Seema Parte

Detection, Characterization, and Spontaneous Differentiation In Vitro of Very Small Embryonic-Like Putative Stem Cells in Adult Mammalian Ovary

The present study was undertaken to detect, characterize, and study differentiation potential of stem cells in adult rabbit, sheep, monkey, and menopausal human ovarian surface epithelium (OSE). Two distinct populations of putative stem cells (PSCs) of variable size were detected in scraped OSE, one being smaller and other similar in size to the surrounding red blood cells in the scraped OSE. The smaller 1-3 μm very small embryonic-like PSCs were pluripotent in nature with nuclear Oct-4 and cell surface SSEA-4, whereas the bigger 4-7 μm cells with cytoplasmic localization of Oct-4 and minimal expression of SSEA-4 were possibly the tissue committed progenitor stem cells. Pluripotent gene transcripts of Oct-4, Oct-4A, Nanog, Sox-2, TERT, and Stat-3 in human and sheep OSE were detected by reverse transcriptase-polymerase chain reaction. The PSCs underwent spontaneous differentiation into oocyte-like structures, parthenote-like structures, embryoid body-like structures, cells with neuronal-like phenotype, and embryonic stem cell-like colonies, whereas the epithelial cells transformed into mesenchymal phenotype by epithelial-mesenchymal transition in 3 weeks of OSE culture. Germ cell markers like c-Kit, DAZL, GDF-9, VASA, and ZP4 were immuno-localized in oocyte-like structures. In conclusion, as opposed to the existing view of OSE being a bipotent source of oocytes and granulosa cells, mammalian ovaries harbor distinct very small embryonic-like PSCs and tissue committed progenitor stem cells population that have the potential to develop into oocyte-like structures in vitro, whereas mesenchymal fibroblasts appear to form supporting granulosa-like somatic cells. Research at the single-cell level, including complete gene expression profiling, is required to further confirm whether postnatal oogenesis is a conserved phenomenon in adult mammals.

Very Small Embryonic-Like Stem Cells: Implications in Reproductive Biology

The most primitive germ cells in adult mammalian testis are the spermatogonial stem cells (SSCs) whereas primordial follicles (PFs) are considered the fundamental functional unit in ovary. However, this central dogma has recently been modified with the identification of a novel population of very small embryonic-like stem cells (VSELs) in the adult mammalian gonads. These stem cells are more primitive to SSCs and are also implicated during postnatal ovarian neo-oogenesis and primordial follicle assembly. VSELs are pluripotent in nature and characterized by nuclear Oct-4A, cell surface SSEA-4, and other pluripotent markers like Nanog, Sox2, and TERT. VSELs are considered to be the descendants of epiblast stem cells and possibly the primordial germ cells that persist into adulthood and undergo asymmetric cell division to replenish the gonadal germ cells throughout life. Elucidation of their role during infertility, endometrial repair, superovulation, and pathogenesis of various reproductive diseases like PCOS, endometriosis, cancer, and so on needs to be addressed. Hence, a detailed review of current understanding of VSEL biology is pertinent, which will hopefully open up new avenues for research to better understand various reproductive processes and cancers. It will also be relevant for future regenerative medicine, translational research, and clinical applications in human reproduction.

Stimulation of ovarian stem cells by follicle stimulating hormone and basic fibroblast growth factor during cortical tissue culture

