A Report on the National Symposium-cum-Workshop on
Pluripotent Stem Cells in Mammalian Gonads
September 6- 7, 2013
National Institute for Research in Reproductive Health, Mumbai
A National Symposium-cum-Workshop Pluripotent Stem Cells in Mammalian Gonads was
organized by the NIRRH, Mumbai under auspices of the Indian Society for the Study of
Reproduction and Fertility (ISSRF). The focus was on a novel population of pluripotent stem cells
termed VSELs (very small embryonic-like stem cells), which exist in various adult body organs
including gonads. More than 100 candidates had responded from all over India and 21 were short
listed to participate. The scientific program included 8 lectures that were delivered during prelunch sessions and lab work was held post-lunch. The main motto of the Workshop was to Believe
What You See.
Dr PS Chauhan was the Chief Guest during the
inaugural function. The inaugural function included
welcome address by Dr SD Kholkute, Director,
NIRRH wherein he welcomed the Guests including
the participants. He mentioned about the two
human embryonic stem cell lines (hES) that were
established at the Institute and the ongoing efforts
to use them for studies involved with diabetes and
myocardial infarcts. He informed the audience that
Dr Bhartiya’s group has reported the presence of
VSELs in adult human testes, ovaries and cord
blood. However, the area remains controversial and rigorous experiments are required. He
highlighted the importance of establishing facts for progression of science, especially for
controversial field or for concepts which challenge the central dogma. He appreciated the group’s
initiative to hold this National Symposium-cum-Workshop to share their work.
This was followed by a welcome address of Dr NK Lohiya, President ISSRF. He emphasized that
as controversies always exist in science, there should be a healthy debate based on evidence to
resolve issues. The thirteenth issue of the ISSRF Newsletter on Stem Cells and Reproductive
Health was formally released on the occasion. Dr Taru Sharma, editor provided a brief about the
Newsletter and also invited everyone to 24th Annual Meeting of the ISSRF at IVRI-Bareilly in
February 2014. This was followed by the announcement by Dr Lalit Singh of Elsevier’s initiative
to come out with International Journal of Reproductive Medicine (IJRM), a peer reviewed
official Journal of ISSRF with Dr Jayshree Sengupta and Dr Debabrata Ghosh as the Chief
Editors. Dr Lohiya urged all scientists to contribute to the Journal.
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Dr Deepa Bhartiya, Scientist E and Head, Stem Cell
Biology Department, NIRRH and Organizing Secretary,
shared the objectives of the Symposium-cum-Workshop
with the audience. She emphasized that pluripotent stem
cells including hES and induced pluripotent stem (iPS) cells
possess maximum regenerative potential as they are the
most primitive stem cells compared to adult stem cells
whose effect [in pilot autologus stem cell trials] appears to
be more of a growth factor/cytokine effect rather than true
regeneration. She stressed that instead of using pluripotent
stem cells grown in a Petri dish for regenerative medicine,
one may also consider exploiting pluripotent stem cells (VSELs) which exist in adult body organs.
She agreed that controversies exist on the very existence of VSELs especially highlighting recent
news in Nature covering a study conducted by Weissman’s group from Stanford. But rather than
blindly accepting opinions made by others, she encouraged on independent thinking, working with
an open mind and believe in what you see with your own eyes. By citing various examples
(mentioned below) she emphasized that it is more of technical issues that make the area
controversial rather than the existence of VSELs.
(i) The four research groups who challenged the existence of VSELs probably failed to detect
them primarily because of their method of sample processing.
(ii) The two papers published in PNAS which completely deny presence of VSELs in adult
mouse ovary also have technical drawbacks. Our group had communicated this in a letter
to PNAS Editor (which was rejected) and now has been peer reviewed in another Journal
and will soon be published.
(iii)A paper in Human Reproduction journal also completely denied presence of VSELs in
adult ovary. But we wrote a letter to Editor which was published and pointed out that the
results are negative because the group has localized OCT-4 on Bouin’s fixed tissue
sections, which is known to result in false negatives.
