Presence of Very Small Embryonic-Like Stem Cells (VSELs) in human semen samples; A novel finding

Presence of Very Small Embryonic-Like Stem Cells (VSELs) in human semen samples; A novel finding | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Presence of Very Small Embryonic-Like Stem Cells (VSELs) in human semen samples; A novel finding DMAB Dissanayake, MD Hettiarachchige, NRY Wijebandara, PS Wijesinghe This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6677204/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Presence of Very Small Embryonic-Like Stem Cells (VSELs) in human semen samples; A novel finding Background: The presence of a very small, quiescent, and pluripotent population of stem cells termed as “VSELs” was first reported in 2006. Those cells have observed in various organs in the mammalian body including bone marrow, peripheral blood, endometrium, ovary and testis so on. But their existence in seminal plasma has not been reported so far. The aim of the present study was to find out whether VSELs are present in human semen samples and if so, study their characteristics and relationship with semen parameters. Semen samples were collected from sub fertile men and analyzed according to WHO guidelines. VSELs in seminal plasma were detected using Giemsa stain. The presence of cells with primitive germ cell properties was confirmed by GPR125 expression and alkaline phosphatase activity. Results: A very small and round cell population, notably smaller than spermatids was observed in all semen samples. The mean (SD) size of cells was 5.1 (0.1) ranging from 3.16 - 6.8 µm, and exhibited a large centrally located nucleus, resulting in a high nucleus-to-cytoplasm (N/C) ratio of 1.75 (0.3). The mean (SEM) concentration of VSELs was 17.21 (4.42) m/ml. VSELs count was significantly high in oligozoospermics compared to normozoospermics (22.71 (5.89) vs 6.22 (1.81), p < 0.05) respectively. There was a positive correlation between VSEL and immature germ cells (r = 0.759, p < 0.001). Two distinct populations of cells were identified according to their color intensity; VSEL dark and pale, 53% and 47% respectively. Minor percentages of sub-populations positive for alkaline phosphatase activity (6.06%) and expressing GPR 125 (5.56%) were also observed. Very slow rate of propagation (3%) and the ability to make embryoid bodies were exhibited by the isolated cells cultured for five weeks. Conclusions: To the best of our knowledge this is the first report on the presence of VSELs in semen samples. Sub-population of cells with primitive germ cell properties would be a good source of stem cells for future studies on in vitro spermatogenesis. Germ cells In vitro spermatogenesis Seminal fluid VSELs Figures Figure 1 Introduction Stem cells are isolated from different sources such as, embryonic and extra embryonic tissues, bone marrow (BM), adipose tissues and different organs of the human body. The capacity to self-renewal and state of potency are the fundamental characteristics to select them for downstream research and clinical applications. Though, pluripotent stem cells highly fulfill the above requirements their usage has been restricted due to ethical, legal and safety issues, especially in therapeutic applications. Shedding light to a new pathway, a rare population of stem cells exhibiting some pluripotent characteristics was described in murine bone marrow by Ratajczak et. al. in 2006. Named as very small embryonic-like stem cells (VSELs) reported to have a broader differentiation potential and display several epiblast and germline markers suggesting their embryonic origin ( 1 ). Those cells are characterized by their small size (3–6 µm), high nuclear/cytoplasmic ratio with a thin rim of cytoplasm and presence of euchromatin. They have been reported to express some pluripotent stem cell markers (Oct-4, Nanog, Rex-1 and SSEA-1), genes specified for both epiblast (Stella, Prdm14, Fragilis, Blimp1, Nanos3 and Dnd1) and migratory primordial germ cells (Dppa2, Dppa4 and Mvh), and negative for hematopoietic lineage markers (Lin − Sca-1 + CD45 − ) ( 1 , 2 , 3 ). VSELs have later been identified in multiple tissues such as, BM, brain, liver, pancreas, kidney, muscles, heart, testes, and thymus, expressing tissue specific and pluripotent markers, and reside in very few numbers in a quiescent state ( 4 , 5 ). Though, remain quiescent in steady states, VSELs are involved in tissue homeostasis by rejuvenation and regeneration after injuries by mobilizing into peripheral blood ( 6 ). Despite their small size VSELs have diploid chromosomes, mitochondria, high telomerase activity, and do not express MHC-1 and HLA-DR antigens ( 2 , 4 ). They have the ability to organize in spheres resembling embryoid bodies when co-cultured with C2C12 cells, and hypo-methylation of somatic imprints helps to maintain their pluripotency ( 7 ). However, the lack of reproducible evidence on certain criteria shown by the embryonic stem cells