Very Small Embryonic-like Stem Cells

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Very Small Embryonic-like Stem Cells (VSELs): Pluripotent Stem Cells in Adult Tissues

Different types of stem cells include (a) pluripotent stem cells (PSCs): embryonic stem cells (hES) and induced pluripotent stem cells (iPS) which are grown from spare human embryos or by reprogramming adult somatic cells like skin fibroblasts & (b) adult stem cells (ASCs) which may be multi- or unipotent. Well studied ASCs are hematopoietic stem cells (HSCs), spermatogonial stem cells (SSCs) in the bone marrow/peripheral blood/cord blood and testes respectively. PSCs have associated concerns like tumorigenicity, immunogenicity and heterogeneity besides their inability to differentiate into adult cell types. HSCs were used in several clinical trials but failed to regenerate since they do not have the potential to transdifferentiate into other lineages. ASCs are lineage restricted and tissue committed in nature like HSCs which may be multipotent and differentiate into red blood cells, white blood cells and platelets or unipotent like SSCs differentiate only into sperm1.

Stem cells which overcome all the above-mentioned drawbacks and have the potential to differentiate into adult cell types of multiple lineages due to their pluripotent state are the very small embryonic-like stem cells (VSELs). Concerns like tumorigenicity, immunogenicity and heterogeneity associated with hES/iPS cells, do not exist with VSELs. VSELs are the PSCs that reside in adult tissues and may be related to other PSCs described in adult tissues including MUSE, MIAMI, MAPCs or spore-like stem cells by different groups who employed different strategies to isolate stem cells from adult tissues2,3,4.

Presence of VSELs in adult tissues was disputed in 2013⁵ but this was essentially because of their small size and scarce nature.  Reasons for the controversy were purely technical and have been overcome over time. However, at present, scRNAseq studies on various adult tissues fail to detect PSCs in adult tissues and reasons for the same have been discussed in literature^6,7. Also lineage tracing studies are unable to track rare and quiescent cell populations.

Globally, buffy coat obtained after density gradient centrifugation of peripheral blood/cord blood/bone marrow is used to study mononuclear cells which are enriched for HSCs whereas a large fraction of VSELs settle down with the red blood cells which are invariably get discarded^8-9.  Similarly, cells suspension obtained after enzymatic digestion of solid tissues, is generally centrifuged at 200-400g to ensure cells do not burst or get damaged while processing for various experiments. However, in the process VSELs, being small, get discarded unknowingly since they pellet down only when centrifuged at 1000g.  This explains why VSELs remain elusive in nature even today¹⁰. With this understanding, VSELs have been successfully enriched from adult mice testes, uterus, ovaries, heart and multiple other tissues^7,10,11,12  

More than 40 independent groups have now confirmed the presence of VSELs since they were first reported in 2006 by a group led by Mariusz Ratajczak from University of Louisville, KY, USA¹⁴. VSELs were recently reviewed by the same group².  Being pluripotent, they express pluripotent markers and can differentiate into three lineages in humans^16,17,18 as well as in mice^14,19.  They do not divide readily in vitro but can now be expanded ex vivo in the presence of nicotinamide or valproic acid or in the presence of the small-molecule UM177 without transduction by DNA or RNA or by employing supportive third-party feeder layer cells^20,21. However, they do not integrate in a developing embryo in assay of blastocyst complementation nor form teratoma upon transplantation.  This is because of their quiescent nature which is well deciphered in murine bone marrow-purified VSELs at molecular level as erasure of parentally imprinted genes^{22, 23}.

Being rare, it is best to enrich VSELs before subjecting to flow cytometry using the gating strategy reported by Ratajczak’s group.  VSELs can be enriched from single cells suspensions obtained from any solid tissue after enzymatic digestion simply be first centrifuging at 200-300g when majority of somatic cells pellet down (Pellet A) and later by centrifuging the supernatant at 1000g to obtain enriched fraction of VSELs (Pellet B)^10,11.

As summarized in a recent review² (Circ Res. 2019;124:208-210) below are major highlights of VSELs biology)

VSEL morphology - seeing is believing. VSELs are small cells, corresponding in size to the cells in the inner cell mass of the blastocyst, and, depending on the measurement conditions (in suspension or after adhesion to slides), they measure ~3–5 µm in mice and ~5–7 µm in humans. Thus, they are slightly smaller than red blood cells and therefore require a special gating strategy during FACS sorting. Transmission electron microscopy analysis revealed that they have large nuclei containing euchromatin and a thin rim of cytoplasm enriched in spherical mitochondria, which are characteristic of early-development cells.

