamnig and Lepperdinger 2012; Villagrán et al. 2016). Endogenous neural stem
cells (NSCs) tend to generate scar tissue that limits further injury but may hamper
neurological activity (Stenudd et al. 2015; Watanabe et al. 2015). Currently, stem
cell pro-healing effects may be due to their ability to secrete trophic factors (Sadan
et al. 2009). In vivo preliminary MSCs studies in other species appear promising as
improved clinical parameters have been demonstrated post-spinal cord injury (Ra
et al. 2011; Bhat et al. 2019). In case of horse, commonly encountered affections are
of spinal cord, peripheral nerves, and laryngeal hemiplegia. However, the relevant
studies remain to be conducted. One study utilizing BM-MSCs as therapeutic for
transected portion of peripheral nerve had failed to demonstrate any observable
improvement (Table 12.11) (Villagrán et al. 2016). An extensive research in the field
is desired to conclude MSCs role, if any in the nerve regeneration. Apart from
peripheral nerve injuries, MSCs role in neurological disorders like equine myeloen-
cephalopathy and equine motor neuron diseases may also be evaluated.
MSCs role has been evaluated in horse endometriosis (Mambelli et al. 2013;
Corradetti et al. 2014; Falomo et al. 2015; Rink et al. 2018). MSCs in utero implan-
tation has been made like that of artificial insemination. MSCs derived from amnion
(AM-MSCs) or endometrium (End-MSCs) may potentially replenish endometrium
in pregnancy failure (Corradetti et al. 2014; Falomo et al. 2015). Intrauterine-
implanted cells had failed to migrate to healthy endometrium while these cells had
migrated endometriosed glandular and periglandular tissues (Mambelli et al. 2013;
320
Table 12.11 In vivo preclinical mesenchymal stem cell study on nerve regeneration in horse
Model defect
Number of size/study Evaluation
Model type animals period Biomaterial used Cell dose/assembly criteria Overall result References
Excision of lateral 3 horses 45 days Allogeneic 10 × 106 Clinical and No evidence of Villagrán et al.
and medial stumps (bilateral BM-MSCs histological nerve (2016)
of ramus incision) examination regeneration at
communicans 45 days. No
difference in
histology of
treated and
control animals
M. B. Gugjoo et al.
12 Equine Mesenchymal Stem Cell Basic Research and Potential Applications 321
Rink et al. 2018). These cells had shown immunomodulation (Falomo et al. 2015)
and decreased inflammation (Rink et al. 2018) in endometritis.
MSCs may also play role to improve fertility by enhancing the follicular growth.
MSCs tend to increase follicular number in ovary, although with an insignificant
effect on dynamism (Grady et al. 2016).
MSCs may also be evaluated in inflammatory lung disease like recurrent airway
obstruction (heaves). Stem cells tend to proliferate pulmonary vessel cells, decrease
apoptotic cell number, and inhibit pulmonary vessel muscularization (Lee et al.
2015). MSCs have been trans-differentiated to other cell types like hepatocyte-like
cells and myocyte-like cells (Reed and Johnson 2008; Lin et al. 2015; Pennington
et al. 2016), but their in vivo therapeutic applications remain to be evaluated.
12.4 Conclusion(S)
Stem cells hold a great potential to act as an all-in-one therapeutic option for diverse
ailments. MSCs generally follow principles of ISCT but may show differences in
cell surface marker expression with respect to their sources. eMSCs from bone mar-
row, adipose tissue, or fetal tissues have mainly been evaluated for therapeutics.
Numerous horse stem cell studies have demonstrated favorable results though with-
out any definitive conclusion(s). MSCs from various tissue sources, together with
variable routes, concentration, and biomaterials have been utilized in these studies.
The conducted studies are uncontrolled, lacking sufficient sample size. Thus, exten-
sive case controlled blinded studies involving uniform cell source, concentration,
and biomaterials are desired in order to arrive at any conclusion.