She concluded that the objective of the Workshop was to share the knowledge with the participants
and create a critical mass who could initiate work in this area. The meeting was unique since
majority of faculty comprised of project staff and students from Stem Cell Biology Dept at NIRRH
who have observed VSELs and demonstrated their presence to the participants by various
approaches including flow cytometry, confocal microscopy, immuno-histochemistry, H & E
staining and qRT-PCR.
The Chief Guest, Dr PS Chauhan, during his
keynote address praised the efforts of Dr Bhartiya’s
group to hold the Workshop. It is an important area
of research and expressed his satisfaction that stem
cell research being carried out at NIRRH follows
the Institute mandate and has direct relevance to
reproductive health. He said that controversies lead
to good science and vice versa. If research from Dr
Bhartiya group calls for a paradigm shift in the field
of
reproductive
health,
multi-institutional
networking is required to further research in the
area.
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The participation of Dr Jayshree Sengupta, Dr D Ghosh, Dr R S Sharma, Dr Dheer Singh, Dr Taru
Sharma, Dr Lalit Singh and all senior scientists and scholars of NIRRH greatly contributed in the
success of the event.
Activities on Day 1
Lecture 1: An Introduction to VSELs by Dr Deepa Bhartiya
VSELs, first reported in 2006 by Prof
Ratajczak’s group at Illinois University
School of Medicine, USA are pluripotent stem
cells in adult body organs and are involved in
maintaining life-long homeostasis, are
mobilized in response to disease and are
possibly the embryonic remnants resulting in
various cancers in adulthood. The lecture
gave an overview of VSELs and described in
detail the work on gonadal VSELs. Dr
Bhartiya’s group was the first to report VSELs
in adult human testes, and in ovaries from rabbit, sheep, monkey and humans. VSELs are
possibly the primordial germ cells that persist not only in gonads but also in various body
organs. Their work provides more information in addition to the latest review by Professor
Tilly’s group from Harvard Medical School on gonadal stem cells as shown below.
The lecture further shared with the participants how VSELs are involved in postnatal
oogenesis and primordial follicle assembly and that ovarian VSELs function is modulated by
FSH via a novel FSH receptor isoform R3 which has earlier also been implicated in cancers
by others. She further showed morphological evidence suggesting that VSELs are mobilized
when 70% of pancreas is surgically removed. She shared data that VSELs biology is affected
when mice pups are exposed to endocrine disruptors resulting in infertility, PCO-like
condition and cancers in uterus and prostate. These results provide a basic mechanism and
may be extrapolated directly to why Punjab has become the Cancer capital of the Country
due to high exposure to pesticides, fertilizers etc. Towards the end she discussed the concept
of fertility restoration in cancer survivors by targeting VSELs.
Lecture resulted in lot of discussion and an interesting question asked by Dr Dheer Singh was
whether the cells described are indeed cells or micro-vesicles? To this Dr Bhartiya replied
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that they stain blue after H & E staining suggestive of presence of nucleus and if it was a
micro-vesicle, it would just stain pink representing only cytoplasm pinched off from cell
surface.
Lecture 2: Old Ovaries and New Eggs: Hatching the Controversy by Dr Deepak Modi
The lecture started with a description of the
ovarian structure and basic biology of follicle
growth and menopause. The development of
follicle in the fetal ovary was explained. The
main focus of the talk was on controversy
surrounding the existing biological paradigm
that adult mammalian females posses a nonrenewable pool of primordial follicles at birth
which does not replenish during adult stage. A
series of reports from Professor Jonathan Tilly
of Harvard Medical School have challenged
the existing paradigm and shown presence of stem cells in adult ovaries similar to testis.
However, their reported discovery of rare population of ovarian stem cells in adult women
and mice and their ability to produce immature oocytes, has initiated controversy among
reproductive biologists. Two recent papers in PNAS contradict the presence of stem cells in
ovary using ‘rainbow mice’ and lineage tracing studies. The pros and cons of these studies
were analyzed and concluded with requirement of further studies to address the existing
controversies.
