Present study aims to describe a simple and robust protocol to delineate the presence of pluripotent, very small embryonic-like stem cells (VSELs) in addition to spermatogonial stem cells (SSCs) in adult mouse testes. Testicular seminiferous tubules were subjected to enzymatic dissociation to obtain single cells suspension. Stem cells were enriched by spinning at different speeds wherein majority of somatic cells were pelleted at 1000 rpm (250 g, Pellet A) and putative stem cells by spinning the supernatant (obtained after separating Pellet A) at 3000 rpm (1000 g, Pellet B). Viable (7AAD-ve), 2–6 μm, LIN-CD45-SCA-1+ VSELs were studied after doublets exclusion by flow cytometry in both Pellets A & B. Almost ten-fold enrichment of VSELs was obtained in Pellet B (0.27 + 0.05%) compared to Pellet A (0.03 + 0.003%). SCA-1 expressing SSCs (>6 μm, 0.18 + 0.06%) were clearly distinguished from VSELs (2–6 μm, 0.07 + 0.003%) by flow cytometry studies on total testicular cells suspension collected by spinning at 3000 rpm. Enriched stem cells from Pellet B were used to study expression of OCT-4, NANOG, SCA-1, SSEA-1, LIFR, GFRa, c-KIT, ERα and ERβ. Cells in Pellet B were also subjected to RT-PCR to study pluripotent (Oct-4a, Sox2, Nanog), primordial germ cells (Stella, Fragilis), SSCs (Oct-4) and estrogen receptors (ERα and ERβ) specific transcripts. qRT-PCR analysis showed >2 folds up-regulation of stem cell markers in Pellet B (Oct-4A, Oct-4, Sox2, Nanog) compared to Pellet A. To conclude, spinning at higher speed led to successful enrichment of pluripotent VSELs from testes which have remained ignored till now. Expression of ERα & β on VSELs/SSCs makes them vulnerable to endocrine disruption.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Sharma, S. J., Pock, T., Schlatt, S., & Neuhaus, N. (2019). Spermatogonial stem cells: Updates from specification to clinical relevance. Human Reproduction Update, 3, 275–297.
Kubota, H., & Brinster, R. L. (2018). Spermatogonial stem cells. Biology of Reproduction, 1, 52–74.
Bhartiya, D., Kasiviswanathan, S., Unni, S. K., Pethe, P., Dhabalia, J. V., Patwardhan, S., & Tongaonkar, H. B. (2010). Newer insights into premeiotic development of germ cells in adult human testis using Oct-4 as a stem cell marker. The Journal of Histochemistry and Cytochemistry, 58(12), 1093–1106.
Anand, S., Bhartiya, D., Sriraman, K., & Mallick, A. (2016). Underlying mechanisms that restore spermatogenesis on transplanting healthy niche cells in busulphan treated mouse testis. Stem Cell Reviews, 12, 682–697.
Patel, H., & Bhartiya, D. (2016). Testicular stem cells express follicle-stimulating hormone receptors and are directly modulated by FSH. Reproductive Sciences, 11, 1493–1508.
Bhartiya, D., Anand, S., Patel, H., Kaushik, A., & Pramodh, S. (2019). Testicular stem cells, spermatogenesis and infertility. In R. Singh (Ed.), Molecular mechanisms in spermatogenesis and infertility. Boca Raton, FL: CRC Press, Taylor & Francis Group ISBN no. 13:978-0-367-19930-2.
Bhartiya, D., Anand, S., & Kaushik, A. (2020). Pluripotent very small embryonic-like stem cells co-exist along with spermatogonial stem cells in adult mammalian testis. Letter to Editor Human Reproduction Update, 26(1), 136–137.
Sharma, S., Wistuba, J., Neuhaus, N., & Schlatt, S. (2020). Reply: Pluripotent very small embryonic-like stem cells co-exist along with spermatogonial stem cells in adult mammalian testis. Human Reproduction Update, 26(1), 138.
