Multiple studies using single-cell RNA sequencing (scRNAseq) have failed to detect stem cells in ... more Multiple studies using single-cell RNA sequencing (scRNAseq) have failed to detect stem cells in adult ovaries. We have maintained that two populations of ovarian stem cells including pluripotent, very small embryonic-like stem cells (VSELs) and tissue-committed ‘progenitors’ termed ovarian stem cells (OSCs) can easily be detected in Hematoxylin and Eosin-stained ovary surface epithelial (OSE) cells smears prepared from both mice and human ovaries. Most likely the stem cells never get subjected to scRNAseq since they pellet down only by centrifuging cells suspension at 1000 g while cells for scRNAseq were invariably prepared by centrifuging at 200-400 g. A recent article provided further explanation for the failure of scRNAseq to detect ovarian stem cells. Extensive reanalysis of data (generated by scRNAseq) using an advanced software successfully detected OSCs and meiotic markers supporting neo-oogenesis in adult human ovaries. But this article remained critical on the biological r...
The concept of dedifferentiation and reprogramming of mature somatic cells holds much promise for... more The concept of dedifferentiation and reprogramming of mature somatic cells holds much promise for the three-front “war” against tissue damage, cancer, and aging. It was hoped that reprogramming human somatic cells into the induced pluripotent state, along with the use of embryonic stem cells, would transform regenerative medicine. However, despite global efforts, clinical applications remain a distant dream, due to associated factors such as genomic instability, tumorigenicity, immunogenicity, and heterogeneity. Meanwhile, the expression of embryonic (pluripotent) markers in multiple cancers has baffled the scientific community, and it has been suggested that somatic cells dedifferentiate and “reprogram” into the pluripotent state in vivo to initiate cancer. It has also been suggested that aging can be reversed by partial reprogramming in vivo. However, better methods are needed; using vectors or Yamanaka factors in vivo, for example, is dangerous, and many potential anti-aging ther...
Background Fertility preservation and restoration in cancer patients/survivors is the need of pre... more Background Fertility preservation and restoration in cancer patients/survivors is the need of present times when increased numbers of patients get cured of cancer but face infertility as a serious side effect. Resveratrol has beneficial effects on chemoablated ovaries and testes in mice but has failed to enter the clinics because of extremely poor bioavailability. The present study was undertaken to evaluate the protective and curative effects of Extremely active Resveratrol (XAR™)- a nano-formulation of resveratrol with significantly improved bioavailability- on mouse ovary and testis after chemotherapy. Effects of XAR™ and FSH were compared on stimulation of follicle growth in adult mice ovaries. XAR™ (25 mg/kg) was administered for two days prior to chemotherapy to study the protective effects on the mouse gonads. XAR™ was also administered for 14 days post chemoablation to study the regenerative effects. Besides effect on numbers of primordial and growing follicles and spermatog...
Dystrophin gene expression during cardiac differentiation of KIND1 hES cells on days 0, 12, and ... more Dystrophin gene expression during cardiac differentiation of KIND1 hES cells on days 0, 12, and 20 during cardiac differentiation of KIND1 hES cell line. Expression of Dystrophin increased in cardiac progenitors and cardiomyocytes compared to undifferentiated KIND1 cells. Results in agreement with earlier reports in DOT1L conditional knockout mice heart concluding Dystrophin as a direct target of DOT1L [35]. Error bars represent ¹SEM. (PDF 329 kb)
A big divide was created among stem cell biologists because of ethical issues associated with hum... more A big divide was created among stem cell biologists because of ethical issues associated with human embryonic stem cells (hESCs). Adult stem cell biologists observed that hematopoietic stem cells (HSCs) in bone marrow possess considerable “plasticity” and could replace hESCs for regenerative medicine. As a result several trials using autologous bone marrow cells were undertaken; however, no significant success has been achieved. Human ESCs and induced pluripotent stem (iPS) cells tend to give rise to fetal counterparts and thus may not regenerate adult organs efficiently. Another novel population of pluripotent stem cells has also been reported in various adult organs termed very small embryonic-like stem cells (VSELs). VSELs explain the “plasticity” of the bone marrow/cord blood cells and have better differentiation potential compared to ES/iPS cells. Present article is an effort to make a case for pluripotent VSELs as ideal stem cell candidate for endogenous regeneration of diseas...