Lecture 3: VSELs in Mammalian Ovary by Ms. Seema Parte
Ms Parte shared her PhD work on the
presence of stem cells in adult mammalian
ovary (rabbit, sheep, monkey and human).
She shared the results of her first paper [cited
more than 50 times till now] that reported the
presence of two distinct populations of stem
cells viz. very small embryonic stem cells
(VSELs) and ovarian germ stem cells
(OGSCs) lodged in the ovary surface
epithelium (OSE). Ovarian stem cells express
characteristic pluripotent and primordial germ
cell specific markers and undergo spontaneous differentiation in vitro into oocyte-like
structures, ES colonies, embryoid bodies, neuronal phenotype cells and parthenote embryos;
whereas epithelial cells undergo transition to mesenchymal phenotype and constitute somatic
niche for the developing oocytes resembling primordial follicle (PF) formation in vivo. She
then discussed the model proposed for PF assembly in postnatal ovary based on the study.
Further she discussed papers from the department showing that these stem cells are
modulated by FSH through a novel FSH receptor isoform (R3). VSELs undergo self-renewal
& OGSCs undergo proliferation & clonal expansion in response to FSH. The work on
ovarian cortical tissue culture [being studied for more than a decade as a model to study
follicle growth and transition] serving as an excellent source of stem cells was also explained.
Results on large numbers of stem cells being shed off on to the cell insert and their potential
to form eggs after extended culture was shared. The studies from the department provide a
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paradigm shift in current understanding of ovarian biology: (i) stem cells exists in ovaries (ii)
in addition to FSH action on granulosa cells of follicles, FSH also acts on ovarian stem cells
(iii) novel isoform of FSH receptor R3 is implicated in stem cell action. (iv) ovarian cysts
representing clonal expansion of ovarian stem cells [hallmark feature of stem cells] are
observed in rabbit, sheep, monkey, human and also mice ovaries contradicting the reports in
PNAS.
Lecture 4: Identification & Characterization of Cord Blood VSELs by Ms. Ambreen
Shaikh
Ms Shaikh described the basics about VSELs
and their characteristics. The lecture
emphasized on how to identify VSELs on
basis of their unique features like size,
morphology and marker expression. Methods
to isolate VSELs using various approaches
like flow cytometry, magnetic sorting and
Ficoll-Hypaque gradient were discussed.
Analyzing VSELs using flow cytometry based
on their unique gating strategy and sequential
gating depending on expression of markers
was dealt in detail with the examples of various studies where VSELs in cord blood were
analyzed. The lecture also described the characterization of VSELs in term of
immunophenotyping, immunostaining and analysis of DNA content after enrichment using
combinational approach of Ficoll-Hypaque gradient and magnetic sorting, which is being
currently employed in the department. Functional aspects of mouse and human VSELs with
a focus on their differentiation potential were also described in the lecture. The proper
understanding of how to identify and isolate VSELs will help in further functional studies on
these cells and also answer the controversies that have arisen recently questioning the
existence of these cells and their pluripotent status.
Activities on Day 2
Lecture 5: Basic Concepts in Testis Biology by Dr Priyanka Parte
Day 2 of the workshop began with a talk by Dr
Parte. Dr Parte orientated the participants towards
understanding
the
intricate
process
of
spermatogenesis covering right from the description
of the basic cytoarchitecture of testis, the various
cell types, their contributions to the process of
spermatogenesis, germ cell nomenclatures, the
theories involved there in and the process of
spermatogenesis per se. The lecture dealt in detail
on spermatogonial stem cells (SSCs) [their rapid
expansion and differences in expansion kinetics between mice and humans], the concept of
symmetric and asymmetric divisions, meiosis and spermiogenesis. She described various stages of
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spermatogenesis and stated that SSCs divide symmetrically. She also introduced the participants to
the concept of ‘stem cell niche’.
Lecture 6: VSELs in Mammalian Testis by Dr Deepa Bhartiya
Dr Bhartiya started her lecture by mentioning that
stem cells biology in testis is not well understood.