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12 Equine Mesenchymal Stem Cell Basic Research and Potential Applications 331
Abstract
13.1 Introduction
Stem cell application remains one of the most researched and sought-in options for
therapeutics. Among all the stem cell types, currently mesenchymal stem cells
(MSCs) mainly contribute to the preclinical and clinical studies. The cell per se is
considered to provide therapeutics in diverse clinical ailments. The cell has innu-
merable sources, easy harvesting/ isolation, and established characterization proce-
dures. The cell also has minimal teratogenic effects and lack ethical issue as is
M. B. Gugjoo (*)
Division of Veterinary Clinical Complex, FVSc & AH, SKUAST-Kashmir,
Jammu and Kashmir, India
A. Pal
Division of Surgery, Indian Veterinary Research Institute, Izatnagar, UP, India
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 333
Springer Nature Singapore Pte Ltd. 2020
M. B. Gugjoo, A. Pal (eds.), Mesenchymal Stem Cell in Veterinary Sciences,
https://doi.org/10.1007/978-981-15-6037-8_13
334 M. B. Gugjoo and A. Pal
usually associated with the pluripotent stem cell, especially in humans. There are
various features attributed to the cell some of which are well established and char-
acterized while others remain to be studied and confirmed. Below are the details of
MSCs features, their applications, and associated complicacies.
Stem cells are characterized by the atypical stemness properties of self-renewal and
differentiation properties. The self-renewal property however, is currently ques-
tioned. These cells have finite culture life as evidenced by decreased population
doubling potential and morphological changes that ensue with extended culturing.
This culture life span also varies with the species type with feline MSCs relatively
bearing limited culture life span. Some of the studies although have shown that
MSCs are able to maintain their pool through the “self-renewal” and “differentiate”
into more advanced and lineage specific cell type. The viral vectors, however, may
be used to induce immortality. However, there is dearth in understanding of the
basic mechanism(s) behind these properties. MSCs have been isolated from almost
all the tissue types including the fetal membranes. Among, MSCs derived from
bone marrow, adipose tissue and fetal membranes have been studied extensively.
The cells are usually being cultured along with the fetal bovine serum (FBS). Due
to the associated limitations of disease transmission, changed cell behavior due to
xenogeneic proteins and variability in preparations, its alternatives are being stud-
ied. Alternatives like allogeneic platelet lysate, autologous/allogeneic serum, or
commercially available serum substitute have provided little success to culture
MSCs in veterinary species, unlike that of human MSCs.
There is lack of a single definition or availability of a specific quantitative assay
to identify MSCs in mixed cell population. Animal source MSCs like humans are
being characterized based upon the recommendations of the International Society
for Cellular Therapy (ISCT). The cells that are plastic adherent, express some sur-
face markers and lack hematopoietic markers (CD105+, CD90+, CD70+, MHC-I+;
CD34-, CD45-, MHC-II-), and are able to differentiate into at least three lineages
(adipogenic, chondrogenic, and osteogenic) are characterized as MSCs. Although,
the cells are plastic adherent and show tri-lineage differentiation but discrepancies
have been demonstrated in their surface marker expression and need to be evalu-
ated. MSCs are considered to be multipotent but recent studies have demonstrated
their extended differentiation potential. The cellular plasticity goes beyond as the
cells also express the pluripotency markers like Oct4, Sox-2, and Nanog, and also tend
to differentiate into germinal cell-like cells, neurocyte-like cells, myocyte-like cells,
and hepatocyte-like cells, among others. Thus, the cellular mechanisms behind such
properties are required to be elucidated. Apart from these properties, these cells are
able to “migrate” and “home” into the distant tissues, and bring about “anti-inflamma-
tory and/ immuno-modulatory” actions. There are numerous studies that have demon-
strated their immune-modulatory and migration/homing properties but consensus
13 Future of Mesenchymal Stem Cell Research 335
lacks among the studies upon the mechanisms involved. Thus, further studies are
desired aimed at the mechanisms involved in these properties.
