Very Small Embryonic-like Stem Cells Research Articles

Murine and Human-Purified very Small Embryonic-like Stem Cells (VSELs) Express Purinergic Receptors and Migrate to Extracellular ATP Gradient.

Purinergic signaling is an ancient primordial signaling system regulating tissue development and specification of various types of stem cells. Thus, functional purinergic receptors are present in several types of cells in the body, including multiple populations of stem cells. However, one stem cell type that has not been evaluated for expression of purinergic receptors is very small embryonic stem cells (VSELs) isolated from postnatal tissues. Herein, we report that human umbilical cord blood (UCB) and murine bone marrow (BM) purified VSELs express mRNA for P1 and P2 purinergic receptors and CD39 and CD73 ectonucleotidases converting extracellular ATP (eATP) into its signaling metabolite extracellular adenosine (eAdo), that antagonizes eATP effects. More importantly, we demonstrate that human and murine VSELs respond by chemotaxis to eATP, and eAdo inhibits this migration. These responses to eATP are mediated by activation of Nlrp3 inflammasome, and exposure of VSELs to its specific inhibitor MCC950 abolished the chemotactic response to ATP. We conclude that purinergic signaling plays an essential, underappreciated role in the biology of these cells and their potential role in response to tissue/organ injuries.

Initiation of Cancer: The Journey From Mutations in Somatic Cells to Epigenetic Changes in Tissue-resident VSELs.

Multiple theories exist to explain cancer initiation, although a consensus on this is crucial for developing effective therapies. 'Somatic mutation theory' suggests that mutations in somatic cells during DNA repair initiates cancer but this concept has several attached paradoxes. Research efforts to identify quiescent cancer stem cells (CSCs) that survive therapy and result in metastasis and recurrence have remained futile. In solid cancers, CSCs are suggested to appear during epithelial-mesenchymal transition by the dedifferentiation and reprogramming of epithelial cells. Pluripotent and quiescent very small embryonic-like stem cells (VSELs) exist in multiple tissues but remain elusive owing to their small size and scarce nature. VSELs are developmentally connected to primordial germ cells, undergo rare, asymmetrical cell divisions and are responsible for the regular turnover of cellsto maintain tissuehomeostasis throughout life. VSELs are directly vulnerable to extrinsic endocrine insults because they express gonadal and gonadotropin hormone receptors. VSELs undergo epigenetic changes due to endocrine insults and transform into CSCs. CSCs exhibit genomic instability and develop mutations due to errors during DNA replication while undergoing excessive proliferation and clonal expansion to formspheroids. Thus tissue-resident VSELs offer a connection between extrinsic insults and variations in cancer incidence reported in various body tissues. To conclude, cancer is indeed a stem cell disease with mutations occurring as a consequence. In addition to immunotherapy, targeting mutations, and Lgr5 + organoids for developing new therapeutics, targeting CSCs (epigenetically altered VSELs) by improving their niche and epigenetic status could serve as a promising strategy to treat cancer.

(Very) Small Stem-like Cells in Human Cell Cultures

Very Small Embryonic-like Stem Cells (VSELSCs) and Very Small Cancer Stem Cells (VSCSCs) are fields of intensive research. Although the presence in vitro of VSELSC and VSCSC cellular stage analogs appear probable, it has yet to be published. Utilizing established human cell cultures with varying populations of primitive cells, stained with CD markers specific to primitive stages, in addition to a fluorescent DNA dye, and following histochemical processing, we have developed a cytological method for detecting Very Small Leukemic Stem-like Cells (VSLSLCs), Very Small Cancer Stem-like Cells (VSCSLCs), and VSELSCs. This detection provides an opportunity to advance research in these areas.

Reversing Uteropathies Including Cancer-Like Changes in Mice by Transplanting Mesenchymal Stromal Cells or XAR Treatment.