such as, complete blastocyst development, and formation of teratomas after inoculation into experimental animals question the true pluripotency state of VSELs ( 8 , 9 ). Overlapping populations of cells sharing similar characteristics have been described in adult tissues namely; multipotent adult progenitor cells (MAPCs), marrow-isolated adult multilineage inducible (MIAMI) cells, multipotent adult stem cells (MASCs) and omnicytes ( 4 ). Considering the pluripotent nature, primitiveness, availability, lack of mitochondrial and genomic mutations, controlled proliferation capacity and broader differentiation capacity, VSELs appear to be particularly a good source compared to other cells for cellular-replacement therapy ( 10 ). In vitro gametogenesis attracted most attention in the recent past but, the reality of a clinically competent gamete is a distant dream with pluripotent stem cells. Being developmentally very related to PGCs, and expressing some migratory germ cell markers there is a great hope for the success of this endeavor with VSELs ( 9 ). In vitro culture of VSELs from ovarian epithelium gave rise to oocyte-like cells expressing pluripotency related genes and releasing Zona-pellucida like structures after sperm injection ( 11 ). Culturing of VSELs collected from busulphan treated mouse testicular tissues were spontaneously differentiated into sperm in the presence of Sertoli cell conditioned medium and follicle stimulation hormone ( 12 ). It is suggested that testicular VSELs expressing nuclear Oct4-A are the true stem cells and SSCs are their progenitors giving rise to continuous sperm production ( 13 ). According to the emerging evidence VSELs in which gene expression and epigenetic status are close to natural germ cells, would be a good source of stem cells for making gametes and treating infertility ( 14 ). In a pilot study, we identified VSELs in all human semen samples including samples with non-obstructive azoospermia for the first time. The present study aimed to reveal further details on this sub population of stem cells. Methodology Identification and enumeration of VSELs Semen samples were obtained from males undergoing subfertility evaluation at the north Colombo teaching hospital Ragama, Sri Lanka. Following liquefaction, 10 µL of semen sample was smeared on a clean glass slide, and stained for 10 minutes with 10% Giemsa stain. Slides were examined under a light microscope at high power field (40x). Immature germ cells and VSELs were assessed using their morphology and size, and different cell components found per 100 sperm were recorded. The size of cells was measured using Capture Pro microscopy image analysis software. Cell viability was assessed in wet mounts using eosin stain. Isolation of VSELs from semen samples This protocol was developed to effectively separate and purify VSELs from semen samples ensuring minimal contamination and maximal yield. A 1.0 mL of liquefied semen sample was diluted with phosphate buffered saline (PBS) 1:2 ratios and centrifuged at 1500 rpm for 10 minutes for pelleting the majority of sperm and other round cells. The supernatant enriched with VSELs was collected leaving 0.5 mL of bottom pellet, and re-suspended in 2.0 mL of PBS. The mixture was thoroughly mixed and centrifuged at 800 rpm for 30 minutes for further removal of remaining sperm and round cells. Following centrifugation, the supernatant was carefully transferred into a new centrifuge tube, and subjected to centrifugation at 2500 rpm for 10 minutes for pelleting the VSELs. For semen samples with high sperm concentrations, the swim-up technique was used as the first step to remove majority of active motile sperm. Culturing of VSELs separated from semen samples The separated VSELs pellet was re-suspended in 3 mL of cell culture media, composed of DMEM/F12 supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% vitamin solution. The cell suspension was replicated into three wells in a 24-well culture plate, and the initial VSELs count per high-power field was recorded after six hours. The culture plate was subsequently incubated at 37°C in 5% CO 2 for five weeks, and cell count per high-power field was taken to assess the growth rate. The procedure was repeated with five semen samples. Assessment of the potential for formation of embryoid bodies VSELs were separated from 1 mL aliquot of normozoospermic samples, and re-suspended in 0.25 mL of complete cell culture media. Small droplets from the cell suspension (10 µL each) were pipetted onto the lid of a 35 mm cell culture dish. The lid with the droplets was carefully inverted and placed over the culture dish containing 1 mL of cell culture media to maintain humidity and prevent drying out the droplets. The culture dishes were then incubated at 37°C in 5% CO 2 . After 24 hours, the droplets were carefully transferred into wells of a 24-well culture plate, and observed for the presence of embryoid bodies. Assessment of primitive germ cell