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

Ovarian stem cells: absence of evidence is not evidence of absence

BACKGROUND: Lei and Spradling in a recent study published in PNAS failed to detect ‘germline cysts’ by elegant studies using lineage tracing approach and thus concluded that adult mouse ovaries lack stem cells. They proposed that primordial follicle pool generated during fetal life is sufficient to sustain oogenesis and that there is no renewal of oocytes during adult life. Contrary to their results, we have reported presence of very small pluripotent, embryonic-like stem cells (VSELs), their immediate descendants (OGSCs) and germ cell ‘cysts’ or ‘nests’ (formed by rapid cell division and incomplete cytokinesis) in surface epithelial cell smears of adult sheep, monkey and human ovaries. METHODS: In the present study, ovaries were collected from adult mouse (treated with 5 IU pregnant mare serum gonadotropin, PMSG) and sheep (from slaughter house) and testis from mouse treated with busulphan (25 mg/Kg). Ovarian surface epithelial (OSE) cells and testicular smears were studied for the presence of cysts. Sheep OSE smears were also used to show cytoplasmic continuity amongst the cyst cells studied by immunolocalization and confocal microscopy of stem cells specific markers OCT-4 and SSEA-4. RESULTS: Cysts were observed and confocal microscopy imaging confirmed cytoplasmic continuity amongst the cells comprising the cysts. CONCLUSIONS: Cysts represent self-renewal and clonal expansion of stem cells with incomplete cytokinesis and are a hallmark feature of stem cells. We suggest the use of PMSG stimulated mouse ovaries and use of more primitive markers like OCT-4 or STELLA rather than MVH for lineage tracing studies to conclusively show presence of stem cells by lineage-tracing studies

Follicle stimulating hormone modulates ovarian stem cells through alternately spliced receptor variant FSH-R3

BACKGROUND: We have earlier reported that follicle stimulating hormone (FSH) modulates ovarian stem cells which include pluripotent, very small embryonic-like stem cells (VSELs) and their immediate descendants ‘progenitors’ termed ovarian germ stem cells (OGSCs), lodged in adult mammalian ovarian surface epithelium (OSE). FSH may exert pleiotropic actions through its alternatively spliced receptor isoforms. Four isoforms of FSH receptors (FSHR) are reported in literature of which FSH-R1 and FSH-R3 have biological activity. Present study was undertaken to identify FSHR isoforms mediating FSH action on ovarian stem cells, using sheep OSE cells culture as the study model. METHODS: Cultures of sheep OSE cells (a mix of epithelial cells, VSELs, OGSCs and few contaminating red blood cells) were established with and without FSH 5IU/ml treatment. Effect of FSH treatment on self-renewal of VSELs and their differentiation into OGSCs was studied after 15 hrs by qRT-PCR using markers specific for VSELs (Oct-4A, Sox-2) and OGSCs (Oct-4). FSH receptors and its specific transcripts (R1 and R3) were studied after 3 and 15 hrs of FSH treatment by immunolocalization, in situ hybridization and qRT-PCR. FSHR and OCT-4 were also immuno-localized on sheep ovarian sections, in vitro matured follicles and early embryos. RESULTS: FSH treatment resulted in increased stem cells self-renewal and clonal expansion evident by the appearance of stem cell clusters. FSH receptors were expressed on ovarian stem cells whereas the epithelial cells were distinctly negative. An increase in R3 mRNA transcripts was noted after 3 hrs of FSH treatment and was reduced to basal levels by 15 hrs, whereas R1 transcript expression remained unaffected. Both FSHR and OCT-4 were immuno-localized in nuclei of stem cells, showed nuclear or ooplasmic localization in oocytes of primordial follicles and in cytoplasm of granulosa cells in growing follicles. CONCLUSIONS: FSH modulates ovarian stem cells via FSH-R3 to undergo potential self-renewal, clonal expansion as ‘cysts’ and differentiation into oocytes. OCT-4 and FSHR proteins (required initially to maintain pluripotent state of VSELs and for FSH action respectively) gradually shift from nuclei to cytoplasm of developing oocytes and are later possibly removed by surrounding granulosa cells as the oocyte prepares itself for fertilization

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

Endogenous, very small embryonic-like stem cells: Critical review, therapeutic potential and a look ahead