Developmental origin of VSELs. It has been proposed that VSELs originate from cells related to the germline, are deposited in developing organs during embryogenesis, and play a role as a backup population for monopotent tissue-committed stem cells. VSELs are quiescent but are activated during stress situations and mobilized into the circulation. The number of these cells decreases with age. Overall, the presence of these early-development cells in postnatal tissues challenges the accepted hierarchy within the adult stem cell compartment in bone marrow.

VSELs and their link to primordial germ cells (PGCs). The germline is immortal from an evolutionary point of view and transfers DNA and mitochondria to the next generation. Living organisms, including their various stem cell compartments, develop from the fusion of gametes derived from PGCs. VSELs express several markers of PGCs, which supports the concept that the most primitive stem cells residing in adult tissues are related to PGCs.

Gene expression analysis. VSELs express some embryonic stem cell markers, such as stage-specific antigen (SSEA), nuclear Oct-4A, Nanog, and Rex1. The true expression of these genes has been confirmed by the open structure of chromatin in their respective promoters, their association with histones promoting transcription, and by the sequencing of RT-PCR products. VSELs also express several markers characteristic of migrating PGCs, such as Stella and Fragilis. Our single-cell cDNA libraries revealed that the gene expression profile in murine BM-isolated VSELs, sorted as very small Sca-1⁺lin^–CD45^– cells, varies.

The quiescent state of VSELs. VSELs residing in adult tissues are highly quiescent due to the erasure of regulatory sequences for certain paternally imprinted genes (e.g., at the Igf2–H19 locus) and thereby protected from insulin/insulin-like growth factor stimulation. They also express bivalent domains at genes encoding transcription factors in the homeobox family. Recent proteomic data have confirmed that genes involved in proliferation and cell signaling are expressed in VSELs at a low level and become upregulated during their expansion.

VSELs in hematopoietic tissues. Evidence has accumulated that VSELs are at the top of the stem cell hierarchy in normal bone marrow, giving rise to HSCs, MSCs, and endothelial progenitor cells (EPCs). VSELs expand in vivo in response to stimulation by pituitary gonadotropins and gonadal sex hormones, which, from a developmental point of view, further links these cells to migrating PGCs.

VSELs in the gonads. It has been convincingly demonstrated that VSELs can be isolated from the ovarian surface epithelium of young and postmenopausal women as well as from testes. Recently, it has been reported that ovary-isolated VSELs differentiate into oocyte-like cells in response to sperm cells and release the zona pellucida, which is the first step in the fertilization process.

VSELs in aging. The number of VSELs correlates with longevity in certain long-living murine strains. Their number can be increased in experimental animals by caloric restriction, regular exercise, and administration of DNA modifiers, such as nicotinamide or valproic acid. By contrast, the exposure of animals to increased insulin/insulin-like growth factor signaling leads to premature aging and depletion of VSELs from the tissues.

VSELs in experimental models of tissue/organ injuries. Several papers have been published showing a contribution by injected purified VSELs to hematopoiesis, osteogenesis, and angiogenesis as well as to myocardium, liver, and pulmonary alveolar epithelium in appropriate in vivo models. The well-demonstrated presence of chimerism in several organs indicates the potential of these cells to differentiate across germ layers.

Ex vivo expansion of VSELs. The most important breakthrough in the potential application of VSELs came with the development of more efficient ex vivo expansion strategies for these rare cells. VSELs can now be expanded ex vivo in the presence of nicotinamide or valproic acid or in the presence of the small-molecule UM177 without transduction by DNA or RNA or by employing supportive third-party feeder layer cells.

The molecular basis behind the expansion of VSELs. To explain our expansion approach, both of the small molecules employed in our expansion medium, nicotinamide and valproic acid, are inhibitors of the histone deacetylase Sirt-1. This enzyme inhibits the activity of the de novo DNA methyl-transferase DnmT3L, which is crucial for methylation of the regulatory regions of paternally imprinted genes. As mentioned above, these loci are demethylated (erased) during early embryogenesis in VSELs, as they are in PGCs migrating to the genital ridges. These epigenetic changes explain why PGCs and VSELs are so quiescent and cannot complement blastocyst development and, what is even more important, do not grow teratomas, despite their pluripotency. The fact that Sirt-1 maintains a low intracellular level of DnmT3L explains why it has beneficial effects on longevity by preventing premature depletion of VSELs from adult tissues. By contrast, downregulation of Sirt-1 by nicotinamide or valproic acid in culture promotes ex vivo expansion of these cells.