properties primitive germ cell properties of VSELs were assessed by the expression of G-protein coupled receptor A3 (GPR-125) and alkaline phosphatase (ALP) activity. Primary antibody (Goat Anti GPR 125 antibody, Sigma, Cat No. SAB2500478) diluted 1:400 in PBS was added to the washed cells and incubated overnight at 4 0 C. Cells were washed in PBS containing 5% BSA, and incubated with a 1:400 diluted secondary antibodies (Anti-Goat IgG (whole molecule)–FITC antibody produced in rabbit, Sigma, Cat. No. F7367) for 1 hr. at room temperature. Cells were washed again with PBS. Negative controls were included in which cells were incubated in the presence of secondary antibody only to the verify specificity of staining. Fluorescence signals emitted by GPR 125 positive cells were detected using Nikon TiU microscope with FITC green filter, (20x). For the assessment of ALP activity, cells were washed with 1x PBS and incubated for six hours at room temperature in ALP staining solution (10 ml: 1 mg sodium a-naphthyl phosphate, 5 mg diazonium salt, 5% borax, and 10% MgCl2). Stained cells were viewed under the inverted microscope (40x), and the percentage of ALP positive cells was estimated ( 15 ). Data analysis was done using IBM SPSS statistics version 26.0 software. Data are represented as mean (SD), and a p value of < 0.05 was considered as statistically significant. Ethical approval for the research project was obtained the institutional ethics committee. Results A population of very small cells was consistently observed in all semen samples (n = 25) including two non-obstructive azoospermic samples. These cells were round and notably smaller than spermatids. The mean (SD) size of cells was 5.1 (0.1) ranging from 3.16–6.8 µm, and exhibited a large centrally located nucleus, resulting in a high nucleus-to-cytoplasm (N/C) ratio 1.75 (0.3). The mean (SEM) concentration of VSELs in semen was 17.21 (4.42) m/ml. VSELs count was significantly high in oligozoospermic group compared to normozoospermics (22.71 (5.89) vs 6.22 (1.81), p < 0.05) respectively. Similar observations were made with immature germ cells too. There was a positive correlation between VSEL and immature germ cells (r = 0.759, p < 0.001). Furthermore, a noticeable amount of VSELs were observed as actively dividing stages or closely contacting two cell stages (around 8%) in severe oligozoospermic samples. But, this occurrence is rare in normozoosperic samples. Multiple cell stages were rarely observed in few samples. Isolated VSELs were stained with eosin, and 97% of the cells did not take up the stain indicating the majority are viable. A clear color difference was observed among cells of similar size stained with Giemsa. Based on the color intensity, two distinct populations were identified: one appearing dark purple (53%) and the other light purple (47%) respectively. Minor subpopulations of VSELs expressing GPR125, with the mean percentage recorded as 5.56%. and positive for alkaline phosphatase activity (6.06%) were also observed. Our protocol developed for the isolation of VSELs was effective with minimal contamination of sperm (15%) and immature germ cells (1%). However, efficacy of separation was poor in samples with high sperm counts. This was partly overcome by reducing the active motile spermatozoa count using swim-up sperm separation at the first step. Figure.1 Upon transfer of hanging drops into 24-well culture plates, loosely assembled cell clumps resembling embryoid bodies were noticed. However, they were not long lasting and gradually dissociated into individual cells with time. Nevertheless, the formation of small embryoid bodies was observed with continuation of culture for five weeks. Some embryoid bodies were positive for ALP activity. The VSELs exhibited a limited growth rate in culture and the cell count was increased only by around 3% within five weeks. Cellular divisions were confirmed with the presence of both asymmetric and symmetric cellular arrangements. Furthermore, 4 and 8 cell stages and clumps with different sizes of cells were noted, indicating active proliferation and differentiation within the VSEL population. Discussion The existence of VSELs and their capacity to fulfill all the criteria utilized to define pluripotency have been an ongoing debate over two decades. However, several authors have shown the existence of very small cell population in both adult and embryonic tissues, expressing specific pattern of pluripotency ( 16 ). The regenerative potential of damaged organs such as spermatogenesis in chemo ablated testes ( 17 ), and restoration of cisplatin induced acute kidney injury by VSELs have also been reported ( 18 ). In addition to the deposition of VSELs in different organs during embryonic development, they are assumed to be mobilized from the bone marrow to peripheral blood during tissue injury and stress. Those cells can facilitate tissue regeneration by giving rise to tissue committed progenitors and stimulating