Pluripotent, very small embryonic-like stem cells (VSELs) and tissue-committed 'progenitors' termed endometrial stem cells (EnSCs) are reported in mouse uterus. They express gonadal and gonadotropin hormone receptors and thus are vulnerable to early-life endocrine insults. Neonatal exposure of mouse pups to endocrine disruption cause stem/progenitor cells to undergo epigenetic changes, excessive self-renewal, and blocked differentiation that results in various uteropathies including non-receptive endometrium, hyperplasia, endometriosis, adenomyosis, and cancer-like changes in adult life. Present study investigated reversal of these uteropathies, by normalizing functions of VSELs and EnSCs. Two strategies were evaluated including (i) transplanting mesenchymal stromal cells (provide paracrine support) on D60 or (ii) oral administration of XAR (epigenetic regulator) daily from days 60-100 and effects were studied later in 100 days old mice. Results show normalization of stem/progenitor cells (Oct-4, Oct-4A, Sox-2, Nanog) and Wnt signalling (Wnt-4, β-catenin, Axin-2) specific transcripts. Flow cytometry results showed reduced numbers of 2-6 µm, LIN-CD45-SCA-1 + VSELs. Hyperplasia (Ki67) of epithelial (Pax-8, Foxa-2) and myometrial (α-Sma, Tgf-β) cells was reduced, adenogenesis (differentiation of glands) was restored, endometrial receptivity and differentiation (LIF, c-KIT, SOX-9, NUMB) and stromal cells niche (CD90, VIMENTIN, Pdgfra, Vimentin) were improved, cancer stem cells markers (OCT-4, CD166) were reduced while tumor suppressor genes (PTEN, P53) and epigenetic regulators (Ezh-2, Sirt-1) were increased. To conclude, normalizing VSELs/EnSCs to manage uteropathies provides a novel basis for initiating clinical studies. The study falls under the umbrella of United Nations Sustainable Development Goal 3 to ensure healthy lives and well-being for all of all ages.

Current Progress in Stem Cell Therapy for Male Infertility.

Infertility has become one of the most common issues worldwide, which has negatively affected society and infertile couples. Meanwhile, male infertility is responsible for about 50% of infertility. Accordingly, a great number of researchers have focused on its treatment during the last few years; however, current therapies such as assisted reproductive technology (ART) are not effective enough in treating male infertility. Because of their self-renewal and differentiation capabilities and unlimited sources, stem cells have recently raised great hope in the treatment of reproductive system disorders. Stem cells are undifferentiated cells that can induce different numbers of specific cells, such as male and female gametes, demonstrating their potential application in the treatment of infertility. The present review aimed at identifying the causes and potential factors that influence male fertility. Besides, we highlighted the recent studies that investigated the efficiency of stem cells such as spermatogonial stem cells (SSCs), embryonic stem cells (ESCs), very small embryonic-like stem cells (VSELs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) in the treatment of various types of male infertility.

The “Mystery Cell Population” Residing in Murine Bone Marrow – A Missing Link Between Very Small Embryonic Like Stem Cells and Hematopoietic Stem Cells?

Bone marrow (BM) contains not only hematopoietic stem cells (HSCs) but also some very rare, early development, small quiescent stem cells that, upon activation, may differentiate across germlines. These small cells, named very small embryonic like stem cells (VSELs), can undergo specification into several types of cells including HSCs. Interestingly, murine BM is also home to a “mystery” population of small CD45+ stem cells with many of the phenotypic characteristics attributed to resting HSCs. Since the size of the “mystery” population cells are between that of VSELs and HSCs, and because CD45− VSELs can be specified into CD45+ HSCs, we hypothesized that the quiescent CD45+ “mystery” population could be a missing developmental link between VSELs and HSCs. To support this hypothesis, we showed that VSELs first became enriched for HSCs after acquiring expression of the CD45 antigen already expressed on “mystery” stem cells. Moreover, VSELs freshly isolated from BM similar to the “mystery” population cells, are quiescent and do not reveal hematopoietic potential in in vitro and in vivo assays. However, we noticed that CD45+ “mystery” population cells, similar to CD45− VSELs, became specified into HSCs after co-culture over OP9 stroma. We also found that mRNA for Oct-4, a pluripotency marker that is highly expressed in VSELs, is also detectable in the “mystery” population cells, albeit at a much lower level. Finally, we determined that the “mystery” population cells specified over OP9 stroma support were able to engraft and establish hematopoietic chimerism in lethally irradiated recipients. Based on these results, we propose that the murine BM “mystery” population could be an intermediate population between BM-residing VSELs and HSCs already specified for lympho-hematopoietic lineages.Graphical

Molecular analysis and comparison of CD34+ and CD133+ very small embryonic-like stem cells purified from umbilical cord blood.