BackgroundCryopreserved ovarian cortical tissue acts as a source of primordial follicles (PF) which can either be auto-transplanted or cultured in vitro to obtain mature oocytes. This offers a good opportunity to attain biological parenthood to individuals with gonadal insufficiency including cancer survivors. However, role of various intra- and extra-ovarian factors during PF growth initiation still remain poorly understood. Ovarian biology has assumed a different dimension due to emerging data on presence of pluripotent very small embryonic-like stem cells (VSELs) and ovarian germ stem cells (OGSCs) in ovary surface epithelium (OSE) and the concept of postnatal oogenesis. The present study was undertaken to decipher effect of follicle stimulating hormone (FSH) and basic fibroblast growth factor (bFGF) on the growth initiation of PF during organ culture with a focus on ovarian stem cells.MethodsSerum-free cultures of marmoset (n=3) and human (young and peri-menopausal) ovarian cortical tissue pieces were established. Cortical tissue pieces stimulated with FSH (0.5 IU/ml) or bFGF (100 ng/ml) were collected on Day 3 for histological and molecular studies. Gene transcripts specific for pluripotency (Oct-4A, Nanog), early germ cells (Oct-4, c-Kit, Vasa) and to reflect PF growth initiation (oocyte-specific Gdf-9 and Lhx8, and granulosa cells specific Amh) were studied by q-RTPCR.ResultsA prominent proliferation of OSE (which harbors stem cells) and transition of PF to primary follicles was observed after FSH and bFGF treatment. Ovarian stem cells were found to be released on the culture inserts and retained the potential to spontaneously differentiate into oocyte-like structures in extended cultures. q-RTPCR analysis revealed an increased expression of gene transcripts specific for VSELs, OGSCs and early germ cells suggestive of follicular transition.ConclusionThe present study shows that both FSH and bFGF stimulate stem cells present in OSE and also lead to PF growth initiation. Thus besides being a source of PF, cryopreserved ovarian cortical tissue could also be a source of stem cells which retain the ability to spontaneously differentiate into oocyte-like structures in vitro. Results provide a paradigm shift in the basic understanding of FSH action and also offer a new perspective to the field of oncofertility research.

Dynamics associated with spontaneous differentiation of ovarian stem cells in vitro

BackgroundRecent studies suggest that ovarian germ line stem cells replenish oocyte-pool in adult stage, and challenge the central doctrine of ‘fixed germ cell pool’ in mammalian reproductive biology. Two distinct populations of spherical stem cells with high nucleo-cytoplasmic ratio have been recently identified in the adult mammalian ovary surface epithelium (OSE) including nuclear OCT-4A positive very small embryonic-like (VSELs) and cytoplasmic OCT-4 expressing ovarian germ stem cells (OGSCs). Three weeks culture of scraped OSE cells results in spontaneous differentiation of the stem cells into oocyte-like, parthenote-like, embryoid body-like structures and also embryonic stem cell-like colonies whereas epithelial cells attach and transform into a bed of mesenchymal cells. Present study was undertaken, to further characterize ovarian stem cells and to comprehend better the process of spontaneous differentiation of ovarian stem cells into oocyte-like structures in vitro.MethodsOvarian stem cells were enriched by immunomagnetic sorting using SSEA-4 as a cell surface marker and were further characterized. Stem cells and clusters of OGSCs (reminiscent of germ cell nests in fetal ovaries), were characterized by immuno-localization for stem and germ cell specific markers and spontaneous differentiation in OSE cultures was studied by live cell imaging.ResultsDifferential expression of markers specific for pluripotent VSELs (nuclear OCT-4A, SSEA-4, CD133), OGSCs (cytoplasmic OCT-4) primordial germ cells (FRAGILIS, STELLA, VASA) and germ cells (DAZL, GDF-9, SCP-3) were studied. Within one week of culture, stem cells became bigger in size, developed abundant cytoplasm, differentiated into germ cells, revealed presence of Balbiani body-like structure (mitochondrial cloud) and exhibited characteristic cytoplasmic streaming.ConclusionsPresence of germ cell nests, Balbiani body-like structures and cytoplasmic streaming extensively described during fetal ovary development, are indeed well recapitulated during in vitro oogenesis in adult OSE cultures along with characteristic expression of stem/germ cell/oocyte markers. Further studies are required to assess the genetic integrity of in vitro derived oocytes before harnessing their clinical potential. Advance in our knowledge about germ cell differentiation from stem cells will enable researchers to design better in vitro strategies which in turn may have relevance to reproductive biology and regenerative medicine.

Stem cell interaction with somatic niche may hold the key to fertility restoration in cancer patients.