mesenchymal stem cells to divide and differentiate ( 19 ). VSELs are reported to be located in the basal layer of seminiferous epithelium and are remains viable in cancer survivors even after chemo/radio therapy ( 20 ). As suggested by different authors VSELs are indeed the backup pool of stem cells which give rise to progenitor spermatogonial stem cells (SSCs) by underg oing asymmetric cell divisions ( 13 , 20 ). Successful differentiation of mouse VSELs into sperm has also been reported ( 21 ). According to our observations VSELs were found in all semen samples with the shedding of a high count of cells into the semen of oligozoospermic men as expected. We assume all the VSELs found in semen are endogenous to the testes, and contamination with peripheral blood VSELs is unlikely due to the blood testes barrier. The other significant observation is presenting a high percentage of dividing or already divided multi cell stages in oligozoospermic samples and a very rare presentation of those stages in normozoospermic samples. Amongst dividing cells majority are symmetrical divisions and occasional asymmetrical divisions. This observation provides a strong support for VSELs as an endogenous dormant stem cells population residing in seminiferous tubules, and activate with stress situations in their biological niche. However, a very low percentage of GPR125 expression and ALP activity suggest that the presence of heterogeneous cell population with different maturation stages. It is well known that ALP activity is excessively presence in migratory and early colonization periods of PGCs, and putative germ cells obtained from induced stem cells ( 22 ). Furthermore, alkaline phosphatase is considered one of the key identification markers of pluripotent embryonic and related cells ( 23 ). Some authors suggest that the VSELs are a population of pluripotent primitive stem cells and serve as a backup pool for tissue committed stem cells ( 24 ). According to our observations majority of cells were non-reactive with ALP stain and only 6% of cells were reactive. In a separate in vitro study, we observed that ALP activity is high up to the primary SSCs and declines towards secondary SSCs levels. From those evidence we suggest that only active state VSELs shows ALP activity and others may be dormant. The presence of both reactive and non-reactive two cell stages in symmetrical cell divisions provided the supporting evidence for this hypothesis. GPR 125 is expressed only in spermatogonial stem cells within the seminiferous tubules in rodents and humans ( 25 , 26 ). ID4 and GPR 125 are accepted as reliable markers for the identification of early spermatogonial subpopulations in the testes ( 27 ). Similar to ALP activity, GPR 125 was expressed only in a small subpopulation of cells. This heterogeneity was also reported with SSCs and suggested that expression of such markers may vary in a dynamic fashion according to the status of the surrounding microenvironment ( 27 ). However, identification of both ALP + and GPR 125 + cells strongly support for the presence of a sub population of VSELs with primitive or early germ cells properties. Whether the negative cells for both markers are in a dormant state or subjected to dynamic changes are questions to be answered. Similar to dark and pale cells available among SSCs we observed dark and pale color VSELs with Giemsa stain. The role of those subtypes is an avenue for further studies. Continuation of culture showed cell aggregates and formation of small embryoid bodies which are positive for ALP enzyme activity. This observation is a supportive evidence for continuous proliferation of cells in in vitro culture conditions. The major drawback in our study was to use of limited markers for characterizing the VSELs. As this is an ongoing study we will rectify all those shortcoming in the next step of the study. Conclusion To the best of our knowledge this is the first report on the presence of VSELs in human semen samples. Sub-population of cells with primitive germ cell properties would be a promising source of stem cells for future studies on in vitro spermatogenesis. Declarations Ethics approval and consent to participate Ethical approval for the research project was obtained the institutional ethics review committee (Ref. No. P/34/04/2021) Consent for publication Not applicable Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding Financial support for this research was provided by the national research council (NRC), Sri Lanka under the grant number 20-066. Authors' contributions Conceptualization: DMABD, Investigation: HMD and NRYW, Supervision: PSW Clinical trial number Not applicable References Ratajczak MZ, Zuba-Surma EK, Shin DM, et al. Very small embryonic-like stem cells (VSELs) represent a real challenge in stem cell biology: recent pros and cons in the midst of a lively debate. Stem Cells Transl Med 2014; 3(2): 140–150. Ratajczak MZ, Zuba-Surma EK, Ratajczak J, et.al. Very Small