This document discusses the role of stem cells in animal reproduction. It defines stem cells as non-specialized cells that can proliferate and renew themselves through cell division, and differentiate into specialized cell types. It describes the different types of stem cells - totipotent, pluripotent, multipotent and unipotent. It discusses sources of stem cells including embryonic stem cells, induced pluripotent stem cells, fetal stem cells and adult stem cells. It summarizes potential applications of stem cells in regenerative medicine and their use in endometrial repair, vaginal reconstruction, erectile dysfunction, and in-vitro gamete production.

1. Role of Stem Cell in Animal
Reproduction
Veterinary Gynaecology & Obstetrics
Lala Lajpat Rai University of Veterinary and Animal Sciences
Hisar
Major advisor :
Dr. J. B. Phogat
Professor
Scholar:
Subhash Chand Gahalot
2017V12D
Doctoral seminar
on
1

2. Stem cells are ………………………
 Non-specialized cells, able to proliferate for
an expanded period of time
 Able to renew themselves through mitotic cell
division
 Able to differentiate into a diverse range of
specialized cell types.
2(Kim et al., 2011)

3. Types of Stem Cells
Stem cell
type
Description Examples
Totipotent
Can form both embryonic and
extra embryonic tissues,
thereby forming the whole
individual
Fertilized oocyte and
blastomeres up to 8 cell
stage
Pluripotent
Cells can form any (over 200)
cell types but not whole
individual
Inner cell mass cells of
blastocyst
And induced pluripotent
cells
Multipotent
Can form a number of other cell
types depending upon origin of
tissue
Fetal tissue, cord blood,
and adult stem cells
(MSC, HSC)
Unipotent
Give rise to only one type of
cells
Tisssue specific
progenitors like
spermatogonial stem
cell
3
(Vaseena et al., 2015)

4. Stem cells :Source
• Embryonic stem cells
Derived from the inner cell mass of
the blastocysts.
1981 - Mouse embryonic stem cells
isolated from the inner cell mass by
Martin Evans, Matthew Kaufman,
and Gail R. Martin.
Gail Martin is attributed for coining
the term "Embryonic Stem Cell".
1998-James Thomson and coworkers
isolated the first human
embryonic stem cell line at
the University of Wisconsin -Madison
4

5. Pluripotent cells have the
ability to differentiate into
derivatives of all three germ
layers (endoderm, mesoderm, and
ectoderm).
The most common assay for
demonstrating pluripotency is
teratoma formation.
Stem cell lines have the ability to
grow indefinitely and express ESC
markers and show ESC-like
morphology.
In addition, the cell line forms
embryonic bodies (in vitro) and/or
teratomas (in vivo) containing all 3
germlayers.
(Yu and Thomson, 2006)
5

6. Induced pluripotent Stem
cells From adult fibroblast
S Yamanaka
Induced pluripotent stem (iPS) cells
are defined as…………..
Differentiated cells that have been
experimentally reprogrammed
to pluripotent cells, to achieve an
embryonic stem cell like character.
( Nobel prize for Physiology/ Medicine , 2012 )
6

7. Both MSC and fibroblasts may be used for these purposes and there are
studies already for the obtaining of iPS to buffalo, cattle, goats, pigs, sheep, and
other farm animals (Kumar et al.,2014)
Share similar characteristics with ESCs: exhibiting morphology of ESCs,
expressing ESCs markers, having normal karyotype, expressing telomerase
activity, and maintaining the developmental potential to differentiate into
derivatives of all three germ layers
1. Fusion of pluripotent cell and somatic
cell
2.SCNT
3. By forced expression of defined genes
4. Using synthetic molecules-- 5-Aza C,
Trichostatin A
Reprogramming strategies
7
 A set of reprogramming factors (also dubbed Yamanaka factors)
the transcription factors Oct4 (Pou5f1), Sox2, cMyc, and Klf4, are
induced in somatic cells
(Kim et al., 2011)