Very small embryonic like stem cells (VSELs) are a dormant population of stem cells that, as proposed, are deposited during embryogenesis in various tissues, including bone marrow (BM). These cells are released under steady state conditions from their tissue locations and circulate at a low level in peripheral blood (PB). Their number increases in response to stressors as well as tissue/organ damage. This increase is evident during neonatal delivery, as delivery stress prompts enrichment of umbilical cord blood (UCB) with VSELs. These cells could be purified from BM, PB, and UCB by multiparameter sorting as a population of very small CXCR4+ Lin- CD45- cells that express the CD34 or CD133 antigen. In this report, we evaluated a number of CD34+ Lin- CD45- and CD133+ Lin- CD45- UCB-derived VSELs. We also performed initial molecular characterization of both cell populations for expression of selected pluripotency markers and compared these cells at the proteomic level. We noticed that CD133+ Lin- CD45- population is more rare and express, at a higher level, mRNA for pluripotency markers Oct-4 and Nanog as well as the stromal-derived factor-1 (SDF-1) CXCR4 receptor that regulates trafficking of these cells, however both cells population did not significantly differ in the expression of proteins assigned to main biological processes.

Candidates for early detection: Effect of pluripotent very small embryonic-like stem cells (VSELs) to transform into cancer stem cells (CSCs) and candidacy for early detection of cancer in a liquid biopsy.

e15024 Background: Prevailing somatic mutation theory (SMT) is mired with several paradoxes and still there is no clarity on how cancer initiates. No distinct pattern of somatic driver mutations specific to cancers affecting solid tissues has yet emerged even after massive NGS studies since 2007. Rather than cancer being a genetic disease, we postulate that endogenous, tissue-resident VSELs get transformed into CSCs to initiate cancer. Under normal conditions, VSELs along with tissue-specific progenitors work in a subtle manner to maintain life-long homeostasis in adult tissues. VSELs express steroid and gonadotropin hormone receptors, are directly vulnerable to environmental insults, and their dysfunction initiates various pathologies, including cancer. Recent mice studies have shown that environmental insults resulted in global hypomethylation, genomic instability and loss of imprinting (LOI) at IGF2/H19 loci that transformed quiescent VSELs into CSCs. Unlike VSELs, CSCs undergo excessive self-renewal, acquire bad-luck mutations during clonal expansion and oncogene activation to initiate cancers, and can also further mobilize to distant sites to cause metastasis. A 1000-sample clinical study based on OCT-4A expression by qRT-PCR suggested that CSCs that get mobilized from affected organs can serve as potential novel candidates for early detection of cancer through liquid biopsy. Methods: CSCs were enriched from the peripheral blood using our proprietary technology. They were enumerated by flow cytometry. VSELs undergo epigenetic changes and LOI to transform into CSCs. TaqMan based qRT-PCR was carried out to study OCT-4A and ALDH-1 positive CSCs and IGF2 expression to monitor LOI at the IGF2/H19 loci. LOI of IGF2 is commonly associated with initiation and also progression of multiple cancers and is also responsible for pushing VSELs out of quiescence and enter the cell cycle as CSCs. Results: Increased numbers of CSCs (epigenetically altered VSELs) were detected in peripheral blood samples of cancer subjects compared to normal subjects (p < 0.0001) by flow cytometry studies. The median absolute count of circulatory VSELs/CSCs was 190 per ml (IQR: 105-295) in normal subjects and 690 per ml (IQR: 370-1265) in cancer subjects. The qRT-PCR study conducted on 100 subjects showed increased expression of OCT-4A, IGF2 and ALDH1 genes in cancer compared to non-cancer subjects. Conclusions: Early detection of cancer by evaluating circulating tumor cells (CTCs) or tumor DNA (ctDNA) holds promise, but their scarce nature and mobilization in late stages only when tumor is > 3 mm in size, are major limitations for widespread translation. Circulating VSELs/CSCs in peripheral blood are evidently promising candidates to screen and classify subjects for cancer absence, likely risk, or presence.