In summary, VSELs, with their broad differentiation potential, have become a new candidate population of stem cells for clinical applications. However, although there is no evidence so far that these cells form teratomas, it remains crucial to assess genomic stability of expanded VSELs. It is also necessary to perform appropriate cell tracking studies. We propose that pluripotent VSELs isolated from adult tissues should be studied further in solid organ injury models, as they may provide a path forward that solves several problems with the use of controversial ESCs and iPSCs in regenerative medicine.

Regenerative Potential of VSELs

·      VSELs are more primitive to HSCs in the hematopoietic system. CD45 negative VSELs isolated from mice bone marrow^24,19 and human cord blood²⁵ when cultured on OP9 stromal cell support differentiation into CD45 positive HSCs.

·      Krause’s group provided first evidence that non-hematopoietic VSELs in the bone marrow rather than the BMDCs differentiate into lung epithelial cells²⁶. Recent study showed that VSELs differentiate into functional bronchioalveolar stem (BASC) and alveolar type 2 (AT2) cells, and this opens up new avenues using VSELs to develop effective cell therapy approaches for patients with lung injury²⁷

·      VSELs are mobilized into peripheral blood under stress conditions like in patients with myocardial infarcts or critical limb ischemia (CLI). Smadja’s group has shown that bone marrow VSELs isolated from CLI patients, after 2 weeks in culture in angiogenic media, produce significant positive effect in pre-clinical mice model for limb ischemia. The cultured VSELs also exhibit the ability to differentiate into endothelial cells. Thus, VSELs have the ability to give rise to cells of the endothelial lineage in humans, in particular in critical limb ischaemia patients²⁸.

·      VSELs are developmentally linked to primordial germ cells which are natural precursors for gametes.  Thus, VSELs are ideal stem cells to differentiate into gametes²⁹.  VSELs are reported to differentiate into oocytes^30,31 as well as sperm³² in culture.  Even VSELs enriched from the bone marrow differentiate into male germ cells in vitro³³.  Oocytes developed in culture from VSELs-like cells in the ovary surface epithelium undergo cortical reaction in presence of sperm³⁴.

VSELs and Cancer

Global scientific community is clueless regarding the underlying patho-mechanisms which initiate various pathologies in adult life like PCOS, endometriosis, fibroids, infertility, and cancer.  Despite large numbers of OMICS studies and GWAS on thousands of patient samples, it is now being suggested in literature that these diseases may not have a genetic basis.  Initial evidence is now available that VSELs being ‘immortal’ can carry epigenetic insults induced by perinatal exposure to endocrine disruption to adult life and initiate various pathologies. But these mechanisms and contributions of stem/progenitor cells towards initiating various pathologies remain masked and will never get highlighted when DNA/RNA/proteins are isolated from intact tissues for OMICS/ GWAS studies since stem/progenitor cells comprise <1% of total cells. Neonatal exposure to estradiol and diethylstilbestrol resulted in testicular dysgenesis syndrome in adult mice due to altered stem cells biology³⁵.  Study highlights novel insight into how exposure to endocrine disruptors is resulting in increased incidence of infertility, reduced sperm counts and testicular cancer in young men.

A study undertaken by Epigeneres Biotech Pvt. Ltd and Tzar Labs Pte. Ltd. to study Oct-4A (marker specific for VSELs in adult tissues) in 1000 participants helped to develop a HrC scale which can detect and stage cancer, predict and monitor treatment outcome, is superior to evaluating circulating tumor cells and can also serve as an early biomarker for cancer³⁶. Virant-Klun’s group has shown the involvement of a population comparable to small-sized VSELs in women with borderline ovarian cancer³⁷.

Tissue resident VSELs serve as a back-up pool for ASCs, help maintain life-long homeostasis, ensure regeneration when required and are also instrumental in initiating various pathologies in adult life including cancers².  VSELs get transformed into cancer stem cells (CSCs) and this is the reason why gene expression profile of stem cell pluripotency and differentiation is conserved across diverse solid and hematopoietic cancers³⁸. Tissue-resident VSELs are unable to regenerate spontaneously since they get altered (epigenetic changes) or their niche gets damaged as a result of the pathology.  Presence of VSELs along with tissue-specific progenitors in adult tissues challenges³⁹ the concept of dedifferentiation of somatic cells into ASCs/CSCs⁴⁰. == References == 1.     Bhartiya D. (March 2019) “Clinical Translation of Stem Cells for Regenerative Medicine”. Circulation Research. 124(6):840-842. doi: 10.1161/CIRCRESAHA.118.313823. PMID: 30870131.

