Stem Cells in the Mammalian Gonads.

Besides spermatogonial stem cells (SSCs) and ovarian stem cells (OSCs), a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) has been reported in both adult mouse and human testes and ovaries. VSELs and SSCs/OSCs are developmentally linked to each other. VSELs are relatively quiescent, small-sized stem cells that undergo asymmetrical cell divisions (ACDs) whereby they self-renew and give rise to the slightly bigger SSCs/OSCs which in turn undergo symmetrical cell divisions (SCDs) and clonal expansion to form germ cell chains/nests before further differentiation into gametes. Comparison of VSELs and SSCs/OSCs for their potential to differentiate into sperm/oocytes is irrelevant since VSELs only undergo ACD to give rise to SSCs/OSCs that further differentiate into gametes. Being relatively quiescent, VSELs survive oncotherapy and can be manipulated to regenerate nonfunctional gonads of cancer survivors, and thus there is possibly no need to bank testicular/ovarian tissue prior to oncotherapy. Being developmentally linked to the primordial germ cells (PGCs) which are the natural precursors to the gametes, VSELs differentiate into haploid sperm/oocyte-like structures in vitro when cultured on appropriate feeder support, in the absence of a cocktail of growth factors. VSELs express receptors for pituitary and sex hormones (FSHR, ER) and thus get directly stimulated/affected by their circulating levels. Excessive self-renewal of VSELs in the gonads may initiate testicular and ovarian cancers. To conclude, VSELs can be targeted to regenerate the gonads of patients with gonadal insufficiency including cancer survivors and are excellent candidates to differentiate into gametes in vitro.

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

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.

Chemoablated mouse seminiferous tubular cells enriched for very small embryonic-like stem cells undergo spontaneous spermatogenesis in vitro.

BACKGROUND: Extensive research is ongoing to empower cancer survivors to have biological parenthood. For this, sperm are cryopreserved prior to therapy and in younger children testicular biopsies are cryopreserved with a hope to mature the germ cells into sperm later on for assisted reproduction. In addition, lot of hope was bestowed on pluripotent embryonic and induced pluripotent stem cells to differentiate into sperm and oocytes. However, obtaining functional gametes from pluripotent stem cells still remains a distant dream and major bottle-neck appears to be their inefficient differentiation into primordial germ cells (PGCs). There exists yet another population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) in adult body organs including gonads. We have earlier reported that busulphan (25 mg/Kg) treatment to 4 weeks old mice destroys actively dividing cells and sperm but VSELs survive and differentiate into sperm when a healthy niche is provided in vivo. METHODS: Mouse testicular VSELs that survived busulphan treatment were cultured for 3 weeks. A mix of surviving cells in seminiferous tubules (VSELs, possibly few spermatogonial stem cells and Sertoli cells) were cultured using Sertoli cells conditioned medium containing fetal bovine serum, follicle stimulating hormone and with no additional growth factors. RESULTS: Stem cells underwent proliferation and clonal expansion in culture and spontaneously differentiated into sperm whereas Sertoli cells attached and provided a somatic support. Transcripts specific for various stages of spermatogenesis were up-regulated by qRT-PCR studies on day 7 suggesting VSELs (Sca1) and SSCs (Gfra) proliferate (Pcna), undergo spermatogenesis (spermatocyte specific marker prohibitin), meiosis (Scp3) and differentiate into sperm (post-meiotic marker protamine). CONCLUSIONS: Process of spermatogenesis and spermiogenesis was replicated in vitro starting with testicular cells that survived busulphan treatment. We have earlier reported similar ability of ovarian VSELs enriched in the ovary surface epithelial cells to form oocyte-like structures in vitro. This striking potential of spontaneous differentiation of primitive testicular cells including VSELs that survive chemotherapy is being described for the first time in the present study.

Effect of Aging and 5-Fluorouracil Treatment on Bone Marrow Stem Cell Dynamics.

Lifelong homeostasis of bone marrow is maintained by the resident stem cells that include the quiescent very small embryonic-like stem cells (VSELs) and lineage restricted, tissue committed 'progenitors' hematopoietic stem cells (HSCs). Niche providing mesenchymal stromal cells (MSCs) regulate the function of VSELs/HSCs by providing crucial paracrine support. Any dysfunction of stem cells and/or their niche leads to disease state. The stem cells biology gets affected with age leading to a myeloid bias in differentiation of HSCs and increased incidence of myeloid leukemia. Present study was undertaken to enumerate VSELs, HSCs and MSCs and evaluate their response on D4 and D10 after chemotherapy with 5-Fluorouracil (5-FU) in young and aged mouse bone marrow. Stem cells were activated in response to 5-FU induced stress in an attempt to restore homeostasis. Although absolute numbers of VSELs and HSCs did not differ much between young and aged mice, their tendency to proliferate was higher on D4 in aged mice. However, ability to revert back to basal numbers and their differentiation was affected on D10 in aged marrow. Stem cells from aged bone marrow showed greater ability to form CFUs on D10 after 5-FU treatment. CD44 positive aged MSCs also showed increased proliferation on D10. Transplanting MSCs from young mice in 5-FU treated aged marrow helped improve hematopoiesis. The results suggest that no significant intrinsic changes occur as proliferative ability of stem cells remains unaffected but the niche gets affected with age leading to excessive self-renewal and compromised differentiation. This may explain increased incidence of leukemia with age.