8. Fetal stem cells from extra-embryonic tissue
Other ----Amniotic
fluid, Amniotic
membrane
(Azari et al., 2011; Cremonesi et al., 2011)
Can be isolated during
gestation also.
Shown multipotent markers
Display negligible immunogenicity
Teratoma formation not observed
No ethical concerns.
(Yadav et al., 2012)
Bubaline AF cells could be cultured and maintained in vitro for a prolonged period
and potential source of multipotent cells for applications like therapeutic
assisted reproduction in animals (Yadav et al., 2011)
8

9.  These are Undifferentiated (unspecialized) cell , in a differentiated
tissue
e.g Bone marrow, adipose tissue, wharton’s jelly, umblical cord blood,
peripheral blood, extraembroyonic tissue, spermatogonial stem cell
Limited Self-renewal , and (with certain limitations) differentiate to
yield all the specialized cell types of the tissue from which it originated
 Most clinical trials studying stem cell therapy have used MSC which
were often derived from bone marrow.
Ease of their isolation from tissues and their extensive capacity for in
vitro expansion
Adult stem cell…..
(Herberts et al., 2011)
Nearly all postnatal organs contain populations of stem cells, which have the
capacity for renewal after damage or ageing (Du and Taylor, 2010)
9

10. Characteristics of Stem Cells Used In Stem Cell Therapy
10
Unipotent
Can
Differentiate in
to only one cell
type

11. Potential Advantages and Disadvantages of Stem Cells in Regenerative Medicine
11

12. A) Donor Recipient relation
 Autologous stem cell therapy
 Isogenic or Syngenic stem cell therapy
 Allogenic stem cell therapy
 Xenogenic stem cell therapy
B) Source of stem cell
 Embryonic stem cell
 Umbilical cord blood derived/ Amniotic stem cell
 Induced pluripotent stem cell
 Adult stem cell
Types of stem cell therapy
12

13. Establishment of Buffalo Embryonic Stem Cell Lines
Embroyos produced by IVF, SCNT
or Parthenogenesis
ICM is removed by either
enzymatic digestion or
mechanically using microblade
ICM seeded separately on
feeder layer
Cultured in embroyonic
stem cell media
Culture medium is changed on
alternate day and primary
colonies observed after 8- 10
days of seeding
Primary colonies are disintigrated using
micrblades and reseeded on new feeder
layer
Colonies showing ES like charactes are
isolated and subcultured on new feeder
layer
Some of the colonies choosen for
characterization using marker,
karyotyping and embryoid body
formation
After confirmation cryopreservation
(Shah et al., 2014)

14. Work done in India
 Bovine ICM derived cells express the Oct4 ortholog (Yadav et al., 2005)
Isolation, culturing and characterization of feeder-independent amniotic
fluid stem cells in buffalo (Bubalus bubalis) (Dev et al., 2007)
 Isolation of ES cells from in vitro-produced buffalo embryos (Verma et al.
2007)
 ES cell lines were established from parthenogenetically produced buffalo
embryos (Sritanaudomchai et al. 2007)
Expression of Pluripotency Genes in Buffalo (Bubalus bubalis) Amniotic Fluid
Cells (Yadav et al., 2011)
Buffalo (Bubalus bubalis) embryonic stem cell-like cells and preimplantation
embryos exhibit expression of pluripotency-related antigens
(Anand et al., 2011)
Expression and quantification of Oct-4 gene in blastocyst and embryonic
stem cells derived from in vitro produced buffalo embryos (Sharma et al.,
2012)
14

15. Cultured buffalo umbilical cord matrix cells exhibit characteristics of
multipotent mesenchymal stem cells (Singh et al., 2013)
Isolation, culture and characterization of caprine mesenchymal stem cells
derived from amniotic fluid (Pratheesh et al., 2013).
 Molecular characterization and xenogenic application of Wharton's jelly
derived caprine mesenchymal stem cells (Pratheesh et al., 2014)
 Selection of appropriate isolation method based on morphology of blastocyst
for efficient derivation of buffalo embryonic stem cells (Kumar et al., 2014)
Isolation and Characterization of Buffalo Wharton’s Jelly Derived
Mesenchymal Stem Cells
(Sreekumar et al., 2014)
15