Lim, J. J., Sung, S. Y., Kim, H. J., Song, S. H., Hong, J. Y., Yoon, T. K., Kim, J. K., Kim, K. S., & Lee, D. R. (2010). Long-term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non-obstructive azoospermia under exogenous feeder-free culture conditions. Cell Proliferation, 43, 405–417.
Izadyar, F., Wong, J., Maki, C., Pacchiarotti, J., Ramos, T., Howerton, K., Yuen, C., Greilach, S., Zhao, H. H., Chow, M., Chow, Y. C., Rao, J., Barritt, J., Bar-Chama, N., & Copperman, A. (2011). Identification and characterization of repopulating spermatogonial stem cells from the adult human testis. Human Reproduction, 6, 1296–1306.
Kurkure, P., Prasad, M., Dhamankar, V., & Bakshi, G. (2015). Very small embryonic-like stem cells (VSELs) detected in azoospermic testicular biopsies of adult survivors of childhood cancer. Reproductive Biology and Endocrinology, 13, 122.
Stimpfel, M., Skutella, T., Kubista, M., Malicev, E., Conrad, S., & Virant-Klun, I. (2012). Potential stemness of frozen-thawed testicular biopsies without sperm in infertile men included into the in vitro fertilization programme. Journal of Biomedicine & Biotechnology, 2012, 291038.
Kucia, M., Reca, R., Campbell, F. R., et al. (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.
Shaikh, A., Anand, S., Kapoor, S., Ganguly, R., & Bhartiya, D. (2017). Mouse bone marrow VSELs exhibit differentiation into three embryonic germ lineages and germ & hematopoietic cells in culture. Stem Cell Reviews and Reports, 13, 202–216.
Ratajczak, M. Z., Ratajczak, J., & Kucia, M. (2019). Very small embryonic-like stem cells (VSELs). Circulation Research, 124, 208–210.
Ratajczak, M. Z., Ratajczak, J., Suszynska, M., Miller, D. M., & Kucia, M. (2017). A novel view of the adult stem cell compartment from the perspective of a quiescent population of very small embryonic-like stem cells. Circulation Research, 1, 166–178.
Lahlil, R., Scrofani, M., Barbet, R., Tancredi, C., Aries, A., & Hénon, P. (2018). VSELs maintain their pluripotency and competence to differentiate after enhanced ex vivo expansion. Stem Cell Reviews, 14, 510–524.
Anand, S., Patel, H., & Bhartiya, D. (2015). Chemoablated mouse seminiferous tubular cells enriched for very small embryonic-like stem cells undergo spontaneous spermatogenesis in vitro. Reproductive Biology and Endocrinology, 13, 33.
Bhartiya, D. (2017). Pluripotent stem cells in adult tissues: Struggling to be acknowledged over two decades. Stem Cell Reviews and Reports, 6, 713–724.
Li, L., & Clevers, H. (2010). Coexistence of quiescent and active adult stem cells in mammals. Science, 5, 327–542.
De Rosa, L., & De Luca, M. (2012). Cell biology: Dormant and restless skin stem cells. Nature, 7415, 489–215.
Oatley, J. M., & Brinster, R. L. (2008). Regulation of spermatogonial stem cell self-renewal in mammals. Annual Review of Cell and Developmental Biology, 24, 263–286.
Bhartiya, D., Patel, H., Ganguly, R., Shaikh, A., Shukla, Y., Sharma, D., & Singh, P. (2018). Novel insights into adult and cancer stem cell biology. Stem Cells and Development, 22, 1527–1539.
Bhartiya, D., Shaikh, A., Anand, S., Patel, H., Kapoor, S., Sriraman, K., Parte, S., & Unni, S. (2016). Endogenous, very small embryonic-like stem cells: Critical review, therapeutic potential and a look ahead. Human Reproduction Update, 23(1), 41–76.
Meyts, E. R. D. (2006). Developmental model for the pathogenesis of testicular carcinoma in situ: Genetic and environmental aspects. Human Reproduction Update, 12(3), 303–323.