2.     Ratajczak MZ, Ratajczak J, Kucia M. (January 2019) “Very Small Embryonic-Like Stem Cells (VSELs)”. Circulation Research. 124(2):208-210. doi: 10.1161/CIRCRESAHA.118.314287. PMCID: PMC6461217. PMID: 30653438.

3.     Ratajczak MZ, Zuba-Surma EK, Wysoczynski M, Ratajczak J, Kucia M. (June 2008) “Very small embryonic-like stem cells: characterization, developmental origin, and biological significance”. Experimental Hematology. 36(6):742-51. doi: 10.1016/j.exphem.2008.03.010. PMCID: PMC2430762. PMID: 18474305.

4.     Bhartiya D. (December 2017) “Pluripotent Stem Cells in Adult Tissues: Struggling To Be Acknowledged Over Two Decades”. Stem Cell Review and Reports.13(6):713-724. doi: 10.1007/s12015-017-9756-y. PMID: 28815441.

5.     Abbott A. (July 2013) “Doubt cast over tiny stem cells”. Nature. 499(7459):390. doi: 10.1038/499390a. PMID: 23887410.

6.     Bhartiya D, Kausik A, Singh P, Sharma D. (February 2021) “Will Single-Cell RNAseq decipher stem cells biology in normal and cancerous tissues?” Human Reproduction Update. 27(2):421. doi: 10.1093/humupd/dmaa058. PMID: 33367724

7.     Bhartiya D, Sharma D. (April 2020) “Ovary does harbor stem cells - size of the cells matter!” Journal of Ovarian Research. 13(1):39. doi: 10.1186/s13048-020-00647-2. PMCID: PMC7164193. PMID: 32303227.

8.     Bhartiya D, Shaikh A, Nagvenkar P, Kasiviswanathan S, Pethe P, Pawani H, Mohanty S, Rao SG, Zaveri K, Hinduja I. (January 2012) “Very small embryonic-like stem cells with maximum regenerative potential get discarded during cord blood banking and bone marrow processing for autologous stem cell therapy. Stem Cells and Development. 21(1):1-6. doi: 10.1089/scd.2011.0311. PMID: 21780911.

9.     Shaikh A, Nagvenkar P, Pethe P, Hinduja I, Bhartiya D. (September 2015) “Molecular and phenotypic characterization of CD133 and SSEA4 enriched very small embryonic-like stem cells in human cord blood”. Leukemia. 29(9):1909-17. doi: 10.1038/leu.2015.100. PMID: 25882698.

10.  Kaushik A, Bhartiya D. (October 2020) “Additional Evidence to Establish Existence of Two Stem Cell Populations Including VSELs and SSCs in Adult Mouse Testes”. Stem Cell Reviews and Reports. 16(5):992-1004. doi: 10.1007/s12015-020-09993-6. PMID: 32578128.

11.  Singh P, Bhartiya D. (January 2021) “Pluripotent Stem (VSELs) and Progenitor (EnSCs) Cells Exist in Adult Mouse Uterus and Show Cyclic Changes Across Estrus Cycle”. Reproductive Sciences. 28(1):278-290. doi: 10.1007/s43032-020-00250-2. PMID: 32710237

12.  Bhartiya D, Flora Y, Sharma D, Mohammad SA. (April 2021) “Two Stem Cell Populations Including VSELs and CSCs Detected in the Pericardium of Adult Mouse Heart”. Stem Cell Review and Reports. 17(2):685-693. doi: 10.1007/s12015-021-10119-9. PMID: 33492626.

13.  Bhartiya D, Ali Mohammad S, Guha A, Singh P, Sharma D, Kaushik A. (June 2019) “Evolving Definition of Adult Stem/Progenitor Cells”. Stem Cell Review and Reports.. 15(3):456-458. doi: 10.1007/s12015-019-09879-2. PMID: 30879243.