© The Author 2016. BACKGROUND: Both pluripotent very small embryonic-like stem cells (VSELs) and induced pluripotent stem (iPS) cells were reported in 2006. In 2012, a Nobel Prize was awarded for iPS technology whereas even today the very existence of VSELs is not well accepted. The underlying reason is that VSELs exist in low numbers, remain dormant under homeostatic conditions, are very small in size and do not pellet down at 250-280g. The VSELs maintain life-long tissue homeostasis, serve as a backup pool for adult stem cells and are mobilized under stress conditions. An imbalance in VSELs function (uncontrolled proliferation) may result in cancer. SEARCH METHODS: The electronic database \u27Medline/Pubmed\u27 was systematically searched with the subject heading term \u27very small embryonic-like stem cells\u27. OBJECTIVE AND RATIONALE: The most primitive stem cells that undergo asymmetric cell divisions to self-renew and give rise to progenitors still remain elusive in the hematopoietic system and testes, while the presence of stem cells in ovary is still being debated. We propose to review the available literature on VSELs, the methods of their isolation and characterization, their ontogeny, how they compare with embryonic stem (ES) cells, primordial germ cells (PGCs) and iPS cells, and their role in maintaining tissue homeostasis. The review includes a look ahead on how VSELs will result in paradigm shifts in basic reproductive biology. OUTCOMES: Adult tissue-specific stem cells including hematopoietic, spermatogonial, ovarian and mesenchymal stem cells have good proliferation potential and are indeed committed progenitors (with cytoplasmic OCT-4), which arise by asymmetric cell divisions of pluripotent VSELs (with nuclear OCT-4). VSELs are the most primitive stem cells and postulated to be an overlapping population with the PGCs. Rather than migrating only to the gonads, PGCs migrate and survive in various adult body organs throughout life as VSELs. VSELs express both pluripotent and PGC-specific markers and are epigenetically and developmentally more mature compared with ES cells obtained from the inner cell mass of a blastocyst-stage embryo. As a result, VSELs readily differentiate into three embryonic germ layers and spontaneously give rise to both sperm and oocytes in vitro. Like PGCs, VSELs do not divide readily in culture, nor produce teratoma or integrate in the developing embryo. But this property of being relatively quiescent allows endogenous VSELs to survive various kinds of toxic insults. VSELs that survive oncotherapy can be targeted to induce endogenous regeneration of non-functional gonads. Transplanting healthy niche (mesenchymal) cells have resulted in improved gonadal function and live births. WIDER IMPLICATIONS: Being quiescent, VSELs possibly do not accumulate genomic (nuclear or mitochondrial) mutations and thus may be ideal endogenous, pluripotent stem cell candidates for regenerative and reproductive medicine. The presence of VSELs in adult gonads and the fact that they survive oncotherapy may obviate the need to bank gonadal tissue for fertility preservation prior to oncotherapy. VSELs and their ability to undergo spermatogenesis/neo-oogenesis in the presence of a healthy niche will help identify newer strategies toward fertility restoration in cancer survivors, delaying menopause and also enabling aged mothers to have better quality eggs

Tumor Microenvironment Enriches the Stemness Features: The Architectural Event of Therapy Resistance and Metastasis

Cancer divergence has many facets other than being considered a genetic term. It is a tremendous challenge to understand the metastasis and therapy response in cancer biology; however, it postulates the opportunity to explore the possible mechanism in the surrounding tumor environment. Most deadly solid malignancies are distinctly characterized by their tumor microenvironment (TME). TME consists of stromal components such as immune, inflammatory, endothelial, adipocytes, and fibroblast cells. Cancer stem cells (CSCs) or cancer stem-like cells are a small sub-set of the population within cancer cells believed to be a responsible player in the self-renewal, metastasis, and therapy response of cancer cells. The correlation between TME and CSCs remains an enigma in understanding the events of metastasis and therapy resistance in cancer biology. Recent evidence suggests that TME dictates the CSCs maintenance to arbitrate cancer progression and metastasis. The immune, inflammatory, endothelial, adipocyte, and fibroblast cells in the TME release growth factors, cytokines, chemokines, microRNAs, and exosomes that provide cues for the gain and maintenance of CSC features. These intricate cross-talks are fueled to evolve into aggressive, invasive, migratory phenotypes for cancer development. In this review, we have abridged the recent developments in the role of the TME factors in CSC maintenance and how these events influence the transition of tumor progression to further translate into metastasis and therapy resistance in cancer

Elevated PAF1-RAD52 Axis Confers Chemoresistance to Human Cancers

Cisplatin- and gemcitabine-based chemotherapeutics represent a mainstay of cancer therapy for most solid tumors; however, resistance limits their curative potential. Here, we identify RNA polymerase II-associated factor 1 (PAF1) as a common driver of cisplatin and gemcitabine resistance in human cancers (ovarian, lung, and pancreas). Mechanistically, cisplatin- and gemcitabine-resistant cells show enhanced DNA repair, which is inhibited by PAF1 silencing. We demonstrate an increased interaction of PAF1 with RAD52 in resistant cells. Targeting the PAF1 and RAD52 axis combined with cisplatin or gemcitabine strongly diminishes the survival potential of resistant cells. Overall, this study shows clinical evidence that the expression of PAF1 contributes to chemotherapy resistance and worse clinical outcome for lethal cancers

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

Abstract Background Stem 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. Methods Stem 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. Results Additional 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. Conclusion Results 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

Making gametes from alternate sources of stem cells: past, present and future

Abstract Infertile couples including cancer survivors stand to benefit from gametes differentiated from embryonic or induced pluripotent stem (ES/iPS) cells. It remains challenging to convert human ES/iPS cells into primordial germ-like cells (PGCLCs) en route to obtaining gametes. Considerable success was achieved in 2016 to obtain fertile offspring starting with mouse ES/iPS cells, however the specification of human ES/iPS cells into PGCLCs in vitro is still not achieved. Human ES cells will not yield patient-specific gametes unless and until hES cells are derived by somatic cell nuclear transfer (therapeutic cloning) whereas iPS cells retain the residual epigenetic memory of the somatic cells from which they are derived and also harbor genomic and mitochondrial DNA mutations. Thus, they may not be ideal starting material to produce autologus gametes, especially for aged couples. Pluripotent, very small embryonic-like stem cells (VSELs) have been reported in adult tissues including gonads, are relatively quiescent in nature, survive oncotherapy and can be detected in aged, non-functional gonads. Being developmentally equivalent to PGCs (natural precursors to gametes), VSELs spontaneously differentiate into gametes in vitro. It is also being understood that gonadal stem cells niche is compromised by oncotherapy and with age. Improving the gonadal somatic niche could regenerate non-functional gonads from endogenous VSELs to restore fertility. Niche cells (Sertoli/mesenchymal cells) can be directly transplanted and restore gonadal function by providing paracrine support to endogenous VSELs. This strategy has been successful in several mice studies already and resulted in live birth in a woman with pre-mature ovarian failure