SSCs undergo symmetric cell division but true stem
cells must undergo asymmetric cell division. She
shared various published reports in literature, which
suggest presence of a sub-population of stem cells
among SSCs. This sub-population of stem cells is
pluripotent in nature and are the VSELs. She
shared their results published in Journal of
Histochem Cytochem that describes this population
as cells with nuclear OCT-4 whereas SSCs have
cytoplasmic OCT-4. The lecture later focused on highlights of work carried out by her group.
VSELs are quiescent in nature and thus remain unaffected by oncotherapy in both busulphan
treated mice testis and also in azoospermic testicular biopsy of adult survivors of childhood
cancers. The existence of VSELs after busulphan treatment has been confirmed by flow cytometry
studies also. Several reports suggest that culture of testicular biopsy gives rise to ES-like colonies
and it is believed that SSCs dedifferentiate to give rise to ES-like colonies. But Dr Bhartiya argued
that even if we accept SSCs dedifferentiate, why only 0.1% cells undergo dedifferentiation in
vitro? Why not all SSCs dedifferentiate? She explained that an alternative and more likely
explanation could be that very few VSELs that may be present in the biopsy and they start growing
as ES-like colonies. She informed that her explanations were published as a paper, making a case
for VSELs. She concluded saying a sub-population of pluripotent stem cells exists in testis, which
is resistant to oncotherapy, express pluripotent markers and starts growing when exposed to
conducive in vitro environment.
Lecture 7: VSELs and Fertility Preservation by Dr Kalpana Sriraman
The main focus of Dr Sriraman’s talk was on
how VSELs can be explored to treat
oncotherapy-induced infertility. Advances in
the treatment of cancer have led to more
number of people surviving cancer but are
rendered infertile as a side effect. This has
increased the demand for fertility preservation
of the cancer patients and hence has led to the
emergence of new field called Oncofertility.
The current scenario of fertility management
of both male and female cancer survivors was
discussed and the challenges associated with it were examined. The question of whether
VSELs can be tapped to restore oncotherapy induced infertility was addressed in detail
through various studies carried out using mouse model. VSELs resist chemotherapy in both
testis and ovary of mice. VSELs have also been observed in testicular biopsies obtained from
childhood cancer survivors. Though the VSELs resist therapy, they cannot repopulate gonads
with germ cells due to a compromised niche. By providing a healthy niche through
transplantation of Sertoli or mesenchymal cells in mouse, fertility is restored from persisting
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VSELs in male mice. VSELs in ovaries that resist chemotherapy show potential to form
oocytes in vitro in response to FSH. The results are exciting and have tremendous clinical
application. It can change the way infertility of cancer survivors is addressed currently and
could completely exclude the need for gonadal tissue banking in future.
Lecture 8: VSELs and Cancer by Dr Deepa Bhartiya
It has been suggested in the past that embryonic remnants in the body give rise to cancers.
VSELs are implicated in various kinds of cancers as suggested in two review articles by
Ratajczak’s group. There are several facts that convince us that this may be true (i) existence
of C/T antigens – why do various types of cancer express testicular antigens? Ratajczak’s
group has reported that VSELs in bone marrow express C/T antigens but not HSCs (ii) Why
do cancers express hCG (iii) The spheres in ovarian cancers have cells with nuclear OCT-4 in
their core as reported by Ahmed et al (2012) (iv) Similarly, Kohane et al from Harvard
reported in 2012 that the gene expression profile of stem cell pluripotentiality and
differentiation is conserved across diverse solid and hematopoietic cancers. (v) CD133 and
OCT-4 are characteristic markers specific for human VSELs and are surprisingly also
expressed by various types of cancers. AC 133 has been reported by FACS in various cancers
including brain tumors, colon cancer, pancreatic cancer, prostate cancer, lungs, liver and skin
in the cancer initiating cells/ cancer stem cells.