The spontaneous return of fertility after bone marrow transplantation or heterotopic grafting of cryopreserved ovarian cortical tissue has surprised many, and a possible link with stem cells has been proposed. We have reviewed the available literature on ovarian stem cells in adult mammalian ovaries and presented a model that proposes that the ovary harbors two distinct populations of stem cells, namely, pluripotent, quiescent, very small embryonic-like stem cells (VSELs), and slightly larger “progenitor” ovarian germ stem cells (OGSCs). Besides compromising the somatic niche, oncotherapy destroys OGSCs since, like tumor cells, they are actively dividing; however VSELs persist since they are relatively quiescent. BMT or transplanted ovarian cortical tissue may help rejuvenate the ovarian niche, which possibly supports differentiation of persisting VSELs resulting in neo-oogenesis and follicular development responsible for successful pregnancies. Postnatal oogenesis in mammalian ovary from VSELs may be exploited for fertility restoration in cancer survivors including those who were earlier deprived of gametes and/or gonadal tissue cryopreservation options.

Isolation and characterization of stem cells in the adult mammalian ovary.

Female mammals are born with a fixed pool of germ cells, which does not replenish during adult life. However, this has been recently challenged and adult ovaries produce oocytes throughout adult life just like sperm in the testes. Evidence is accumulating on the presence of ovarian stem cells, but the need for robust protocols to isolate, identify, further characterize, and subject them to various functionality tests is essential. Knowledge about the function and potential of ovarian stem cells is well demonstrated by various groups, but their true identity remains elusive because of the variability in the approaches used to identify them by different groups. In order to address this we have made attempts to compile our protocols to isolate, identify, characterize, and culture the stem cells using different animal models including human. Two distinct populations of stem cells exist in adult mammalian ovary, including very small embryonic-like stem cells (VSELs) and the progenitors termed ovarian germ stem cells (OGSCs). VSELs are relatively quiescent and undergo asymmetric cell division to give rise to OGSCs, which divide rapidly, occasionally form germ cell nests and undergo meiosis and differentiation into oocytes, which are surrounded by granulosa cells to assemble as primordial follicles.

VSELs may obviate cryobanking of gonadal tissue in cancer patients for fertility preservation

BackgroundInfertility is an undesirable side effect and gonadal tissue banking is advocated in young cancer patients who are unable to preserve embryos or gametes prior to oncotherapy to achieve biological parenthood later on. Banking gonadal tissue is challenging and protocols to mature gametes in vitro are not yet clinically established. Transplanting ovarian cortical tissue at hetero-or orthotopic sites in women and bone marrow transplantation (BMT) in both men and women has resulted in spontaneous recovery of fertility, pregnancy and live births. Various studies in humans and mice suggest that genetic origin of offspring after BMT is similar to transplanted patient and not the donor. Thus the source of oocytes/sperm which result in spontaneous pregnancies still remains contentious.FindingsVery small embryonic-like stem cells (VSELs) have been reported in adult human testis and ovary, in azoospermic testicular biopsies from survivors of childhood cancer and also in women with premature ovarian failure and menopause. VSELs survive chemotherapy because of their quiescent nature and can be detected in chemoablated mice gonads at protein and mRNA level and also by flow cytometry. Surviving VSELs spontaneously differentiate into oocyte-like structures and sperm when inhibitory factors are overcome in vitro. Transplantation of mesenchymal cells (isolated from different sources) has led to regeneration of chemoablated mouse gonads and also live births. Spermatogenesis is also restored from endogenous stem cells on inter-tubular transplantation of Sertoli cells in chemoablated mouse testis.ConclusionsEndogenous VSELs (which survive oncotherapy) can possibly regenerate non-functional gonads in cancer survivors when exposed to a healthy niche in vitro or in vivo (by way of transplanting mesenchymal cells which secrete trophic factors required for endogenous VSELs to differentiate into gametes). Presence of VSELs can also explain spontaneous pregnancies after BMT and cortical tissue transplantation (at heterotopic or orthotopic sites). This understanding once verified and accepted by the scientific community could obviate the need to remove whole ovary or testicular biopsy for cryopreservation prior to oncotherapy.