Embryonic Like (VSEL) stem cells – characterization, developmental origin and biological significance. Exp Hematol 2008; 36(6): 742–751 Taichman RS, Wang Z, Shiozawa Y, Jung Y, Song J, Balduino A, Wang J, Patel LR, Havens AM, Kucia M, Ratajczak MZ, Krebsbach PH. Prospective identification and skeletal localization of cells capable of multilineage differentiation in vivo. Stem Cells Dev 2010;19 (10): 1557–70. doi: 10.1089/scd.2009.0445 . Ratajczak MZ, Kucia M, Shin DM, et.al. A unique population of mobile very small embryonic/epiblast like (VSEL) stem cells resides in adult tissues: physiological and pathological consequences. Cellular Therapy and Transplant 2008; l (2): 36–43. doi: 10.3205/ctt-2008-en-000023.01 Zuba-Surma EK, Wojakowski W, Ratajczak MZ, Dawn B. Very small embryonic-like stem cells: biology and therapeutic potential for heart repair. Antioxid Redox Signal 2011; 15(7): 1821–34. doi: 10.1089/ars.2010.3817 . Mariusz Z. Ratajczak, Rui Liu, Wojciech Marlicz, Wojciech Blogowski, Teresa Starzynska, Wojciech Wojakowski, Ewa Zuba-Surma, Chap. 3 - Identification of Very Small Embryonic/Epiblast-Like Stem cells (VSELs) circulating in peripheral blood during organ/tissue Injuries, Editor(s): Zbigniew Darzynkiewicz, Elena Holden, Alberto Orfao, William Telford, Donald Wlodkowic, Methods in Cell Biology, Academic Press, Volume 103, 2011, p 31–54 Shin DM, Zuba-Surma EK, Ratajczak MZ, Kucia M. The unique pattern of somatic imprint in Oct-4 + Very Small Embryonic Like (VSEL) stem cells isolated from adult tissues further supports both their epiblast/germ line origin and explains quiescent status: Potential modification of somatic imprint as a key to longevity?, Blood 2008; 112 (11): 385. doi.org/10.1182/blood.V112.11.385.385 . Ratajczak MZ, Liu R, Ratajczak J, Kucia M, Shin DM. The role of pluripotent embryonic-like stem cells residing in adult tissues in regeneration and longevity. Differentiation 2011; 81(3): 153–61. doi: 10.1016/j.diff.2011.01.006 . Bhartiya D, Jha N, Tripathi A, Tripathi A. Very small embryonic-like stem cells have the potential to win the three-front war on tissue damage, cancer, and aging. Front Cell Dev Biol 2023; 10: https://doi.org/10.3389/fcell.2022.1061022 Bhartiya D. Pluripotent stem cells in adult tissues: Struggling to be acknowledged over two decades. Stem Cell Rev and Rep 2017; 13: 713–724 Virant-Klun I. 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; 14(5): 715–721. doi: 10.1007/s12015-018-9832-y . Anand S, Patel H, Bhartiya D. Chemoablated mouse seminiferous tubular cells enriched for very small embryonic-like stem cells undergo spontaneous spermatogenesis in vitro. Reprod Biol Endocrinol 2015; 13: 33–43. doi: 10.1186/s12958-015-0031-2 . Bhartiya D, Unni S, Parte S, Anand S. Very small embryonic-like stem cells: implications in reproductive biology. Biomed Res Int 2013; 2013: 682326. doi: 10.1155/2013/682326 . Bhartiya D, Anand S, Patel H. et. al. Making gametes from alternate sources of stem cells: past, present and future. Reprod Biol Endocrinol 2017; 15: 89. https://doi.org/10.1186/s12958-017-0308-8 Linher K, Dyce P, Li J. Primordial germ cell-like cells differentiated in vitro from skin-derived stem cells. PLoS ONE 2009; 4(12): e8263. doi: 10.1371/journal.pone.0008263 Virant-Klun I. Very small embryonic-like stem cells: A potential developmental link between germinal lineage and hematopoiesis in humans. Stem Cells and Devel 2016; 25(2): 101–13. https://doi.org/10.1089/scd.2015.0275 Bhatiya D. Stem cells survive oncotherapy can regenerate non-functional gonads: A paradigm shift for oncofertility. Indian J Med Res 2018; 148 (1): S38-49. DOI: 10.4103/ijmr.IJMR 2065 17 Monti M, Imberti B, Bianchi N, Pezzotta A, Morigi M, Del Fante C, Redi CA and Perotti C. A Novel Method for Isolation of Pluripotent Stem Cells from Human Umbilical Cord Blood. Stem Cells and Development 2017; 26(17): 1258–69 Sanna P, Hadi LA, Jimenez RAR, Hernandez AAB, Aya Z, et al. Very Small Embryonic Like Stem Cells: A Review of Basic Science, Applications, and Potential Use in Orthopedics. Am J Biomed Sci & Res 2022; 17(4): 442–49. DOI: 10.34297/AJBSR.2022.17.002377 Kurkure P, Prasad M, Dhamankar V. et. al. Very small embryonic-like stem cells (VSELs) detected in azoospermic testicular biopsies of adult survivors of childhood cancer. Reprod Biol Endocrinol 2015; 13 (122): https://doi.org/10.1186/s12958-015-0121-1 Bhartiya D, Anand S, Patel H. Making gametes from pluripotent stem cells: embryonic stem cells or very small embryonic-like stem cells?. Stem Cell Investigat 2016; 3: 57 Talaei-khozani T, Zarei fard N, Bahmanpour S, Jaberipour M, Hosseini A, Esmaeilpour T. Romanian Journal of Morphology and Embryology 2014; 55(2): 297–303 Stefková K, Prochazkova J, Pachernik J. Alkaline phosphatase in stem cells. Stem Cells Internat 2015; doi: 10.1155/2015/628368 . Bhartiya D, Shaikh A, Anand S, Patel H, Kapoor S, Sriraman K, Parte1 S, Unni1 S. Endogenous, very small embryonic-like stem cells: critical