Embryonic Stem Cell Marker Expression in Cancers
OCT-4, an embryonic stem cell marker, is
highly expressed in bladder cancer
Atlasi et al, 2011
The expression of pluripotency marker
OCT-3/4 in prostate cancer and benign
prostate hyperplasia Monsef et al, 2009
Specific detection of OCT-3/4 isoform
expression in solid germ cell tumors
Looijenga et al, 2011
Specific detection of OCT-3/4 isoform
A/B/B1 expression in solid (germ cell)
tumours and cell lines: confirmation of
OCT-3/4 specificity for germ cell tumours
Rijlaarsdam et al, 2011
OCT-3/4 is a dose dependent oncogenic
fate determinant Gidekel et al, 2003
Human embryonic genes re-expressed in
cancer cells Monk and Holding, 2001
Residual embryonic cells as precursors of a
Barrett's-like metaplasia
Wang et al, 2011
Expression profile of embryonic stem cellassociated genes OCT-4, SOX-2 and Nanog in
human gliomas Guo et al, 2011
Embryonic stem cells markers SOX-2, OCT-4 and
Nanog expression in nasopharyngeal carcinoma
Luo et al, 2013
Attributes of OCT-4 in stem cell biology:
perspectives on cancer stem cells of the ovary
Samardzija et al, 2012
OCT-3/4 is a dose dependent oncogenic fate
determinant Gidekel et al, 2003
Embryonic stem cell markers expression in
cancers Schoenhals et al, 2009
Adult tissues may contain embryonic remnants
that normally lie dormant, but that can be
“activated” to become cancerous Virchow,1858
VSELs with nuclear OCT-4 are possibly responsible for various cancers in the body
Thus work done by various groups suggest that VSELs may be implicated in various cancers
as the quiescent cancer stem cells and it was proposed in the talk that with age the
microenvironment of stem cells gets compromised, is unable to keep the VSELs under
quiescent state and hence they multiply rapidly giving rise to cancers. In the last part of
lecture, Dr Bhartiya shared their study wherein they show that VSELs biology is altered on
exposing mice pups to endocrine disruptors. The homeostasis gets altered more in favor of
proliferation rather than differentiation leading to PCO-like condition in ovary, compromised
spermatogenesis, absent glands in uterus and signs of cancers in both uterus and prostate. She
also provided direct evidence of VSELs being implicated in testicular cancer. This work has
recently been accepted for publication.
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Practicals on Day 1
Kalpana and Ambreen demonstrated how VSELs could be studied in mouse testis by flow
cytometry as LIN-ve/CD45-ve/SCA-1 positive population. Kalpana, Hiren and Seema
showed VSELs by confocal microscopy as nuclear OCT-4 positive cells compared to
progenitors with cytoplasmic OCT-4 and explained the protocol for immuno-localization.
This was followed by demonstration of isolation of sheep ovarian surface epithelial cells to
study VSELs by Seema and Hiren. The enzymatic digestion method of mouse ovaries to
isolate surface epithelial cells and stem cells was discussed by Jarnail Singh with the
participants. The OSE smears were viewed under microscope after H & E staining to see
VSELs, progenitors and epithelial cells.
Practicals on Day 2
Ambreen first showed how to analyze data acquired by flow cytometry confirming the
presence of LIN-ve/CD45-ve/SCA-1 positive cells. Then Prasad discussed how to use qRTPCR approach to characterize pluripotent state of VSELs. Later Hiren showed VSELs in
testicular smears after H & E staining. Seema shared her videos where she has observed
cytoplasmic streaming in oocytes, which spontaneously differentiate from VSELs in culture
Valedictory Function
The participation certificates were presented to all candidates with compliments and hope that
the knowledge gained will be of immense use in their future research pursuits. Some of the
participants also shared their views about the workshop and desired that ISSRF should
continue organizing such events covering various aspects of reproductive health issues in
different parts of the country. Dr Lohiya complimented Dr Kholkute, Dr Deepa Bhartiya and
entire team for successful organization & wished good luck to all participants. The financial
support of NIRRH, ICMR and ISSRF was gratefully acknowledged.
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