Dynamics associated with spontaneous differentiation of ovarian stem cells in vitro

Abstract

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.

Mouse Ovarian Very Small Embryonic-Like Stem Cells Resist Chemotherapy and Retain Ability to Initiate Oocyte-Specific Differentiation

“…Based on these results and our earlier studies, 2,17,18 we propose in mouse ovary that small nuclear OCT-4 positive cells represent VSELs, while the slightly larger cytoplasmic OCT-4 positive cells represent OGSCs. To further confirm that VSELs are present in mouse ovary, flow cytometry experiment based on SCA-1þ/LinÀ/CD45À expression unique to VSELs 24 was carried out.…”

“…More explanations for existing criticisms against the concept of ovarian stem cells and postnatal oogenesis were recently reviewed. 15,16 Our group has been working in the area of gonadal stem cells, and we have shown that they include 2 distinct populations, namely, the relatively ''quiescent'' very small embryonic-like stem cells (VSELs) and the more committed ''active'' progenitor stem cells, which include ovary germ stem cells (OGSCs) in ovary 2,17 and spermatogonial stem cells in testis. 18 The concept is very similar to the presence of quiescent and active stem cell population proposed in other organs like gut, skin, and bone marrow.…”

“…2,29 Our detailed protocols to isolate both the population of ovarian stem cells from adult mammalian ovaries are published recently. 30 Our group has shown that ovarian stem cells (1) spontaneously differentiate into oocyte-like structures in culture; 2 (2) exhibit process of cytoplasmic streaming, germ cell nest, and Balbiani body formation in vitro 17 ; (3) express follicle-stimulating hormone receptors (FSHRs) and are modulated by FSH and bFGF. 31,32 We have also shown that the alternative FSHR isoform-FSHR3 (a growth factor type 1 receptor) 33 is involved in the activation of ovarian stem cells by FSH and results in germ cell differentiation 31,34 and pregnant mare serum gonadotropin (PMSG-a FSH analog) treatment augments the process of neo-oogenesis and primordial follicle assembly in mice.…”

FSH–FSHR3–stem cells in ovary surface epithelium: basis for adult ovarian biology, failure, aging, and cancer

“…Similarly, others have also described the presence of stem cells in post-menopausal women OSE, which differentiate into oocytes and parthenote embryos in culture (Virant-Klun & Skutella 2010). Various events such as formation of germ cell nests, Balbiani body-like structures, and cytoplasmic streaming, which are extensively studied in fetal ovary, are recapitulated during in vitro oogenesis in adult OSE cultures along with characteristic expression of stem/ germ cell/oocyte markers (Parte et al 2014).…”

Embryonic Stem Cells and Infertility

“…The authors in a separate study reported that they observed the formation of germ cell nests and Balbiani bodylike structures during fetal ovary development. 51 In another recent study by Sriraman et al, 52 VSELs were demonstrated to survive chemotherapy in mouse ovaries.…”