14.  Kucia, M., Reca, R., Campbell, F. R., et al. (February 2006). A population of very small embryonic-like (VSEL) CXCR4 (+) SSEA-1(+) Oct-4+ stem cells identified in adult bone marrow. Leukemia, 20(5), 857–869

15.  Wagner M, Yoshihara M, Douagi I, Damdimopoulos A, Panula S, Petropoulos S, Lu H, Pettersson K, Palm K, Katayama S, Hovatta O, Kere J, Lanner F, Damdimopoulou P. (March 2020).  Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells. Nat Commun. 11(1):1147. doi: 10.1038/s41467-020-14936-3.

16.  Lahlil R, M Scrofani, R Barbet, C Tancredi, A Aries and P He´non. (2018). VSELs maintain their pluripotency and competence to differentiate after enhanced ex vivo expansion. Stem Cell Rev. [Epub ahead of print]; DOI:10.1007/s12015-018-9821-1.

17.  Havens AM, H Sun, Y Shiozawa, Y Jung, J Wang, A Mishra, Y Jiang, DW O’Neill, PH Krebsbach, DO Rodgerson and RS Taichman. (2014). Human and murine very small embryonic-like cells represent multipotent tissue progenitors, in vitro and in vivo. Stem Cells Dev 23:689–701.

18.  Monti M, B Imberti, N Bianchi, A Pezzotta, M Morigi, C Del Fante, CA Redi and C Perotti. (2017). A novel method for isolation of pluripotent stem cells from human umbilical cord blood. Stem Cells Dev 26:1258–1269.

19.  Shaikh A, Anand S, Kapoor S, Ganguly R, Bhartiya D. (April 2017) “Mouse Bone Marrow VSELs Exhibit Differentiation into Three Embryonic Germ Lineages and Germ & Hematopoietic Cells in Culture”. Stem Cell Reviews and Reports. 13(2):202-216. doi: 10.1007/s12015-016-9714-0. PMID: 28070859.

20.  Lahlil R, Scrofani M, Barbet R, Tancredi C, Aries A, Hénon P. (August 2018). VSELs Maintain their Pluripotency and Competence to Differentiate after Enhanced Ex Vivo Expansion. Stem Cell Rev. 14(4):510-524. doi: 10.1007/s12015-018-9821-1.

21.  Ratajczak MZ, Ratajczak J, Suszynska M, Miller DM, Kucia M, Shin DM. (2017). A Novel View of the Adult Stem Cell Compartment From the Perspective of a Quiescent Population of Very Small Embryonic-Like Stem Cells. Circ Res. 2017;120: 166–178. doi: 10.1161/CIRCRESAHA.116.309362.

22.  Shin DM, Zuba-Surma EK, Wu W, Ratajczak J, Wysoczynski M, Ratajczak MZ, Kucia M. (November 2009). “Novel epigenetic mechanisms that control pluripotency and quiescence of adult bone marrow-derived Oct4(+) very small embryonic-like stem cells”. Leukemia. 23(11):2042-51. doi: 10.1038/leu.2009.153. PMCID: PMC2783188. PMID: 19641521.

23.  Mierzejewska K, Heo J, Kang JW, Kang H, Ratajczak J, Ratajczak MZ, Kucia M, Shin DM. (August 2013) “Genome-wide analysis of murine bone marrow‑derived very small embryonic-like stem cells reveals that mitogenic growth factor signaling pathways play a crucial role in the quiescence and ageing of these cells”. International Journal of Molecular Medicine. 32(2):281-90. doi: 10.3892/ijmm.2013.1389. PMCID: PMC3776718. PMID: 23708325.

24.  Ratajczak J, Wysoczynski M, Zuba-Surma E, Wan W, Kucia M, Yoder MC, Ratajczak MZ. (February 2011). Adult Murine Bone Marrow-Derived Very Small Embryonic-Like Stem Cells Differentiate into The Hematopoietic Lineage After Coculture Over OP9 Stromal Cells. Exp Hematol. 39(2):225-37.doi: 10.1016/j.exphem.2010.10.007.

25.  Ratajczak J, Zuba-Surma E, Klich I, Liu R, Wysoczynski M, Greco N, Kucia M, Laughlin MJ,  Ratajczak MZ. (August 2011). Hematopoietic Differentiation of Umbilical Cord Blood-Derived Very Small Embryonic/Epiblast-Like Stem Cells. Leukemia.  25(8):1278-85.doi: 10.1038/leu.2011.73.