Altered Biology of Testicular VSELs and SSCs by Neonatal Endocrine Disruption Results in Defective Spermatogenesis, Reduced Fertility and Tumor Initiation in Adult Mice.

Reproductive health of men has declined in recent past with reduced sperm count and increased incidence of infertility and testicular cancers mainly attributed to endocrine disruption in early life. Present study aims to evaluate whether testicular stem cells including very small embryonic-like stem cells (VSELs) and spermatogonial stem cells (SSCs) get affected by endocrine disruption and result in pathologies in adult life. Effect of treatment on mice pups with estradiol (20 µg on days 5-7) and diethylstilbestrol (DES, 2 µg on days 1-5) was studied on VSELs, SSCs and spermatogonial cells in adult life. Treatment affected spermatogenesis, tubules in Stage VIII & sperm count were reduced along with reduction of meiotic (4n) cells and markers (Prohibitin, Scp3, Protamine). Enumeration of VSELs by flow cytometry (2-6 µm, 7AAD-, LIN-CD45-SCA-1+) and qRT-PCR using specific transcripts for VSELs (Oct-4a, Sox-2, Nanog, Stella, Fragilis), SSCs (tOct-4, Gfra-1, Gpr-125) and early germ cells (Mvh, Dazl) showed several-fold increase but transition from c-Kit negative to c-Kit positive spermatogonial cells was blocked on D100 after treatment. Transcripts specific for apoptosis (Bcl2, Bax) remained unaffected but tumor suppressor (p53) and epigenetic regulator (NP95) transcripts showed marked disruption. 9 of 10 mice exposed to DES showed tumor-like changes. To conclude, endocrine disruption resulted in a tilt towards excessive self-renewal of VSELs (leading to testicular cancer after DES treatment) and blocked differentiation (reduced numbers of c-Kit positive cells, meiosis, sperm count and fertility). Understanding the underlying basis for infertility and cancer initiation from endogenous stem cells through murine modelling will hopefully improve human therapies in future.

Mouse Bone Marrow VSELs Exhibit Differentiation into Three Embryonic Germ Lineages and Germ & Hematopoietic Cells in Culture.

Very small embryonic-like stem cells (VSELs) have been reported in various adult tissues, express pluripotent and primordial germ cells (PGCs) specific markers, are mobilized under stress/disease conditions, give rise to tissue committed progenitors and thus help regenerate and maintain homeostasis. The aim of the present study was to evaluate in vitro differentiation potential of VSELs using a quantitative approach. VSELs were collected from mouse bone marrow after 4 days of 5-fluorouracil (5-FU, 150 mg/Kg) treatment, further enriched by size based filtration and cultured on a feeder support in the presence of specific differentiation media. Cultured VSELs were found to differentiate into all three embryonic germ cell lineages, germ and hematopoietic cells after 14 days in culture. This was confirmed by studying Nestin, PDX-1, NKX2.5, DAZL, CD45 and other markers expression by various approaches. Very small, CD45 negative cells collected and enriched from GFP positive 5-FU treated mice bone marrow transitioned into CD45 positive cells in vitro thus demonstrating that VSELs can give rise to hematopoietic stem cells (HSCs). We envision that VSELs may be responsible for plasticity and ability of bone marrow cells to give rise to non-hematopoietic tissue progenitors of all 3 germ layers. Moreover the ability of VSELs to differentiate into germ cells as well as all the three lineages provides further evidence to support their pluripotent state and confirms developmental link between bone marrow VSELs and PGCs. The property of quiescence, no risk of teratoma formation and autologus source, make pluripotent VSELs a potential candidate to facilitate endogenous regeneration compared to cell replacement strategy envisioned using embryonic and induced pluripotent stem cells.

Underlying Mechanisms that Restore Spermatogenesis on Transplanting Healthy Niche Cells in Busulphan Treated Mouse Testis.