16. 16

17. Stem cells in Endometrial Repair
Three kinds of stem cells exist in the human endometrium: epithelial stem
cells, mesenchymal stem cells, and endothelial stem cells
(Xu et al., 2015)
MSC functionally contribute to human endometrial regeneration in vitro and
in vivo (Cervello et al., 2010)
BMDSC infusion might improve endometrial regeneration in a murine model
of Asherman’ s syndrome
(Zhao et al., 2015).
17

18. Stem cells can play important role in many vaginal pathologies like Mayer
Rokitansky Kuster Hauser syndrome (MRHK), vaginal prolapse, vaginal fistula,
cancer and other types of trauma
In mouse models it was shown that MDSC are able to improve vaginal
regeneration by reducing fibrosis and enhancing epithelial tissue formation
(Ho et al., 2009)
The stem cell injections brought benefits in terms of restored erectile function and penile
physiology (Zhang et al., 2012)
The majority of studies were done using MSC, neural crest stem cells, ESC, endothelial
progenitor cells and MDSC.
The improvement in erectile function seems to be due to the paracrine factors secreted
by the injected cells (cytoprotective, anti-fibrotic and anti-apoptotic molecules), rather
than direct grafting/differentiation (Albersen et al., 2010).
Stem Cells and Vaginal Reconstruction
Stem Cells and Erectile Dysfunction
18

19. In-vitro Gamete Production From Stem Cells-
--male gamet
(Volarevic et al., 2014)
ESc
iPSCs in vitro
differentiation
into advanced,
haploid cell
products 19Sterile mice

20. 20
In vitro differentiated embryonic stem cells give rise to male gametes
that can generate offspring in mice (Nayernia et al., 2006)
Differentiation of male germ cells from human ESC has also been
demonstrated (Chen et al., 2007)
So far, functional male gametes from human iPSCs have not been
obtained.
Spermatozoa generated from iPSCs were capable of fertilizing the oocytes
after intracytoplasmatic injection and giving rise to fertile offspring following
embryo transfer in mouse (Hayashi et al., 2012)
SSC are adult stem cells, but SSC-derived cells, called multipotent adult
germline stem cells (maGSC), have differentiation potential similar to ESCs.
Nolte and coworkers showed that maGSC are able to undergo meiosis and
form haploid male germ cells in vitro (Nolte et al., 2010)

21. In-vitro Gamete Production From Stem Cells---follicle containig
oocytes
Eguizabal and coworkers generate haploid female cells from human pluripotent
stem cells, but neither of them resembled an oocyte nor is predicated to possess a
functional ooplasm capable of being fertilized
(Eguizabal et al., 2011)
Mouse pluripotent stem cells could be differentiated in an in-vitro/in vivo
system into oocyte-like cells that are capable of being fertilized by spermatozoa
and generating normal progeny (Hayashi et al., 2012)
21
First reported the successful Derivation of oocytes from mouse embryonic
stem cells in vitro (Hubner et al.,2003)

22. Applications of spermatogonial stem cells
(i) Testis tissue from immature animals transplanted ectopically into
immunodeficient mice is able to respond to mouse gonadotropins and to
initiate and complete differentiation to the level where fertilization-
competent sperm are obtained ------Testis xenografting
(ii) Isolated spermtogonial stem cells are able to organize and rearrange
into seminiferous cords that subsequently undergo complete
development, including production of viable sperm…….Spermatogonial
stem cell transplantation
22

23. Testis xenografting
Application is in—
To preserve the breeding potential of a
genetically valuable pre-pubertal male
animal (Pukazhenthi et al., 2006)
Spermatogenesis is suppressed
outside the breeding season
(Blottner et al., 1995)
Azoospermia by disease or and patient
ongoing cytotoxic therapy for treatment
of cancer
Preservation of endangered species
(Pukazhenthi et al. 2006) 23

24. Salient
feature of
Testis
xenografting
Survival of xenografts decreases with the
degree of maturity of the donor tissue.
Presence of an appropriate surrounding somatic
compartment therefore seems to be necessary for
germcells to proliferate and differentiate.
24
The functionally immature sperm can help
generate off spring only through intracytoplasmic
sperm injection (ICSI).
Morphologically mature sperm have been produced in
xenografts from rabbits, pigs , goats, hamsters
, rhesus macaques ,sheep , cats ,and dogs.
Viable off spring has been produced in allografted
mouse and xenografted rabbit and pig .