Transformation of tissue-resident, very small embryonic-like stem cells (VSELs) into cancer stem cells (CSCs) and its role in cancer initiation via epigenetic changes.

e15084 Background: CSCs are postulated to initiate cancer but still no consensus on their surface markers and origin. Pluripotent VSELs exist in adult tissues, express receptors for steroid and gonadotropin hormones and are possible ‘embryonic remnants’ that could initiate cancer. Methods: Neonatal exposure to diethylstilbestrol (DES) or estradiol (E2) resulted in tumor-like changes in uterus and testes of 100 days old mice. Various studies included histological changes, VSELs enumeration by flow cytometry, and a carefully selected panel of transcripts by qRT-PCR to delineate various cellular events. Mice neonatally exposed to endocrine disruption were administered XAR (a nano-formulation of resveratrol) or transplanted niche-providing mesenchymal stromal cells on D60 and studied later on D100 for reversal/prevention of cancer phenotype. Results: Both E2 and DES resulted in increased numbers of VSELs in testis (Ctrl:0.2%; E2:0.4%; DES:1.3%) and uterus (Ctrl:18.52%; E2:35.42%; DES:40.57%). An up-regulation of pluripotent transcripts ( Oct-4, Sox-2, Nanog) was detected. Oct4A (specific marker for pluripotent VSELs) was upregulated after both DES (testis > 8 folds; uterus > 15 folds) and E2 (testis > 3 folds and uterus > 10 folds) treatment. Excessive self-renewal of VSELs was associated with dysregulated expression of DNA methyltransferases (Dnmts). Dnmt1 was downregulated while Dnmt3a, Dnmt3b, and Dnmt3L were upregulated in the testis. In the uterus, Dnmts showed increased expression after both E2 and DES treatment. Marked reduction of 5mC suggested global hypomethylation and increased Igf2 and Dlk-1 suggested affected/loss of imprinting at IGF2/H19 and DLK-1/MEG3 loci. VSELs acquired CSCs phenotype with increased expression of CD166 and ALDH1. Genomic instability in CSCs was suggested by dysregulated mismatch repair axis (DNMT-1/NP95/PCNA) and repair enzymes ( Brca-1, Rad-51, Mlh-1). Reduced expression of Pten and p53 was associated with blocked differentiation. Both reversal strategies resulted in normalization of stem cells compartment, improved differentiation (spermatogenesis/adenogenesis), reduced CSC markers, and increased p53, Pten, Np95, Sirt-1 expression. Interestingly, XAR treatment resulted in downregulation of Igf2 and Dlk1. Stem cells niche was also improved as evident by increased expression of SOX9 in testis and CD90, VIMENTIN in endometrium. Conclusions: Only immortal VSELs can transmit developmental insults to adult cells. Altered epigenetic state of VSELs resulted in cancer initiation and it was possible to prevent/reverse cancer by normalizing epigenetic state of VSELs and their niche. Targeting CSCs and pushing them back into quiescent VSELs and facilitating their normal differentiation offers novel method to win the war against cancer.

Beyond brain injury biomarkers: chemoattractants and circulating progenitor cells as biomarkers of endogenous rehabilitation effort in preterm neonates with encephalopathy.

Preclinical work and studies in adults have shown that endogenous regeneration efforts that involve mobilization of progenitor cells take place after brain injury. However, kinetics of endogenous circulating progenitor cells (CPCs) in preterm neonates is not well described, particularly their possible role regarding brain injury and regeneration. We aimed to assess the kinetics of CPCs in neonates with encephalopathy of prematurity in relation to brain injury biomarkers, chemoattractants and relevant antenatal and postanal clinical factors, in an effort to outline the related pathophysiology. 47 preterm neonates (of 28-33 weeks GA) were enrolled: 31 newborns with no or minimal brain injury (grade I IVH) and 16 prematures with encephalopathy (grade III or IV IVH, PVL or infarct). Peripheral blood samples obtained on days 1, 3, 9, 18 and 45 after birth were analyzed using flow cytometry, focusing on EPCs (early and late Endothelial Progenitor Cells), HSCs (Hematopoietic Stem Cells) and VSELs (Very Small Embryonic-Like Stem Cells). At the same time-points serum levels of S100B, Neuron-specific Enolase (NSE), Erythropoietin (EPO), Insulin-like growth factor-1 (IGF-1) and SDF-1 were also measured. Neonates were assessed postnatally with brain MRI, and with Bayley III developmental test at 2 years of corrected age. Preterms with brain injury proved to have significant increase of S100B and NSE, followed by increase of EPO and enhanced mobilization mainly of HSCs, eEPCs and lEPCs. IGF-1 was rather decreased in this group of neonates. IGF-1 and most CPCs were intense decreased in cases of antenatal or postnatal inflammation. S100B and NSE correlated with neuroimaging and language scale in Bayley III test, providing good prognostic ability. The observed pattern of CPCs' mobilization and its association with neurotrophic factors following preterm brain injury indicate the existence of an endogenous brain regeneration process. Kinetics of different biomarkers and associations with clinical factors contribute to the understanding of the related pathophysiology and might help to early discriminate neonates with adverse outcome. Timely appropriate enhancement of the endogenous regeneration effort, when it is suppressed and insufficient, using neurotrophic factors and exogenous progenitor cells might be a powerful therapeutic strategy in the future to restore brain damage and improve the neurodevelopmental outcome in premature infants with brain injury.