MSCs concentration varies in tissue sources; peripheral blood harbors minimal
cell concentration, and adipose tissue harbors better cell concentration than the bone
marrow. The cell from the particular source but at different locations may or may
not have comparable culture characteristics. Even the mechanisms involved in their
differentiation may also vary with tissue source. Age of the donor also affects their
properties especially population doubling and differentiation properties. The popu-
lation doubling and differentiation potential of MSCs decrease with aging of donor.
The donor health status also has bearing on cellular characteristics. Diseases like
equine metabolic syndrome (insulin resistance disease in equines) and feline foamy
virus affect MSCs properties that become senescent and carry limited differentia-
tion potential. Prion disease in sheep and osteoarthritis in horse though do not affect
MSCs growth but may decrease their neurogenic and chondrogenic potential,
respectively. Even the reproductive cyclicity may also affect properties of the endo-
metrium derived MSCs. It becomes imperative to elucidate the mechanisms behind
such discrepancies for their effective utilization.
MSCs aspiration and injection method also affect their viability. The vigorous
aspiration appears more damaging than that of the injection. Even needle bore size
affects the MSCs viability. The large bore needle tends to have limited adverse
effect on cell viability as compared to the small bore needles. The type of storage
container whether glass or plastic although has no obvious effect on cell viability.
The storage type affects MSCs viability with cryopreservation better able to pre-
serve cell viability for longer time as compared to the biological fluids. For transit
storage of shorter duration (8–12 hrs) phosphate buffer saline appears useful clini-
cally as cells do not show any adverse effect of residual proteins of foreign materials
like FBS or dimethylsulfoxide (DMSO). Sedatives (detomidine and butorphanol),
antibiotics (aminoglycosides), and steroids (methylprednisolone and triamcinolone)
at therapeutic concentrations tend to deleteriously affect MSCs viability.
Environmental factors involving different scaffolds, growth factors, and cell type
into the culture environment affect MSCs proliferation and determine their fate.
Apart from these factors mechanics also have bearing on their proliferation and dif-
ferentiation. Currently, the effect of these factors has been studied under isolated
experiments. The results under in vivo environment may be altogether variable as
the in vivo conditions have simultaneous effect on various factors. Thus, ex vivo
systems should ape the environment available under in vivo conditions.
The ultimate aim of harvesting and culture expansion of MSCs is to utilize them in
research and under clinical settings. MSCs under preclinical studies and clinical
trials have variably improved the outcome without any major adverse effect except
few minor reactions. There are various mechanism attributed to their therapeutic
applications. The undesirable reactions reported including limited inflammation
336 M. B. Gugjoo and A. Pal
and pulmonary parenchymal edema and hemorrhage are self-limiting. Such effects
however, may be controlled by proper care and applications of the anticoagulants
like heparin may be required in disseminated intravascular coagulation. Currently,
MSCs are considered to provide therapeutic benefits mainly through their paracrine
secretion, although their in vivo survival and subsequent differentiation if achieved
may provide enhanced and effective results. Immuno-compromised and/ or immuno-
modulatory properties have made their allogeneic and or xenogeneic applications
possible. Mostly no observable immune reactions to the allogeneic/ xenogeneic
cells have been reported, barring few studies and thus, further studies for their safety
are desired before cells are utilized in clinical settings.
The cells have been evaluated in both musculoskeletal as well as non-
musculoskeletal tissues for their therapeutic effects. However, MSCs application
mostly remains under experimental phase. The limited understanding of their basic
physiological properties, poorly understood cellular interactions with respect to the
microenvironment restricts their definitive clinical applications. Further, variable
culture conditions make understanding more complicated. The combined use of
cells and other biomaterials like scaffolds and growth factor makes understanding
even more complex.
In case of musculoskeletal system, MSCs have been studied both in preclinical
experimental models (sheep, goat, dog, cat, horse, and cattle) as well as in clinical
trials (dog, cat, and horse). MSCs irrespective of the source (allogeneic or autolo-
gous) have been utilized without any fatality. Applications of MSCs in musculoskel-
etal affections like osteoarthritis, bone fractures, muscle damage, cardiac infarctions,
periodontal defects, ligaments and tendon injuries have been studied with an overall
positive response.