Adult mammalian heart is considered to be one of the least regenerative organs as it is not able ... more Adult mammalian heart is considered to be one of the least regenerative organs as it is not able to initiate endogenous regeneration in response to injury unlike in lower vertebrates and neonatal mammals. Evidence is now accumulating to suggest normal renewal and replacement of cardiomyocytes occurs even in middle-aged and old individuals. But underlying mechanisms leading to this are not yet clear. Do tissue-resident stem cells exist or somatic cells dedifferentiate leading to regeneration? Lot of attention is currently being focused on epicardium as it is involved in cardiac development, lodges multipotent progenitors and is a source of growth factors. Present study was undertaken to study the presence of stem cells in the pericardium. Intact adult mouse heart was subjected to partial enzymatic digestion to collect the pericardial cells dislodged from the surface. Pericardial cells suspension was processed to enrich the stem cells using our recently published protocol. Two populat...
Objective True identity and specific set of markers to enrich endometrial stem cells still remain... more Objective True identity and specific set of markers to enrich endometrial stem cells still remains elusive. Present study was undertaken to further substantiate that very small embryonic-like stem cells (VSELs) are the true and elusive stem cells in adult mice endometrium. Methods This was achieved by undertaking three sets of experiments. Firstly, SSEA-1+ and Oct-4 + positive VSELs, sorted from GFP mice, were transplanted into the uterine horns of wild-type Swiss mice and GFP uptake was studied within the same estrus cycle. Secondly, uterine lumen was scratched surgically and OCT-4 expressing stem/progenitor cells were studied at the site of injury after 24–72 h. Thirdly, OCT-4 expression was studied in the endometrium and myometrium of adult mice after neonatal exposure to estradiol (20 µg/pup/day on days 5–7 after birth). Results GFP + ve VSELs expressing SSEA-1 and Oct-4 engrafted and differentiated into the epithelial cells lining the lumen as well as the glands during the est...
Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduct... more Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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 repl...
Present study aims to describe a simple and robust protocol to delineate the presence of pluripot... more Present study aims to describe a simple and robust protocol to delineate the presence of pluripotent, very small embryonic-like stem cells (VSELs) in addition to spermatogonial stem cells (SSCs) in adult mouse testes. Testicular seminiferous tubules were subjected to enzymatic dissociation to obtain single cells suspension. Stem cells were enriched by spinning at different speeds wherein majority of somatic cells were pelleted at 1000 rpm (250 g, Pellet A) and putative stem cells by spinning the supernatant (obtained after separating Pellet A) at 3000 rpm (1000 g, Pellet B). Viable (7AAD-ve), 2–6 μm, LIN-CD45-SCA-1+ VSELs were studied after doublets exclusion by flow cytometry in both Pellets A & B. Almost ten-fold enrichment of VSELs was obtained in Pellet B (0.27 + 0.05%) compared to Pellet A (0.03 + 0.003%). SCA-1 expressing SSCs (>6 μm, 0.18 + 0.06%) were clearly distinguished from VSELs (2–6 μm, 0.07 + 0.003%) by flow cytometry studies on total testicular cells suspension collected by spinning at 3000 rpm. Enriched stem cells from Pellet B were used to study expression of OCT-4, NANOG, SCA-1, SSEA-1, LIFR, GFRa, c-KIT, ERα and ERβ. Cells in Pellet B were also subjected to RT-PCR to study pluripotent (Oct-4a, Sox2, Nanog), primordial germ cells (Stella, Fragilis), SSCs (Oct-4) and estrogen receptors (ERα and ERβ) specific transcripts. qRT-PCR analysis showed >2 folds up-regulation of stem cell markers in Pellet B (Oct-4A, Oct-4, Sox2, Nanog) compared to Pellet A. To conclude, spinning at higher speed led to successful enrichment of pluripotent VSELs from testes which have remained ignored till now. Expression of ERα & β on VSELs/SSCs makes them vulnerable to endocrine disruption.