Very Small Embryonic-Like Stem Cells

Continuous and growing research studies regarding the clinical applications of the pluripotent or multipo-tent stem cells with their potential to differentiate into three germ layers are very well conducted in regenerative medicine (RM). In this review, we report the recent clinical applications and potential use of very small embryonic-like stem cells (VSELs) in orthopedics. VSELs are nonhematopoietic (CD45 - / Lin -), rare, and very small cells; they were reported as “dormant” cells in the bone marrow (BM), but are also found in cord blood, peripheral blood (PB), and in adult organs. Based on their capability to express markers of pluripotency (such as Oct-4 +/Nanog +/SSEA-1/4+/CXCR4+), it has been hypothesized that these cells could be early deposited during the embryonic development as descendants of epiblast-derived stem cells and perhaps from some primordial germ cells. VSELs can be released or mobilized from the BM to the PB during tissue injury and stress, facilitating the regeneration of damaged tissues. As well as mesenchymal stem cells, nowadays VSELs can be expanded ex vivo. Their pluripotency could be suitable for applications in RM, solving several problems regarding the use of both controversial embryonic stem cells and induced pluripotent stem cells. VSELs studies will hopefully open new frontiers to better understand their potential that would be relevant for future applications in RM and translational research.

Very Small Embryonic-Like Stem Cells Transform Into Cancer Stem Cells and are Novel Candidates for Detecting/Monitoring Cancer by a Simple Blood Test.

Cancer continues to remain a "Black Box" as there is no consensus on how it initiates, progresses, metastasizes, or recurs. Many imponderables exist about whether somatic mutations initiate cancer, do cancer stem cells (CSCs) exist, and if yes, are they a result of de-differentiation or originate from tissue-resident stem cells; why do cancer cells express embryonic markers, and what leads to metastasis and recurrence. Currently, the detection of multiple solid cancers through liquid biopsy is based on circulating tumor cells (CTCs) or clusters, or circulating tumor DNA (ctDNA). However, quantity of starting material is usually adequate only when the tumor has grown beyond a certain size. We posit that pluripotent, endogenous, tissue-resident, very small embryonic-like stem cells (VSELs) that exist in small numbers in all adult tissues, exit from their quiescent state due to epigenetic changes in response to various insults and transform into CSCs to initiate cancer. VSELs and CSCs share properties like quiescence, pluripotency, self-renewal, immortality, plasticity, enrichment in side-population, mobilization, and resistance to oncotherapy. HrC test, developed by Epigeneres, offers the potential for early detection of cancer using a common set of VSEL/CSC specific bio-markers in peripheral blood. In addition, NGS studies on VSELs/CSCs/tissue-specific progenitors using the All Organ Biopsy (AOB) test provide exomic and transcriptomic information regarding impacted organ(s), cancer type/subtype, germline/somatic mutations, altered gene expressions, and dysregulated pathways. To conclude, HrC and AOB tests can confirm the absence of cancer and categorize the rest of subjects into low/moderate/high risk of cancer, and also monitor response to therapy, remission, and recurrence.