Zuba-Surma, E. K., Kucia, M., Ratajczak, J., & Ratajczak, M. Z. (2009). “Small stem cells” in adult tissues: Very small embryonic-like stem cells stand up! Cytometry. Part A, 1, 4–13.
Anway, M. D., Folmer, J., Wright, W. W., & Zirkin, B. R. (2003). Isolation of Sertoli cells from adult rat testes: An approach to ex vivo studies of Sertoli cell function. Biology of Reproduction, 3, 996–1002.
Liedtke, S., Enczmann, J., Waclawczyk, S., Wernet, P., & Kogler, G. (2007). Oct-4 and its pseudogenes confuse stem cell research. Cell Stem Cell, 4, 364–366.
Abbott, A. (2013). Doubt cast over tiny stem cells. Nature, 499, 390–499.
Mohammad, S. A., Bhartiya, D., & Metkar, S. (2019). Mouse pancreas stem/progenitor cells get augmented by streptozotocin and regenerate diabetic pancreas after partial pancreatectomy. Stem Cell Reviews and Reports, 16, 144–158. https://doi.org/10.1007/s12015-019-09919-x.
Zhou, Q., & Melton, D. A. (2018). Pancreas regeneration. Nature, 557, 351–358.
Kubota, H., Avarbock, M. R., & Brinster, R. L. (2003). Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells. Proceedings of the National Academy of Sciences of the United States of America, 100(11), 6487–6492.
Van Bragt, M. P., Ciliberti, N., Stanford, W. L., de Rooij, D. G., & Van Pelt, A. M. (2005). LY6A/E (SCA-1) expression in the mouse testis. Biology of Reproduction, 73(4), 634–638.
Ratajczak, M. Z., Zuba-Surma, E., Kucia, M., Poniewierska, A., Suszynska, M., & Ratajczak, J. (2012). Pluripotent and multipotent stem cells in adult tissues. Advances in Medical Sciences, 57(1), 1–17.
Kaushik, A., Anand, S., & Bhartiya, D. (2020). Altered biology of testicular VSELs and SSCs by neonatal endocrine disruption results in defective spermatogenesis, reduced fertility and tumor initiation in adult mice. In press.
Oduwole, O. O., Peltoketo, H., & Huhtaniemi, I. T. (2018). Role of follicle-stimulating hormone in spermatogenesis. Frontiers in Endocrinology, 9, 763. https://doi.org/10.3389/fendo.2018.00763.
Guercio, G., Saraco, N., Costanzo, M., Marino, R., Ramirez, P., Berensztein, E., Rivarola, M. A., & Belgorosky, A. (2020). Estrogens in human male gonadotropin secretion and testicular physiology from infancy to late puberty. Frontiers in Endocrinology, 11, 72. https://doi.org/10.3389/fendo.2020.00072.
Clevers, H., & Watt, F. M. (2018). Defining adult stem cells by function, not by phenotype. Annual Review of Biochemistry, 87, 1015–1027.
Azizi, H., Asgari, B., & Skutella, T. (2019). Pluripotency potential of embryonic stem cell-like cells derived from mouse testis. Cell Journal, 21(3), 281–289.
De Rooij, D. G. (2017). The nature and dynamics of spermatogonial stem cells. Develop., 17, 3022–3030.
We are thankful to Indian Council of Medical Research for providing financial support and to UGC fellowship (11-04-2016-329600) to AK. Authors are thankful to the Central Facilities at the Institute for their help [NIRRH/MS/RA/851/12-2019].
All information and detailed protocols are provided in the main article itself.
The authors declare no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
About this article
Cite this article
Kaushik, A., Bhartiya, D. Additional Evidence to Establish Existence of Two Stem Cell Populations Including VSELs and SSCs in Adult Mouse Testes. Stem Cell Rev and Rep (2020). https://doi.org/10.1007/s12015-020-09993-6
- Stem cells
- Spermatogonial stem cells
- Very small embryonic-like stem cells
- Primordial germ cells