26.  Kassmer SH, Jin H, Zhang PX, Bruscia EM, Heydari K, Lee JH, Kim CF, Kassmer SH, Krause DS. (December 2013). Very small embryonic-like stem cells from the murine bone marrow differentiate into epithelial cells of the lung.  Stem Cells. 31(12):2759-66. doi: 10.1002/stem.1413.

27.  Ciechanowicz AK, Sielatycka K, Cymer M, Skoda M, Suszyńska M, Bujko K, Ratajczak

MZ, Krause DS, Kucia M. (June 2021). Bone Marrow-Derived VSELs Engraft as Lung Epithelial Progenitor Cells after Bleomycin-Induced Lung Injury. Cells. 10(7):1570. doi: 10.3390/cells10071570.

28.  Guerin CL, Loyer X, Vilar J, Cras A, Mirault T, Gaussem P, Silvestre JS, Smadja DM. (May 2015). Bone-marrow-derived very small embryonic-like stem cells in patients with critical leg ischaemia: evidence of vasculogenic potential. Thromb Haemost. 113(5):1084-94. doi: 10.1160/TH14-09-0748.

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30.  Virant-Klun I, Zech N, Rozman P, Vogler A, Cvjeticanin B, Klemenc P, Malicev E, Meden-Vrtovec H. (October 2008). Putative stem cells with an embryonic character isolated from the ovarian surface epithelium of women with no naturally present follicles and oocytes. Differentiation 76(8):843-56. doi: 10.1111/j.1432-0436.

31.  Parte S, Bhartiya D, Telang J, Daithankar V, Salvi V, Zaveri K, Hinduja I. (August 2011). Detection, characterization, and spontaneous differentiation in vitro of very small embryonic-like putative stem cells in adult mammalian ovary. Stem Cells Dev 20(8):1451-64. doi: 10.1089/scd.2010.0461.

32.  Anand S, Patel H, Bhartiya D. (April 2015). Chemoablated mouse seminiferous tubular cells enriched for very small embryonic-like stem cells undergo spontaneous spermatogenesis in vitro. Reprod Biol Endocrinol 13:33. doi: 10.1186/s12958-015-0031-2.

33.  Shaikh A, Anand S, Kapoor S, Ganguly R, Bhartiya D. (April 2017). Mouse Bone Marrow VSELs Exhibit Differentiation into Three Embryonic Germ Lineages and Germ & Hematopoietic Cells in Culture.  Stem Cell Rev Rep.13(2):202-216. doi: 10.1007/s12015-016-9714-0.

34.  Virant-Klun I. (October 2018). Functional Testing of Primitive Oocyte-like Cells Developed in Ovarian Surface Epithelium Cell Culture from Small VSEL-like Stem Cells: Can They Be Fertilized One Day? Stem Cell Rev Rep. 2018 Oct;14(5):715-721. doi: 10.1007/s12015-018-9832-y.

35.  Kaushik A, Anand S, Bhartiya D. (October 2020) “Altered Biology of Testicular VSELs and SSCs by Neonatal Endocrine Disruption Results in Defective Spermatogenesis, Reduced Fertility and Tumor Initiation in Adult Mice”. Stem Cell Reviews and Reports. 16(5):893-908. doi: 10.1007/s12015-020-09996-3. PMID: 32592162

36.  Tripathi V, Bhartiya D, Vaid A, Chhabria S, Sharma N, Chand B, Takle V, Palahe P, Tripathi A. (May 2021) “Quest for Pan-Cancer Diagnosis/Prognosis Ends with HrC Test Measuring Oct4A in Peripheral Blood”. Stem Cell Reviews and Reports. doi: 10.1007/s12015-021-10167-1. PMID: 33954878.

37.  Virant-Klun I, Stimpfel M. (October 2016) Novel population of small tumour-initiating stem cells in the ovaries of women with borderline ovarian cancer. Scientific Reports 6:34730. doi: 10.1038/srep34730

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39.  Bhartiya D. (January 2021) “Adult tissue-resident stem cells-fact or fiction?” Stem Cell Research and Therapy. 12(1):73. doi: 10.1186/s13287-021-02142-x. PMCID: PMC7819245. PMID: 33478531.

40.  Shivdesani AR, Clevers H, de Sauvage FJ. (February 2021) “Tissue regeneration: Reserve or reverse?” 371(6531): 784-786. DOI: 10.1126/science.abb6848

Very Small Embryonic like Stem Cells

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