VSELs and OSCs together sustain oogenesis in adult ovaries and their dysfunction results in age-related senescence, PCOS, POI and cancer

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 relevance of VSELs and their relationship with OSCs. By carefully studying the OSE cells smears (which hold VSELs, OSCs and germ cell nests GCNs), prepared by partial trypsin digestion of intact mice ovaries during different stages of estrus cycle, we have successfully delineated novel functions of VSELs/OSCs in vivo under physiological conditions. VSELs undergo asymmetrical divisions to self-renew and give rise to slightly bigger OSCs which in turn undergo symmetrical divisions and clonal expansion to form GCNs, regular neo-oogenesis and follicle assembly. GCNs have been earlier described in fetal ovaries and during OSE cells culture (from adult ovaries) in response to FSH treatment. Dysfunction of VSELs/OSCs (which express ERα, ERβ, FSHR) due to neonatal exposure to endocrine disruption results in ovarian insufficiency and polycystic ovaries. VSELs have also been implicated in ovarian cancer. Age-related ovarian senescence/menopause is also due to dysfunction and blocked differentiation of VSELs/OSCs. These novel findings in vivo along with abundant in vitro and lineage tracing studies data in published literature provides huge scope for further research, offers novel avenues to manage ovarian pathologies and calls for re-writing of textbooks.

Culture of leukocyte-derived cells from human peripheral blood: Increased expression of pluripotent genes OCT4, NANOG, SOX2, self-renewal gene TERT and plasticity.

There are few stem cells in human peripheral blood (PB). Increasing the population and plasticity of stem cells in PB and applying it to regenerative medicine require suitable culture methods. In this study, leukocyte populations 250 mL of PB were collected using a blood separator before that were cultured in optimal cell culture medium for 4 to 7 days. After culturing, stemness characteristics were analyzed, and red blood cells were removed from the cultured cells. In our results, stemness markers of the leukocyte populations Sca-1+ CD45+, CD117+ CD45+, and very small embryonic-like stem cells CD34+ Lin- CD45- and CXCR4+ Lin- CD45- were significantly increased. Furthermore, the expression of stem cell genes OCT4 (POU5F1), NANOG, SOX2, and the self-renewal gene TERT was analyzed by quantitative real-time polymerase chain reaction in these cells, and it showed a significant increase. These cells could be candidates for multi-potential cells and were further induced using trans-differentiation culture methods. These cells showed multiple differentiation potentials for osteocytes, nerve cells, cardiomyocytes, and hepatocytes. These results indicate that appropriate culture methods can be applied to increase expression of pluripotent genes and plasticity. Leukocytes of human PB can be induced to trans-differentiate into pluripotent potential cells, which will be an important breakthrough in regenerative medicine.

Very small embryonic-like stem cells have the potential to win the three-front war on tissue damage, cancer, and aging.

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 therapies carry the same risks as those using induced pluripotent cells, as described above. The present perspective examines the potential of endogenous, pluripotent very small embryonic-like stem cells (VSELs). These cells are naturally present in multiple tissues; they routinely replace diseased tissue and ensure regeneration to maintain life-long homeostasis, and they have the ability to differentiate into adult counterparts. Recent evidence suggests that cancers initiate due to the selective expansion of epigenetically altered VSELs and their blocked differentiation. Furthermore, VSEL numbers have been directly linked to lifespan in studies of long- and short-lived transgenic mice, and VSEL dysfunction has been found in the ovaries of aged mice. To conclude, a greater interest in VSELs, with their potential to address all three fronts of this war, could be the "light at the end of the tunnel."

Mouse uterine stem cells are affected by endocrine disruption and initiate uteropathies.

Incidence of uteropathies has increased in recent times, possibly due to exposure to endocrine-disrupting chemicals during early development. The present study shows that various uteropathies like endometrial cancer, adenomyosis, and endometriosis are interlinked and occur due to the dysfunction of tissue-resident, very small embryonic-like stem cells (VSELs). Underlying pathomechanisms leading to the initiation of uteropathies including non-receptive endometrium, hyperplasia, adenomyosis, endometriosis, fibroids, and cancer remain elusive. Two populations of stem cells exist in mouse uterus including pluripotent VSELs and 'progenitors' termed endometrial stem cells (EnSCs) which express ERα, ERβ, PR, and FSHR, participate in the regular remodelling, and maintain life-long homeostasis. The present study aimed to delineate possible stem cell origins for various uteropathies. For this, mouse pups were treated with oestradiol or diethylstilbestrol and were studied for adult onset of various uteropathies. Treatment resulted in disrupted oestrous cycles, reduced uterine weights, and marked hyperplasia in both epithelial and myometrial compartments, and the stromal compartment was also affected. VSELs were increased in numbers as judged by flow cytometry and increased expression of transcripts specific for Oct-4A, Sox-2, and Nanog, but their further differentiation into a receptive endometrium was affected. Reduced 5-methyl cytosine expression suggested global hypomethylation and was associated with several oncogenic events including loss of tumour-suppressor genes (Pten, p53), dysregulated DNA mismatch repair axis, and repair enzymes. Stem cells were epigenetically altered and showed increased expression of DNMTs, loss of imprinting loci (Igf2-H19, Dlk1-Meg3), and Ezh2. Increased co-expression of CD166 and ALDHA1 with OCT-4 in stem cells was associated with increased Esr-2 and reduced Pr in the endometrium, while both were several folds upregulated in the myometrium. Study results suggest that various uteropathies ensue due to the dysfunction of tissue-resident stem cells and provide huge scope for further research.