Multiple studies using single-cell RNA sequencing (scRNAseq) have failed to detect stem cells in ... more Multiple studies using single-cell RNA sequencing (scRNAseq) have failed to detect stem cells in adult ovaries. We have maintained that two populations of ovarian stem cells including pluripotent, very small embryonic-like stem cells (VSELs) and tissue-committed ‘progenitors’ termed ovarian stem cells (OSCs) can easily be detected in Hematoxylin and Eosin-stained ovary surface epithelial (OSE) cells smears prepared from both mice and human ovaries. Most likely the stem cells never get subjected to scRNAseq since they pellet down only by centrifuging cells suspension at 1000 g while cells for scRNAseq were invariably prepared by centrifuging at 200-400 g. A recent article provided further explanation for the failure of scRNAseq to detect ovarian stem cells. Extensive reanalysis of data (generated by scRNAseq) using an advanced software successfully detected OSCs and meiotic markers supporting neo-oogenesis in adult human ovaries. But this article remained critical on the biological r...
The concept of dedifferentiation and reprogramming of mature somatic cells holds much promise for... more The concept of dedifferentiation and reprogramming of mature somatic cells holds much promise for the three-front “war” against tissue damage, cancer, and aging. It was hoped that reprogramming human somatic cells into the induced pluripotent state, along with the use of embryonic stem cells, would transform regenerative medicine. However, despite global efforts, clinical applications remain a distant dream, due to associated factors such as genomic instability, tumorigenicity, immunogenicity, and heterogeneity. Meanwhile, the expression of embryonic (pluripotent) markers in multiple cancers has baffled the scientific community, and it has been suggested that somatic cells dedifferentiate and “reprogram” into the pluripotent state in vivo to initiate cancer. It has also been suggested that aging can be reversed by partial reprogramming in vivo. However, better methods are needed; using vectors or Yamanaka factors in vivo, for example, is dangerous, and many potential anti-aging ther...
Background Fertility preservation and restoration in cancer patients/survivors is the need of pre... more Background Fertility preservation and restoration in cancer patients/survivors is the need of present times when increased numbers of patients get cured of cancer but face infertility as a serious side effect. Resveratrol has beneficial effects on chemoablated ovaries and testes in mice but has failed to enter the clinics because of extremely poor bioavailability. The present study was undertaken to evaluate the protective and curative effects of Extremely active Resveratrol (XAR™)- a nano-formulation of resveratrol with significantly improved bioavailability- on mouse ovary and testis after chemotherapy. Effects of XAR™ and FSH were compared on stimulation of follicle growth in adult mice ovaries. XAR™ (25 mg/kg) was administered for two days prior to chemotherapy to study the protective effects on the mouse gonads. XAR™ was also administered for 14 days post chemoablation to study the regenerative effects. Besides effect on numbers of primordial and growing follicles and spermatog...
Dystrophin gene expression during cardiac differentiation of KIND1 hES cells on days 0, 12, and ... more Dystrophin gene expression during cardiac differentiation of KIND1 hES cells on days 0, 12, and 20 during cardiac differentiation of KIND1 hES cell line. Expression of Dystrophin increased in cardiac progenitors and cardiomyocytes compared to undifferentiated KIND1 cells. Results in agreement with earlier reports in DOT1L conditional knockout mice heart concluding Dystrophin as a direct target of DOT1L [35]. Error bars represent ¹SEM. (PDF 329 kb)
A big divide was created among stem cell biologists because of ethical issues associated with hum... more A big divide was created among stem cell biologists because of ethical issues associated with human embryonic stem cells (hESCs). Adult stem cell biologists observed that hematopoietic stem cells (HSCs) in bone marrow possess considerable “plasticity” and could replace hESCs for regenerative medicine. As a result several trials using autologous bone marrow cells were undertaken; however, no significant success has been achieved. Human ESCs and induced pluripotent stem (iPS) cells tend to give rise to fetal counterparts and thus may not regenerate adult organs efficiently. Another novel population of pluripotent stem cells has also been reported in various adult organs termed very small embryonic-like stem cells (VSELs). VSELs explain the “plasticity” of the bone marrow/cord blood cells and have better differentiation potential compared to ES/iPS cells. Present article is an effort to make a case for pluripotent VSELs as ideal stem cell candidate for endogenous regeneration of diseas...