Very small embryonic-like stem cells (VSELs) exist among spermatogonial stem cells and survive chemotherapy in both mice and human testes because of their relatively quiescent nature. Our earlier study revealed that inter-tubular transplantation of niche (Sertoli or bone marrow derived mesenchymal) cells can restore spermatogenesis from endogenous surviving VSELs. Present study was undertaken to delineate the effect of busulphan on testicular stem/germ/Sertoli cells and to comprehend the underlying mechanisms of how transplanted niche cells restore spermatogenesis. Ploidy analysis showed an increase in diploid cells on D7 and VSELs (2-6 µm; LIN-/CD45-/SCA-1+) were detected at all time-points studied and were maximum on D15 after busulphan treatment. They were visualized in cell smears, expressed nuclear NANOG and SOX2 and BrdU uptake on D15 suggested they were proliferating in response to stress induced by busulphan. Verapamil-sensitive side population detected comprised SCA-1 positive stem cells (5 ± 0.02 % in normal and 8.6 ± 2.02 % in chemoablated testis). Adverse effects of busulphan on Sertoli cells by transcriptome analysis included altered expression of Gdnf, Scf, Fgf, Bmp4, androgen binding protein, components of blood-testis-barrier and also stem cells related signaling pathways including Wnt. GFP positive transplanted cells aligned themselves as 'neo-tubules' and were visualized adjacent to 'native' germ cells depleted tubules. 'Neo-tubules' provide paracrine support to endogenous VSELs to undergo spermatogenesis. Quantitative analysis was done to track proliferation (PCNA) and differentiation (MVH) of stem cells by immuno-localization studies at different time intervals. Results provide an alternative strategy to restore spermatogenesis in cancer survivors from endogenous stem cells which needs to be further researched.

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

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 'Medline/Pubmed' was systematically searched with the subject heading term 'very small embryonic-like stem cells'. 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.

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

BACKGROUND: Infertility 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. FINDINGS: Very 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. CONCLUSIONS: Endogenous 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.

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

This study was undertaken to investigate stem cells in adult mouse ovary, the effect of chemotherapy on them and their potential to differentiate into germ cells. Very small embryonic-like stem cells (VSELs) that were SCA-1+/Lin-/CD45-, positive for nuclear octamer-binding transforming factor 4 (OCT-4), Nanog, and cell surface stage-specific embryonic antigen 1, were identified in adult mouse ovary. Chemotherapy resulted in complete loss of follicular reserve and cytoplasmic OCT-4 positive progenitors (ovarian germ stem cells) but VSELs survived. In ovarian surface epithelial (OSE) cell cultures from chemoablated ovary, proliferating germ cell clusters and mouse vasa homolog/growth differentiation factor 9-positive oocyte-like structure were observed by day 6, probably arising as a result of differentiation of the surviving VSELs. Follicle-stimulating hormone (FSH) exerted a direct stimulatory action on the OSE and induced stem cells proliferation and differentiation into premeiotic germ cell clusters during intact chemoablated ovaries culture. The FSH analog pregnant mare serum gonadotropin treatment to chemoablated mice increased the percentage of surviving VSELs in ovary. The results of this study provide evidence for the presence of potential VSELs in mouse ovaries and show that they survive chemotherapy, are modulated by FSH, and retain the ability to undergo oocyte-specific differentiation. These results show relevance to women who undergo premature ovarian failure because of oncotherapy.

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.

Quiescent very small embryonic-like stem cells resist oncotherapy and can restore spermatogenesis in germ cell depleted mammalian testis.

Adult mouse and human testes harbor relatively quiescent, pluripotent very small embryonic-like stem cells (VSELs), in addition to actively dividing spermatogonial stem cells (SSCs). Here we report that various oncotherapy regimens in human cancer patients (n=7) and busulphan treatment (25mg/Kg body weight) in eight weeks old male mice (n=15) selectively affects actively dividing SSCs, spermatogonia, haploid germ cells and somatic microenvironment resulting in germ cell aplasia, whereas VSELs are unaffected and persist in otherwise germ cell depleted testis. Testicular VSELs are 2-5 µm in size, have high nucleo-cytoplasmic ratio, SCA-1+/CD45-/LIN- (mice), CD133+/CD45-/LIN- (human survivors of childhood cancer) and express various pluripotent transcripts including OCT-4A. SCA-1 sorted cells from busulphan treated mice testes in vitro formed small clusters suggestive of self-renewal and differentiation into progenitors, which divide rapidly. Inter-tubular random injections of syngeneic Sertoli cells (105 cells per testis, n=14) or bone marrow derived mesenchymal cells (104 cells per testis, n=16) into the germ cell depleted busulphan treated mice testes, were able to restore spermatogenesis from persisting VSELs. Transplanted Sertoli or mesenchymal cells possibly were a source of growth factors essential for VSELs differentiation. Since sperm formation occurred in situ, various epigenetic concerns associated with the 'synthetic gametes' may be eliminated in our approach. Ability of mesenchymal cells to restore spermatogenesis may benefit existing azoospermic survivors of childhood cancer who were otherwise deprived of testicular tissue cryopreservation prior to oncotherapy. Further studies are warranted to delineate the underlying mechanisms and to study quality and potential of sperm generated by this approach.