Single-cell RNA sequencing reveals different signatures of mesenchymal stromal cell pluripotent-like and multipotent populations

Somatic stem cells are advantageous research targets for understanding the properties required to maintain stemness. Human bone marrow-mesenchymal stromal cells (BM-MSCs) were separated into pluripotent-like SSEA-3(+) Muse cells (Muse-MSCs) and multipotent SSEA-3(-) MSCs (MSCs) and were subjected to single-cell RNA sequencing analysis. Compared with MSCs, Muse-MSCs exhibited higher expression levels of the p53 repressor MDM2; signal acceptance-related genes EGF, VEGF, PDGF, WNT, TGFB, INHB, and CSF; ribosomal protein; and glycolysis and oxidative phosphorylation. Conversely, MSCs had higher expression levels of FGF and ANGPT; Rho family and caveola-related genes; amino acid and cofactor metabolism; MHC class I/II, and lysosomal enzyme genes than Muse-MSCs. Unsupervised clustering further divided Muse-MSCs into two clusters stratified by the expression of cell cycle-related genes, and MSCs into three clusters stratified by the expression of cell cycle-, cytoskeleton-, and extracellular matrix-related genes. This study evaluating the differentiation ability of BM-MSC subpopulations provides intriguing insights for understanding stemness.

Proteomic Analysis of Murine Bone Marrow Very Small Embryonic-like Stem Cells at Steady-State Conditions and after In Vivo Stimulation by Nicotinamide and Follicle-Stimulating Factor Reflects their Germ-Lineage Origin and Multi Germ Layer Differentiation Potential

Very small embryonic-like stem cells (VSELs) are a dormant population of development early stem cells deposited in adult tissues that as demonstrated contribute to tissue/organ repair and regeneration. We postulated developmental relationship of these cells to migrating primordial germ cells (PGCs) and explained the quiescent state of these cells by the erasure of differently methylated regions (DMRs) at some of the paternally imprinted genes involved in embryogenesis. Recently, we reported that VSELs began to proliferate and expand in vivo in murine bone marrow (BM) after exposure to nicotinamide (NAM) and selected pituitary and gonadal sex hormones. In the current report, we performed proteomic analysis of VSELs purified from murine bone marrow (BM) after repeated injections of NAM + Follicle-Stimulating Hormone (FSH) that in our previous studies turned out to be an effective combination to expand these cells. By employing the Gene Ontology (GO) resources, we have performed a combination of standard GO annotations (GO-CAM) to produce a network between BM steady-state conditions VSELs (SSC-VSELS) and FSH + NAM expanded VSELs (FSH + NAM VSELs). We have identified several GO biological processes regulating development, organogenesis, gene expression, signal transduction, Wnt signaling, insulin signaling, cytoskeleton organization, cell adhesion, inhibiting apoptosis, responses to extra- and intracellular stimuli, protein transport and stabilization, protein phosphorylation and ubiquitination, DNA repair, immune response, and regulation of circadian rhythm. We report that VSELs express a unique panel of proteins that only partially overlapped with the proteome of BM – derived hematopoietic stem cells (HSCs) and hematopoietic mononuclear cells (MNCs) and respond to FSH + NAM stimulation by expressing proteins involved in the development of all three germ layers. Thus, our current data supports further germ-lineage origin and multi germ layer differentiation potential of these cells.Graphical abstract

Dysfunctional Ovarian Stem Cells Due to Neonatal Endocrine Disruption Result in PCOS and Ovarian Insufficiency in Adult Mice.