review, therapeutic potential and a look ahead. Hum Reprod Update 2017; 23 (1): 41–76 He Z, Kokkinaki M, Jiang J, Dobrinski I, Dym M. Isolation, characterization, and culture of human spermatogonia. Biol Reprod 2010; 82(2): 363–72. doi: 10.1095/biolreprod.109.078550 . Seandel M, James D, Shmelkov SV, Falciatori I, Kim J, Chavala S, Scherr DS, Zhang F, Torres R, Gale NW, Yancopoulos GD, Murphy A, Valenzuela DM, Hobbs RM, Pandolfi PP, Rafii S. Generation of functional multipotent adult stem cells from GPR125 + germline progenitors. Nature 2007; 449(7160): 346–50. doi: 10.1038/nature06129 . Sachs C, Robinson BD, Andres Martin L, Webster T, Gilbert M, Lo HY, Rafii S, Ng CK, Seandel M. Evaluation of candidate spermatogonial markers ID4 and GPR125 in testes of adult human cadaveric organ donors. Andrology 2014; 2(4): 607–14. doi: 10.1111/j.2047-2927.2014.00226.x . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6677204","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":466463521,"identity":"59b3e8de-1c68-4853-a106-32fda800c0bc","order_by":0,"name":"DMAB Dissanayake","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYNACNgkgwXyY4QGYx9xMrBa2ZIYEMI+RKC0ggseYOC38s3vMPvwos7CXbz/z2SBxB4M8fwNjswE+LRJ3zhjP7DknkbjhTO7mhMQzDIYzDjA2J+C15kaOMQNvm0SCAUPu5gOJbQyMG4AOO4BPhzxQC+PfNgl7+f43j0Fa7AlqMQBqYQbawthwI4c5AaglEaQFr8MMb6QVM8uA/HLjmbFBYptE8ozDBLwvdyN5M+Obsjqgw5IfS3xss7Htb28+LIFPCzoAKmYmRf0oGAWjYBSMAqwAAGMdRfXlgO+vAAAAAElFTkSuQmCC","orcid":"","institution":"University of Kelaniya","correspondingAuthor":true,"prefix":"","firstName":"DMAB","middleName":"","lastName":"Dissanayake","suffix":""},{"id":466463522,"identity":"9f1058e7-e945-49f8-9710-2886af494fca","order_by":1,"name":"MD Hettiarachchige","email":"","orcid":"","institution":"University of Kelaniya","correspondingAuthor":false,"prefix":"","firstName":"MD","middleName":"","lastName":"Hettiarachchige","suffix":""},{"id":466463523,"identity":"06d3e3ae-7681-4647-b01e-108bec4592e5","order_by":2,"name":"NRY Wijebandara","email":"","orcid":"","institution":"University of Kelaniya","correspondingAuthor":false,"prefix":"","firstName":"NRY","middleName":"","lastName":"Wijebandara","suffix":""},{"id":466463524,"identity":"963d7fe6-8c32-4635-8128-338fbd05ec95","order_by":3,"name":"PS Wijesinghe","email":"","orcid":"","institution":"University of Kelaniya","correspondingAuthor":false,"prefix":"","firstName":"PS","middleName":"","lastName":"Wijesinghe","suffix":""}],"badges":[],"createdAt":"2025-05-16 05:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6677204/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6677204/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84010141,"identity":"88e44077-2864-4740-9410-70fcb17cbb07","added_by":"auto","created_at":"2025-06-05 16:19:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":103067,"visible":true,"origin":"","legend":"\u003cp\u003eVery Small Embryonic-like Stem cells (VSELs) in human semen.\u003c/p\u003e\n\u003cp\u003ea) different sizes of VSELs in isolated germ cell fraction (arrow). b) highly purified VSELs from semen samples. c) \u003cem\u003edark\u003c/em\u003e (arrow) and \u003cem\u003epale \u003c/em\u003e(arrow head) colored cells stained with Giemsa. d) symmetrically dividing cells (one cell is ALP positive). e) asymmetrically dividing cells (one cell is ALP positive). f) VSELs aggregates. g) ALP positive cell aggregates. h) embryoid bodies. i) ALP positive EBs after five weeks of culture. J) GPR-125 positive cells.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6677204/v1/3d3eaace36ec81a06f4afe5f.jpg"},{"id":84229020,"identity":"13617a2d-1639-44d0-b91e-32fcc6507ddc","added_by":"auto","created_at":"2025-06-09 13:32:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":643874,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6677204/v1/9101a117-2493-44d6-a99b-03688141b1cb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Presence of Very Small Embryonic-Like Stem Cells (VSELs) in human semen samples; A novel finding","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStem cells are isolated from different sources such as, embryonic and extra embryonic tissues, bone marrow (BM), adipose tissues and different organs of the human body. The capacity to self-renewal and state of potency are the fundamental characteristics to select them for downstream research and clinical applications. Though, pluripotent stem cells highly fulfill the above requirements their usage has been restricted due to ethical, legal and safety issues, especially in therapeutic applications. Shedding light to a new pathway, a rare population of stem cells exhibiting some pluripotent characteristics was described in murine bone marrow by Ratajczak \u003cem\u003eet. al.