Adult mammalian heart is considered to be one of the least regenerative organs as it is not able ... more Adult mammalian heart is considered to be one of the least regenerative organs as it is not able to initiate endogenous regeneration in response to injury unlike in lower vertebrates and neonatal mammals. Evidence is now accumulating to suggest normal renewal and replacement of cardiomyocytes occurs even in middle-aged and old individuals. But underlying mechanisms leading to this are not yet clear. Do tissue-resident stem cells exist or somatic cells dedifferentiate leading to regeneration? Lot of attention is currently being focused on epicardium as it is involved in cardiac development, lodges multipotent progenitors and is a source of growth factors. Present study was undertaken to study the presence of stem cells in the pericardium. Intact adult mouse heart was subjected to partial enzymatic digestion to collect the pericardial cells dislodged from the surface. Pericardial cells suspension was processed to enrich the stem cells using our recently published protocol. Two populat...
Objective True identity and specific set of markers to enrich endometrial stem cells still remain... more Objective True identity and specific set of markers to enrich endometrial stem cells still remains elusive. Present study was undertaken to further substantiate that very small embryonic-like stem cells (VSELs) are the true and elusive stem cells in adult mice endometrium. Methods This was achieved by undertaking three sets of experiments. Firstly, SSEA-1+ and Oct-4 + positive VSELs, sorted from GFP mice, were transplanted into the uterine horns of wild-type Swiss mice and GFP uptake was studied within the same estrus cycle. Secondly, uterine lumen was scratched surgically and OCT-4 expressing stem/progenitor cells were studied at the site of injury after 24–72 h. Thirdly, OCT-4 expression was studied in the endometrium and myometrium of adult mice after neonatal exposure to estradiol (20 µg/pup/day on days 5–7 after birth). Results GFP + ve VSELs expressing SSEA-1 and Oct-4 engrafted and differentiated into the epithelial cells lining the lumen as well as the glands during the est...
Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduct... more Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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 repl...
Present study aims to describe a simple and robust protocol to delineate the presence of pluripot... more Present study aims to describe a simple and robust protocol to delineate the presence of pluripotent, very small embryonic-like stem cells (VSELs) in addition to spermatogonial stem cells (SSCs) in adult mouse testes. Testicular seminiferous tubules were subjected to enzymatic dissociation to obtain single cells suspension. Stem cells were enriched by spinning at different speeds wherein majority of somatic cells were pelleted at 1000 rpm (250 g, Pellet A) and putative stem cells by spinning the supernatant (obtained after separating Pellet A) at 3000 rpm (1000 g, Pellet B). Viable (7AAD-ve), 2–6 μm, LIN-CD45-SCA-1+ VSELs were studied after doublets exclusion by flow cytometry in both Pellets A & B. Almost ten-fold enrichment of VSELs was obtained in Pellet B (0.27 + 0.05%) compared to Pellet A (0.03 + 0.003%). SCA-1 expressing SSCs (>6 μm, 0.18 + 0.06%) were clearly distinguished from VSELs (2–6 μm, 0.07 + 0.003%) by flow cytometry studies on total testicular cells suspension collected by spinning at 3000 rpm. Enriched stem cells from Pellet B were used to study expression of OCT-4, NANOG, SCA-1, SSEA-1, LIFR, GFRa, c-KIT, ERα and ERβ. Cells in Pellet B were also subjected to RT-PCR to study pluripotent (Oct-4a, Sox2, Nanog), primordial germ cells (Stella, Fragilis), SSCs (Oct-4) and estrogen receptors (ERα and ERβ) specific transcripts. qRT-PCR analysis showed >2 folds up-regulation of stem cell markers in Pellet B (Oct-4A, Oct-4, Sox2, Nanog) compared to Pellet A. To conclude, spinning at higher speed led to successful enrichment of pluripotent VSELs from testes which have remained ignored till now. Expression of ERα & β on VSELs/SSCs makes them vulnerable to endocrine disruption.
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