Polycystic ovarian syndrome (PCOS) is a common global cause of anovulatory infertility but underlying etiology leading to PCOS still remains elusive. Fetal and perinatal endocrine disruption reportedly affects germ cell nests (GCN) breakdown, meiosis, and primordial follicle (PF) assembly with unassembled oocytes in neonatal ovaries. We recently reported that very small embryonic-like stem cells (VSELs) and ovarian stem cells (OSCs) express ERα, ERβ and FSHR, undergo distinct cyclic changes and neo-oogenesis encompassing GCN formation, meiosis, and primordial follicle (PF) assembly on regular basis in adult mice ovaries and these GCN are arrested in pre-meiotic or early meiotic stage in aged ovaries. Present study was undertaken to evaluate whether neonatal exposure to endocrine disruption (estradiol E2 or diethylstilbestrol DES) affects ovarian stem cells and their differentiation (neo-oogenesis) and PF assembly in adult 100 days old ovaries. Neonatal exposure to E2 resulted in typical features of PCOS including hyperandrogenism, infertility, increased stromal compartment, absent corpus lutea, and cystic follicles whereas DES treated ovaries showed rapid recruitment of follicles in young ovaries and multi-ovular/cystic follicles. Ovary surface epithelial cells smears showed large numbers of growth-arrested GCN in zygotene/pachytene with increased expression of Mlh-1 and Scp-1 suggesting defects at synapsis and recombination stages during prophase-1 of meiosis. Being immortal and expression of ERα and ERβ makes VSELs directly vulnerable to carry developmental endocrine insults to adult life. Dysfunction of VSELs/OSCs possibly results in oocyte defects observedin our study in PCOS/POI besides the widely reported defects in granulosa cells.

Testicular cancer in mice: interplay between stem cells and endocrine insults.

BackgroundIncidence of type II germ cell tumors (T2GCT) has increased in young men possibly due to fetal/perinatal exposure to estrogenic compounds. Three-fold increased incidence of T2GCT was reported in men exposed in utero to diethylstilbestrol (DES). T2GCT is a development-related disease arising due to blocked differentiation of gonocytes into spermatogonia in fetal testes which survive as germ cell neoplasia in situ (GCNIS) and initiate T2GCT. In our earlier study, T2GCT-like features were observed in 9 out of 10 adult, 100-day-old mice testes upon neonatal exposure to DES (2 μg/pup/day on days 1–5). Neonatal DES exposure affected testicular very small embryonic-like stem cells (VSELs) and spermatogonial stem cells and resulted in infertility, reduced sperm counts and tumor-like changes leading to our postulate that testicular dysgenesis syndrome possibly has a stem cell basis. The present study was undertaken to further characterize testicular tumor in mice testes.MethodsDES-exposed mice pups (n = 70) were studied on D100 and after 12 months to understand how T2GCT progresses. Besides histological studies, a carefully selected panel of markers were studied by immuno-fluorescence and qRT-PCR.ResultsDES resulted in either atrophied or highly vascularized, big-sized testes and extra-testicular growth was also observed. GCNIS-like cells with big, vacuolated cytoplasm and increased expression of OCT-4, SSEA-1, SCA-1 and CD166 (cancer stem cells marker) along with reduced c-KIT, MVH and PTEN were evident. Global hypomethylation was found associated with altered expression of Dnmts, Igf2-H19 and Dlk-Meg3 imprinted genes along with reduced expression of Ezh2, cell cycle regulator p57KIP2 and Meg3; however, Pten remained unaltered. Increased expression of PCNA and Ki67 was observed in concert with complete lack of SOX-9 suggesting Sertoli cells independent proliferation.ConclusionsMouse model for T2GCT is described which will have immense potential to understand cancer initiation, cancer stem cells and also to develop effective therapies in future. T2GCT initiates from tissue-resident, pluripotent VSELs due to their altered epigenome. Neonatal exposure to DES blocks differentiation (spermatogenesis) and VSELs get transformed into CD166 positive cancer stem cells that undergo excessive self-renewal and initiate cancer in adult life challenging existing concept of fetal origin of T2GCT.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-022-02784-5.