\u003c/em\u003e in 2006. Named as very small embryonic-like stem cells (VSELs) reported to have a broader differentiation potential and display several epiblast and germline markers suggesting their embryonic origin (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Those cells are characterized by their small size (3\u0026ndash;6 \u0026micro;m), high nuclear/cytoplasmic ratio with a thin rim of cytoplasm and presence of euchromatin. They have been reported to express some pluripotent stem cell markers (Oct-4, Nanog, Rex-1 and SSEA-1), genes specified for both epiblast (Stella, Prdm14, Fragilis, Blimp1, Nanos3 and Dnd1) and migratory primordial germ cells (Dppa2, Dppa4 and Mvh), and negative for hematopoietic lineage markers (Lin\u003csup\u003e\u0026minus;\u003c/sup\u003eSca-1\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003e\u0026minus;\u003c/sup\u003e) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVSELs have later been identified in multiple tissues such as, BM, brain, liver, pancreas, kidney, muscles, heart, testes, and thymus, expressing tissue specific and pluripotent markers, and reside in very few numbers in a quiescent state (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Though, remain quiescent in steady states, VSELs are involved in tissue homeostasis by rejuvenation and regeneration after injuries by mobilizing into peripheral blood (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Despite their small size VSELs have diploid chromosomes, mitochondria, high telomerase activity, and do not express MHC-1 and HLA-DR antigens (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). They have the ability to organize in spheres resembling embryoid bodies when co-cultured with C2C12 cells, and hypo-methylation of somatic imprints helps to maintain their pluripotency (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). However, the lack of reproducible evidence on certain criteria shown by the embryonic stem cells such as, complete blastocyst development, and formation of teratomas after inoculation into experimental animals question the true pluripotency state of VSELs (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOverlapping populations of cells sharing similar characteristics have been described in adult tissues namely; multipotent adult progenitor cells (MAPCs), marrow-isolated adult multilineage inducible (MIAMI) cells, multipotent adult stem cells (MASCs) and omnicytes (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Considering the pluripotent nature, primitiveness, availability, lack of mitochondrial and genomic mutations, controlled proliferation capacity and broader differentiation capacity, VSELs appear to be particularly a good source compared to other cells for cellular-replacement therapy (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e gametogenesis attracted most attention in the recent past but, the reality of a clinically competent gamete is a distant dream with pluripotent stem cells. Being developmentally very related to PGCs, and expressing some migratory germ cell markers there is a great hope for the success of this endeavor with VSELs (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). \u003cem\u003eIn vitro\u003c/em\u003e culture of VSELs from ovarian epithelium gave rise to oocyte-like cells expressing pluripotency related genes and releasing Zona-pellucida like structures after sperm injection (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Culturing of VSELs collected from busulphan treated mouse testicular tissues were spontaneously differentiated into sperm in the presence of Sertoli cell conditioned medium and follicle stimulation hormone (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). It is suggested that testicular VSELs expressing nuclear Oct4-A are the true stem cells and SSCs are their progenitors giving rise to continuous sperm production (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). According to the emerging evidence VSELs in which gene expression and epigenetic status are close to natural germ cells, would be a good source of stem cells for making gametes and treating infertility (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn a pilot study, we identified VSELs in all human semen samples including samples with non-obstructive azoospermia for the first time. The present study aimed to reveal further details on this sub population of stem cells.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and enumeration of VSELs\u003c/h2\u003e \u003cp\u003eSemen samples were obtained from males undergoing subfertility evaluation at the north Colombo teaching hospital Ragama, Sri Lanka. Following liquefaction, 10 \u0026micro;L of semen sample was smeared on a clean glass slide, and stained for 10 minutes with 10% Giemsa stain. Slides were examined under a light microscope at high power field (40x). Immature germ cells and VSELs were assessed using their morphology and size, and different cell components found per 100 sperm were recorded. The size of cells was measured using Capture Pro microscopy image analysis software. Cell viability was assessed in wet mounts using eosin stain.