Further characterization of adult sheep ovarian stem cells and their involvement in neo-oogenesis and follicle assembly

BackgroundStem cells in the ovary comprise of two distinct populations including very small embryonic-like stem cells (VSELs) and slightly bigger progenitors termed ovarian stem cells (OSCs). They are lodged in ovary surface epithelium (OSE) and are expected to undergo neo-oogenesis and primordial follicle (PF) assembly in adult ovaries. The ovarian stem cells express follicle stimulating hormone (FSH) receptors and are directly activated by FSH resulting in formation of germ cell nests (GCN) in vitro. Present study was undertaken to further characterize adult sheep OSCs and to understand their role during neo-oogenesis and PF assembly.MethodsStem cells were collected by gently scraping the OSE cells and were characterized by H&E staining, immuno-localization, immuno-phenotyping and RT-PCR studies. Expression of FSH receptors and markers specific for stem cells (OCT-4, SSEA-4) and proliferation (PCNA) were studied on stem/progenitor cells in OSE culture and on adult sheep ovarian cortical tissue sections. Effect of FSH on stem cells was also studied in vitro. Asymmetric cell division (ACD) was monitored by studying expression of OCT-4 and NUMB.ResultsAdditional evidence was generated on the presence of two populations of stem cells in the OSE including VSELs and OSCs. FSHR expression was observed on both VSELs and OSCs by immuno-localization and immuno-phenotyping studies. FSH treatment in vitro stimulated VSELs that underwent ACD to self-renew and give rise to OSCs which divided rapidly by symmetric cell divisions (SCD) and clonal expansion with incomplete cytokinesis to form GCN. ACD was further confirmed by differential expression of OCT-4 in VSELs and NUMB in the OSCs. Immuno-histochemical expression of OCT-4, PCNA and FSHR was noted on stem cells located in the OSE in sheep ovarian sections. GCN and cohort of PF were observed in the ovarian cortex and provided evidence in support of neo-oogenesis from the stem cells.ConclusionResults of present study provide further evidence in support of two stem cells populations in adult sheep ovary. Both VSELs, OSCs and GCN express FSH receptors and FSH possibly regulates their function to undergo neo-oogenesis and primordial follicle assembly.

Pluripotent Very Small Embryonic-like Stem Cells in Adult Mammalian Gonads

The presence of very small embryonic-like stem cells (VSELs) in adult mammalian gonads is set to disrupt several existing paradigms in the field of reproductive biology. Being pluripotent, VSELs are present at the top of hierarchy among the tissue-specific stem cells. In the testis, they exist as a sub-population of small spherical cells with high nucleo-cytoplasmic ratio among the spermatogonial stem cells (SSCs) along the basement membrane of the seminiferous tubules. They undergo asymmetric cell division to self-renew, give rise to the SSCs and may also be responsible for the embryonic stem (ES) cell-like colonies observed on culturing testicular biopsy. The SSCs undergo rapid division (clonal expansion as chains), meiosis, and further differentiate into sperm. In the ovary, VSELs are lodged in the ovary surface epithelium (OSE) along with immediate progenitors termed ovarian germ stem cells (OGSCs). Ovarian VSELs also undergo asymmetric cell division to give rise to OGSCs which undergo clonal expansion to form nests (cysts) and further differentiate into oocytes which assemble as primordial follicles below the OSE. Stem cell function in the adult mammalian ovary is modulated by follicle stimulating hormone (FSH) via a novel FSH receptor isoform R3. These results are in contradiction to the existing paradigm that initial primordial follicle growth is independent of FSH and that it acts on the granulosa cells of growing follicles through a G protein-coupled FSH receptor. Being relatively quiescent in nature, VSELs survive oncotherapy in both testis and ovary but are unable to differentiate because of a compromised niche. Their functionality may be restored by providing a healthy niche. This newer understanding of VSELs biology in mammalian gonads will provide deep insight in various fields of reproductive health.