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.

Blastocyst-like Structures in the Peripheral Retina of Young Adult Beagles.

In this immunohistological study on the peripheral retina of 3-year-old beagle dogs, excised retina specimens were immunostained with antibodies against nestin, Oct4, Nanog, Sox2, CDX2, cytokeratin 18 (CK 18), RPE65, and YAP1, as well as hematoxylin and DAPI, two nuclear stains. Our findings revealed solitary cysts of various sizes in the inner retina. Intriguingly, a mass of small round cells with scant cytoplasms was observed in the cavity of small cysts, while many disorganized cells partially occupied the cavity of the large cysts. The small cysts were strongly positive for nestin, Oct4, Nanog, Sox2, CDX2, CK18, and YAP1. RPE65-positive cells were exclusively observed in the tissue surrounding the cysts. Since RPE65 is a specific marker of retinal pigment epithelial (RPE) cells, the surrounding cells of the peripheral cysts were presumably derived from RPE cells that migrated intraretinally. In the small cysts, intense positive staining for nestin, a marker of retinal stem cells, seemed to indicate that they were derived from retinal stem cells. The morphology and positive staining for markers of blastocyst and RPE cells indicated that the small cysts may have formed structures resembling the blastocyst, possibly caused by the interaction between retinal stem cells and migrated RPE cells.

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.

Stem Cell Responsiveness to Imatinib in Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease characterized by the presence of the BCR-ABL fusion gene, which results from the Philadelphia chromosome. Since the introduction of tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM), the clinical outcomes for patients with CML have improved significantly. However, IM resistance remains the major clinical challenge for many patients, underlining the need to develop new drugs for the treatment of CML. The basis of CML cell resistance to this drug is unclear, but the appearance of additional genetic alterations in leukemic stem cells (LSCs) is the most common cause of patient relapse. However, several groups have identified a rare subpopulation of CD34+ stem cells in adult patients that is present mainly in the bone marrow and is more immature and pluripotent; these cells are also known as very small embryonic-like stem cells (VSELs). The uncontrolled proliferation and a compromised differentiation possibly initiate their transformation to leukemic VSELs (LVSELs). Their nature and possible involvement in carcinogenesis suggest that they cannot be completely eradicated with IM treatment. In this study, we demonstrated that cells from CML patients with the VSELs phenotype (LVSELs) similarly harbor the fusion protein BCR-ABL and are less sensitive to apoptosis than leukemic HSCs after IM treatment. Thus, IM induces apoptosis and reduces the proliferation and mRNA expression of Ki67 more efficiently in LHSCs than in leukemic LVSELs. Finally, we found that the expression levels of some miRNAs are affected in LVSELs. In addition to the tumor suppressor miR-451, both miR-126 and miR-21, known to be responsible for LSC leukemia-initiating capacity, quiescence, and growth, appear to be involved in IM insensitivity of LVSELs CML cell population. Targeting IM-resistant CML leukemic stem cells by acting via the miRNA pathways may represent a promising therapeutic option.

Stem and Progenitor Cells in the Pathogenesis and Treatment of Digestive Diseases.

The global epidemic of chronic degenerative diseases expands rapidly. The pathogenesis of these noncommunicable disorders revolves around innate immunity, microbiome, and stem cell alterations. Understanding the mechanisms behind stem cell biology and their regulatory pathways is a key to understanding the origin of human disease. Stem cells are involved in tissue and organ damage and regeneration. The evidence is mounting that not only eukaryotic cells but also gut microbiota may release extracellular microvesicles that are absorbed from the gut into the portal and systemic circulation. Linking the fields of stem cells, innate immunity and microbiome research opens up new avenues to develop novel diagnostic (e.g., biomarkers), therapeutic (e.g., microbiome modulation, stem cell-based medicines), and prognostic (personalized diets) tools. In this chapter, we present the short overview of various stem and progenitor cells of adult tissues circulating in peripheral blood and their role in the pathogenesis and treatment of digestive diseases. We also briefly discuss the role of host-stem cell-microbial interactions as a new frontier of research in gastroenterology.