25. Spermatogonial stem cell transplantation
Isolation of a mixed germ cell population from a donor testis (preferably
enriched in SSC if markers are known for that species).
Treatment with focal testicular irradiation or systemic busulfan to reduce
their endogenous SSC and transfer to recipient
Time is allowed for colonization, proliferation and spermatogenesis,
Semen is collected and assessed for the relative percentage that is of
donor origin
25

26. In adult mice, only 0.02–0.03% of the total germ cells have stem cell capacity
it is necessary to isolate and enrich SSCs with high viability and purity, for the sake of
subsequent culture or manipulation of these cells.
Fluorescence activated cell sorting (FACS) or magnetic-
activated cell sorting (MACS).
Differential plating, velocity sedimentation, or density
gradient centrifugation
The highest purity (90%) of type A spermatogonia from buffalo testes is achieved by
Ahmad et al. (2013), who adopted the selection by Percoll gradient separation.
At present DMEM (high glucose) and DMEM/F12 are the most widely used media in
cultures of SSCs from domestic animals
Enrichment
(Zheng et al. 2013b)
(Tegelenbosch and De Rooij, 1993).
26

27. SSC are a potential tool for the treatment of male infertility due to
their ability to differentiate into male gametes in vitro and capacity to
restore male fertility in vivo (Brinster, 2007)
In indian contex it may have a potential benefit , as large number of
scrub bull can be converted in to potential fertile bull
The SSC identity of the cultured cells is verified by the expression of
molecular markers (such as UCHL1, ZBTB16, GFRa1, NANOG2, POU5F1,
CSF1R and THY1)
Busulfan used to create sterile mice for transplt.
Lac-Z gene used as marker-successful germ line propagation observed
Ultrasound guided microinjection into efferent ductules and rete-testis
27

28. Spermatogonial stem cell injection
The efferent bundle is dissected and the tip of the needle is
placed into it and guided to the rete. Then pressure is applied to
the cell suspension in the needle and it is pushed into the rete and
then into the tubules
28
A—Rat
B—Farm
animal

29. Summary of germ cell transplantation in different donors of domestic
animals and recipient species
29
(Zeng et al., 2014)

30. Stem Cell-Derived Oocytes: Current Knowledge and Future Perspectives
Dilemma regarding the presence of ovarian stem cells in adult mammalian
ovaries
Zou and his coworkers successfully established long-persisting pluripotent/
multipotent ovarian stem cell lines in neonatal and adult mice (Zou et al., 2009).
White et al. identified a rare population of mitotically active germ cells in
human ovaries that can be purified and cultured in vitro to spontaneously
form oocytes (White et al., 2012).
These cells, named as germ stem cells (GSCs), were isolated from
reproductive-aged human ovaries using fluorescence-activated cell sorting
(FACS) with an antibody against the carboxyl (−COOH) terminus of the germ
cell-specific marker Ddx4, which is expressed on the cell surface of GSCs.
30
Further, GSCs were capable of forming oocyte-like structures and
incorporating into follicles under specific in vitro and in vivo
conditions

31. Ovarian stem cells (MVH+BrdU+ cells) residing within the ovarian surface
epithelium of neonatal and adult mice express –
high telomerase activity, Oct4, and Nanog and have a capacity to generate
functional oocytes when transplanted back into sterile recipient mice
(Zou et al., 2009)
31

32. Role of stem cells in cloning
Cloning using somatic
cells
High abortion and fetal
mortality rates are commonly
observed
Incomplete reprogramming of
the somatic nuclei
Currently, the efficiency for nuclear transfer is between 0–10%, i.e., 0–10 live
births after transfer of 100 cloned embryos.
Epigenetic Alterations.
Gross karyotypic alterations
32