In case of osteoarthritis, the cells may show improvement with respect to the pain
relief. However, no obvious and actual hyaline cartilage seems to develop that inte-
grates to the native hyaline tissue. Inflammation induced MSC’s increased expres-
sion of adhesion molecules together with their decreased production of
glycosaminoglycans may be one of the reasons for their insignificant positive results.
To compensate these lacunas MSCs may be genetically engineered.
Bone regenerative medicine may be required in case of extensive damages that
otherwise has good healing potential. In case of extensive bone fractures, or other
relevant problems like delayed/non-union, regenerative medicine may be employed
involving MSCs and other biomaterials. In general MSCs, along with other factors
like scaffolds and growth/humoral factors promotes bone healing in shorter time
interval. However, the cellular posology, scaffold type, and growth factors incorpo-
rated remain to be standardized.
Muscle damage/injury is a problem as post-injury muscles are repaired by fibrous
tissue. The preliminary reports on the application of MSCs in these ailments favor
their application but intricacies in definitive therapy for the management of such
ailments is yet to be elucidated.
In case of ligament/tendon injuries, MSCs especially in equines have been fruit-
ful as represented by ability of animals to return to the normal activity. However, the
13 Future of Mesenchymal Stem Cell Research 337
sole MSCs application may not be the reason behind but along with the other stan-
dard managemental practices.
Cardiac ailments like infarction is extensively studied area in the stem cell
research. The preclinical experimental studies conducted on sheep/goat favor MSCs
application. A lot more needs to be studied to confirm their feasibility, safety, and
beneficial effect on cardiac infarctions. However, no beneficial effect on dilatation
cardiomyopathy has been demonstrated in a dog study.
In relation to the neurological system, preliminary reports show MSCs beneficial
effects to some extent. But the cells under in vivo conditions have not been reported
to actually differentiate into the neurons and the possible beneficial effect may be
through the release of trophic factors. Currently, the study area is in its infancy.
Apart from the musculoskeletal tissues, the cells have been utilized in non-
musculoskeletal tissues like dermal wounds, vocal fold injuries, kerato-conjunctivitis
sicca (KCS), anal fistula, inflammatory bowel disease (IBD), chronic kidney disease
(CKD), asthma, feline eosinophilic keratitis (FEK). MSCs in general promote heal-
ing in dermal wounds, vocal fold injuries, and anal fistula. However, some of the
reports show recurrence in cases like anal fistula which need to be studied. In case
of KCS, asthma, FEK, CKD, and IBD in dogs and cats, improved results have vari-
ably been reported following the MSCs application. Apart from these conditions,
studies on MSCs have also been started to evaluate effect on diabetes, liver affec-
tions, tumors, etc.
Available in vivo literature in veterinary science has frequently relied on study
designs that do not incorporate blinded randomized control trials to act as evidence
based medicine. The experimental power of these studies also suffers due to the
limited sample size enhancing inter-animal variability. Furthermore, these studies
lack sufficient follow-up period to determine long term effects.
13.4 Conclusion(S)
MSCs are adult stem cells being harvested from almost all the body tissues includ-
ing fetal membranes. These cells lack any specific marker and are characterized on
the basis of certain characteristic features being recommended by ISCT. The cell
lines from different sources have shown variations in relation to the surface markers
and differentiation properties. The cells from aged and unhealthy donors tend to be
less effective. These variations need further incites for their effective utilization.
The cells are being cultured and cryopreserved in FBS that may pose adverse reac-
tions and as such FBS alternatives are being evaluated. MSCs therapeutic effects are
being evaluated in various models and even in clinical trials in dog, cat, and horse.
However, further extensive studies aimed at their actual involved mechanisms need
to be recognized. The current lack of blinded randomized control trials and insuffi-
cient sample size make their application even more complicated. The studies should
be aimed at the MSCs posology that can help to determine their dose, route, and
frequency of application.