Plausible Links Between Metabolic Networks, Stem Cells, and Longevity.

Aging is an inevitable consequence of life, and all multicellular organisms undergo a decline in tissue and organ functions as they age. Several well-known risk factors, such as obesity, diabetes, and lack of physical activity that lead to the cardiovascular system, decline and impede the function of vital organs, ultimately limit overall life span. Over recent years, aging research has experienced an unparalleled growth, particularly with the discovery and recognition of genetic pathways and biochemical processes that control to some extent the rate of aging.In this chapter, we focus on several aspects of stem cell biology and aging, beginning with major cellular hallmarks of aging, endocrine regulation of aging and its impact on stem cell compartment, and mechanisms of increased longevity. We then discuss the role of epigenetic modifications associated with aging and provide an overview on a most recent search of antiaging modalities.

Stem cells survive oncotherapy & can regenerate non-functional gonads: A paradigm shift for oncofertility.

A large proportion of patients who survive cancer are rendered infertile as an unwanted side effect of oncotherapy. Currently accepted approaches for fertility preservation involve banking eggs/sperm/embryos or ovarian/testicular tissue before oncotherapy for future use. Such approaches are invasive, expensive, technically challenging and depend on assisted reproductive technologies (ART). Establishing a gonadal tissue bank (for cancer patients) is also fraught with ethical, legal and safety issues. Most importantly, patients who find it difficult to meet expenses towards cancer treatment will find it difficult to meet expenses towards gonadal tissue banking and ART to achieve parenthood later on. In this review an alternative strategy to regenerate non-functional gonads in cancer survivors by targeting endogenous stem cells that survive oncotherapy is discussed. A novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs), developmentally equivalent to late migratory primordial germ cells, exists in adult gonads and survives oncotherapy due to their quiescent nature. However, the stem-cell niche gets compromised by oncotherapy. Transplanting niche cells (Sertoli or mesenchymal cells) can regenerate the non-functional gonads. This approach is safe, has resulted in the birth of fertile offspring in mice and could restore gonadal function early in life to support proper growth and later serve as a source of gametes. This newly emerging understanding on stem cells biology can obviate the need to bank gonadal tissue and fertility may also be restored in existing cancer survivors who were earlier deprived of gonadal tissue banking before oncotherapy.

Ovarian stem cells-resolving controversies.

A recent review on ovarian stem cells by Horan and Williams entitled "Oocyte Stem Cells: Fact or Fantasy?" suggests that the debate on ovarian stem cells (OSCs) is still not over. They did not even discuss the presence of two distinct populations of stem cells in the ovary in their review. OSCs are located in the ovary surface epithelium and Tilly's group reported them in the size range of 5-8 µm whereas Virant-Klun's group has reported pluripotent, 2-4 µm OSCs. Our group reported OSCs of two distinct sizes including pluripotent very small embryonic-like stem cells (VSELs) which are smaller in size than RBCs (similar to those reported by Virant-Klun's group) and slightly bigger (similar to those reported by Tilly's group) tissue committed progenitors (OSCs) that presumably differentiate from VSELs. These stem/progenitor cells express receptors for follicle stimulating hormone (FSH) and are activated by FSH. Our opinion article provides explanation to several open-ended questions raised in the review on OSCs by Horan and Williams. VSELs survive chemotherapy; maintain life-long homeostasis; loss of their function due to a compromised niche results in age-related senescence and presence of overlapping pluripotent markers suggest that they may also be implicated in epithelial ovarian cancers.

Human haematopoietic stem/progenitor cells express several functional sex hormone receptors.