33. Cloning using
embryonic stem
cells
Unusual karyotypic stability
Can be cultured in vitro for many passages
without showing mutation
Exhibit developmental pluripotency
More controlled genetic modification can be done
On Aug 22, 2010, a cloned calf of
female buffalo ‘Garima-2’ nicknamed
Gamini was born from embryonic stem
cell; she is the mother of a calf named
‘Mahima’
Dr. Srivastava and his team of scientists, including M.S. Chauhan, S.K.
Singla, R.S. Manik, Shiv Prasad and Aman George, feel that embryonic
stem cells have a better cloning ability as compared to somatic cells (used
in earlier cloning) that are lineage committed
33

34. Reproductive cloning using embryonic stem cells
34

35. Stem cell and Transgenesis
Animals which have been
genetically engineered to contain
one or more genes from an
exogenous source.
35

36. One big advantage of using ES cells over microinjection is the ability to
select for transgene integration through the use of selectable markers.
(Hodge and Stice, 2003)
ES-mediated gene transfer is the method of choice for gene inactivation,
the so called knock-out method. (Capecchi , 1989)
Embryonic stem cells are relatively efficient at homologous recombination.
 Recombination between homologous sequence in the vector DNA and the
genome is used to target the insertion of the foreign DNA to a specific
sequence in the genome. (Pichova, X)
ES cells enable the researcher to place new genes in advantageous places in
the genome or to remove deleterious gene.
36

37.  Fewer genetically valuable embryos are used, unlike by
microinjection method where 1000 to 3000 embryos/
transgenic calf are required
 Transferring non transgenic embryos to recipient
females is avoided ,so no wastage of time, labour and
assets.
 More than 200 surrogate mothers/transgenic calf are
needed in other methods
 Significant reduction of cost of production 2 to 20
million dollars/transgenic calf by other methods
Commercial aspects of using embryonic stem cells for
Transgenesis
(Stice, x)
37

38. 38
Transgenesis using spermatogonial stem cells
ES cells, after genetically intervened, are injected into blastocysts with the
hope of successful integration and contribution to the germline, which not
always can be guaranteed.
By contrast, SSCs are already constituents of the germline
SSC genetic and epigenetic stability keep these cells committed to the
germline phenotype so that they do not tend to differentiate into other lineages
Efficiency is 5-10 times better than ES
Transmit transgenes from one generation to the next
Do not produce teratomas
(Aponte, 2015)

39. A) FAVOURS NEOPLASTIC GROWTH
Boy treated with allogenic human fetal neural
SCs diagnosed with a multifocal brain tumor after four
years
(Amariglio et. al. 2009)
b) Teratoma Formation
Embryonic stem cells – Parkinson’s disease - teratoma
(Sonntag et al. 2006 )
c)Graft-versus-host disease
Immunosuppressive therapy: chance of diseases
d) Ectopic grafting: Grafting in the non target sites
Undesirable effects……
39

40. b) Embryonic stem cells: difficultto control growth,
ethical issues
c) Donor-to-recipient transmission of pathogens
d) Unwanted differentiation; Encapsulated structures in
infarcated areas of heart in mice
(Breitbach et al., 2007)
E) Lack of standardised protocols for culture and therapy
Challenges………..
40
a) Adult stem cells : difficult to isolate , short storage life in
culture

41. a) Autograft or isograft: less chance of rejection
b) Use of stem cells at the point of injury
c) Use of multipotentstem cells, undergone prolonged in vitro
differentiation for a specific injury
d)While characterization of differentiated cells immunogenicity and
tumorogenicity should be checked
e)Commercial production of allogenic adult stem cell lines and
devlopment of standardized protocols for therapy
Reduction of undesirable effects
41

42. 42
Conclusion
At the moment, clinically validated stem cell treatments for
reproductive diseases & alterations are not available
The development of ES cell lines of domestic livestock, such as
cattle, buffalo, sheep and goat, would greatly facilitate
reproductive performance and prolificacy.
Use of stem cells can be best tool in restoration of fertility and
preservation of endangered species.

43. 43