Evidence has accumulated that murine haematopoietic stem/progenitor cells (HSPCs) share several markers with the germline, a connection supported by recent reports that pituitary and gonadal sex hormones (SexHs) regulate development of murine HSPCs. It has also been reported that human HSPCs, like their murine counterparts, respond to certain SexHs (e.g. androgens). However, to better address the effects of SexHs, particularly pituitary SexHs, on human haematopoiesis, we tested for expression of receptors for pituitary SexHs, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL), as well as the receptors for gonadal SexHs, including progesterone, oestrogens, and androgen, on HSPCs purified from human umbilical cord blood (UCB) and peripheral blood (PB). We then tested the functionality of these receptors in ex vivo signal transduction studies and in vitro clonogenic assays. In parallel, we tested the effect of SexHs on human mesenchymal stromal cells (MSCs). Finally, based on our observation that at least some of the UCB-derived, CD45(-) very small embryonic-like stem cells (VSELs) become specified into CD45(+) HSPCs, we also evaluated the expression of pituitary and gonadal SexH receptors on these cells. We report for the first time that human HSPCs and VSELs, like their murine counterparts, express pituitary and gonadal SexH receptors at the mRNA and protein levels. Most importantly, SexH if added to suboptimal doses of haematopoietic cytokines and growth factors enhance clonogenic growth of human HSPCs as well as directly stimulate proliferation of MSCs.

Evidence that the population of quiescent bone marrow-residing very small embryonic/epiblast-like stem cells (VSELs) expands in response to neurotoxic treatment.

The concept that bone marrow (BM)-derived cells may participate in neural regeneration remains controversial, and the identity of the specific cell type(s) involved remains unknown. We recently reported that the adult murine BM contains a highly mobile population of Sca-1(+) Lin(-) CD45(-) cells known as very small embryonic/epiblast-like stem cells (VSELs) that express several markers of pluripotency such as Oct-4. In the BM microenvironment, these cells are kept quiescent because of epigenetic modification of certain paternally imprinted genes. However, as reported, these cells can be mobilized in mice in an experimental model of stroke and express several genes involved in neurogenesis while circulating in peripheral blood (PB). In the current work, we employed a model of toxic brain damage, which is induced by administration of kainic acid, to see not only whether VSELs can be mobilized into PB in response to this neurotoxin, but, more importantly, whether they proliferate and expand in BM tissue. We report here for the first time that brain damage leads to activation and expansion of the BM pool of quiescent VSELs, which precedes their subsequent egress into PB. Harnessing these cells in neural tissue regeneration is currently one of the challenges in regenerative medicine.

Non-haematopoietic Sca-1+ Cells in the Retina of Adult Mice Express Functional TLR2.

The mammal retina does not have the capacity to regenerate throughout life, although some stem and progenitor cells persist in the adult retina and might retain multipotentiality, as previously described in many tissues. In this work we demonstrate the presence of a small lineage- Sca-1+ cell population in the adult mouse retina which expresses functional TLR2 receptors as in vitro challenge with the pure TLR2 agonist Pam3CSK4 increases cell number and upregulates TLR2. Therefore, this population could be of interest in neuroregeneration studies to elucidate its role in these processes.

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, ß-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-ß) 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.

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 cells to maintain tissue homeostasis 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 form spheroids. 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.

The Phoenix of stem cells: pluripotent cells in adult tissues and peripheral blood.

Pluripotent stem cells are defined as cells that can generate cells of lineages from all three germ layers, ectoderm, mesoderm, and endoderm. On the contrary, unipotent and multipotent stem cells develop into one or more cell types respectively, but their differentiation is limited to the cells present in the tissue of origin or, at most, from the same germ layer. Multipotent and unipotent stem cells have been isolated from a variety of adult tissues, Instead, the presence in adult tissues of pluripotent stem cells is a very debated issue. In the early embryos, all cells are pluripotent. In mammalians, after birth, pluripotent cells are maintained in the bone-marrow and possibly in gonads. In fact, pluripotent cells were isolated from marrow aspirates and cord blood and from cultured bone-marrow stromal cells (MSCs). Only in few cases, pluripotent cells were isolated from other tissues. In addition to have the potential to differentiate toward lineages derived from all three germ layers, the isolated pluripotent cells shared other properties, including the expression of cell surface stage specific embryonic antigen (SSEA) and of transcription factors active in the early embryos, but they were variously described and named. However, it is likely that they are part of the same cell population and that observed diversities were the results of different isolation and expansion strategies. Adult pluripotent stem cells are quiescent and self-renew at very low rate. They are maintained in that state under the influence of the "niche" inside which they are located. Any tissue damage causes the release in the blood of inflammatory cytokines and molecules that activate the stem cells and their mobilization and homing in the injured tissue. The inflammatory response could also determine the dedifferentiation of mature cells and their reversion to a progenitor stage and at the same time stimulate the progenitors to proliferate and differentiate to replace the damaged cells. In this review we rate articles reporting isolation and characterization of tissue resident pluripotent cells. In the attempt to reconcile observations made by different authors, we propose a unifying picture that could represent a starting point for future experiments.

Quo Vadis medycyno regeneracyjna?: Quo Vadis Regenerative Medicine?

There are presented the most important sources of pluripotent stem cells for potential application in the regenerative medicine. This review summarizes also advantages and disadvantages for potential application of these cells in clinical medicine.

Stem Cell Therapy, Artificial Heart or Xenotransplantation: What will be New "Regenerative" Strategies in Heart Failure during the Next Decade?

The main limitation of allotransplantation and in particular heart transplantation is the insufficient supply of donor organs. As alternative strategies to heart transplantation, stem cells opened the way of regenerative medicine in early 2000. While new biotechnologies tried to minimize side effects due to hemocompatibility in artificial hearts, progress in xenotransplantation allowed in 2022 to realize the first pig-to-human heart transplant on a compassionate use basis. This xenotransplantation has been successful thanks to genetically modified pigs using the CRISPR-Cas9 technology. Indeed, gene editing allowed modifications of immune responses and thrombotic potential to modulate graft and systemic reaction. Academic research and preclinical studies of xenogeneic tissues already used in clinic such as bioprosthesis valve and of new xenotransplantation options will be necessary to evaluate immune-thrombosis and organ/vascular damages more deeply to make this hope of xenotransplantation a clinical reality. Stem cells, artificial heart and xenotransplantation are all in line to overcome the lack of donor hearts. Combination of stem cell approaches and/or xenogeneic tissue and/or artificial organs are probably part of the research objectives to make these projects real in the short term.

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.

Preventing/Reversing Adverse Effects of Endocrine Disruption on Mouse Testes by Normalizing Tissue Resident VSELs.

Reproductive health of men is declining in today's world due to increased developmental exposure to endocrine-disrupting chemicals (EDCs). We earlier reported that neonatal exposure to endocrine disruption resulted in reduced numbers of seminiferous tubules in Stage VIII, decreased sperm count, and infertility along with testicular tumors in 65% of diethylstilbestrol (DES) treated mice. Epigenetic changes due to EDCs, pushed the VSELs out of a quiescent state to enter cell cycle and undergo excessive self-renewal while transition of c-KIT- stem cells into c-KIT + germ cells was blocked due to altered MMR axis (Np95, Pcna, Dnmts), global hypomethylation (reduced expression of 5-methylcytosine) and loss of imprinting at Igf2-H19 and Dlk1-Meg3 loci. The present study was undertaken to firstly show similar defects in FACS sorted VSELs from DES treated testis and to further explore the reversal of these testicular pathologies by (i) oral administration of XAR (a nano-formulation of resveratrol) or (ii) inter-tubular transplantation of mesenchymal stromal cells (MSCs). Similar defects as reported earlier in the testes were evident, based on RNAseq data, on FACS sorted VSELs from DES treated mice. Both strategies were found effective, improved spermatogenesis, increased number of tubules in Stage VIII, normalized numbers of VSELs and c-KIT + cells, improved epigenetic status of VSELs to restore quiescent state, and reduced cancer incidence from 65% after DES to 13.33% and 20% after XAR treatment or MSCs transplantation respectively. Results provide a basis for initiating clinical studies and 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.