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIsolation of VSELs from semen samples\u003c/h3\u003e\n\u003cp\u003eThis protocol was developed to effectively separate and purify VSELs from semen samples ensuring minimal contamination and maximal yield. A 1.0 mL of liquefied semen sample was diluted with phosphate buffered saline (PBS) 1:2 ratios and centrifuged at 1500 rpm for 10 minutes for pelleting the majority of sperm and other round cells. The supernatant enriched with VSELs was collected leaving 0.5 mL of bottom pellet, and re-suspended in 2.0 mL of PBS. The mixture was thoroughly mixed and centrifuged at 800 rpm for 30 minutes for further removal of remaining sperm and round cells. Following centrifugation, the supernatant was carefully transferred into a new centrifuge tube, and subjected to centrifugation at 2500 rpm for 10 minutes for pelleting the VSELs. For semen samples with high sperm concentrations, the swim-up technique was used as the first step to remove majority of active motile sperm.\u003c/p\u003e\n\u003ch3\u003eCulturing of VSELs separated from semen samples\u003c/h3\u003e\n\u003cp\u003eThe separated VSELs pellet was re-suspended in 3 mL of cell culture media, composed of DMEM/F12 supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% vitamin solution. The cell suspension was replicated into three wells in a 24-well culture plate, and the initial VSELs count per high-power field was recorded after six hours. The culture plate was subsequently incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e for five weeks, and cell count per high-power field was taken to assess the growth rate. The procedure was repeated with five semen samples.\u003c/p\u003e\n\u003ch3\u003eAssessment of the potential for formation of embryoid bodies\u003c/h3\u003e\n\u003cp\u003eVSELs were separated from 1 mL aliquot of normozoospermic samples, and re-suspended in 0.25 mL of complete cell culture media. Small droplets from the cell suspension (10 \u0026micro;L each) were pipetted onto the lid of a 35 mm cell culture dish. The lid with the droplets was carefully inverted and placed over the culture dish containing 1 mL of cell culture media to maintain humidity and prevent drying out the droplets. The culture dishes were then incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. After 24 hours, the droplets were carefully transferred into wells of a 24-well culture plate, and observed for the presence of embryoid bodies.\u003c/p\u003e\n\u003ch3\u003eAssessment of primitive germ cell properties\u003c/h3\u003e\n\u003cp\u003eprimitive germ cell properties of VSELs were assessed by the expression of G-protein coupled receptor A3 (GPR-125) and alkaline phosphatase (ALP) activity. Primary antibody (Goat Anti GPR 125 antibody, Sigma, Cat No. SAB2500478) diluted 1:400 in PBS was added to the washed cells and incubated overnight at 4 \u003csup\u003e0\u003c/sup\u003eC. Cells were washed in PBS containing 5% BSA, and incubated with a 1:400 diluted secondary antibodies (Anti-Goat IgG (whole molecule)\u0026ndash;FITC antibody produced in rabbit, Sigma, Cat. No. F7367) for 1 hr. at room temperature. Cells were washed again with PBS. Negative controls were included in which cells were incubated in the presence of secondary antibody only to the verify specificity of staining. Fluorescence signals emitted by GPR 125 positive cells were detected using Nikon TiU microscope with FITC green filter, (20x).\u003c/p\u003e \u003cp\u003eFor the assessment of ALP activity, cells were washed with 1x PBS and incubated for six hours at room temperature in ALP staining solution (10 ml: 1 mg sodium a-naphthyl phosphate, 5 mg diazonium salt, 5% borax, and 10% MgCl2). Stained cells were viewed under the inverted microscope (40x), and the percentage of ALP positive cells was estimated (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eData analysis was done using IBM SPSS statistics version 26.0 software. Data are represented as mean (SD), and a p value of \u0026lt;\u0026thinsp;0.05 was considered as statistically significant.\u003c/p\u003e \u003cp\u003e Ethical approval for the research project was obtained the institutional ethics committee.\u003c/p\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA population of very small cells was consistently observed in all semen samples (n\u0026thinsp;=\u0026thinsp;25) including two non-obstructive azoospermic samples. These cells were round and notably smaller than spermatids. The mean (SD) size of cells was 5.1 (0.1) ranging from 3.16\u0026ndash;6.8 \u0026micro;m, and exhibited a large centrally located nucleus, resulting in a high nucleus-to-cytoplasm (N/C) ratio 1.75 (0.3). The mean (SEM) concentration of VSELs in semen was 17.21 (4.42) m/ml. VSELs count was significantly high in oligozoospermic group compared to normozoospermics (22.71 (5.89) vs 6.22 (1.81), p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) respectively. Similar observations were made with immature germ cells too. There was a positive correlation between VSEL and immature germ cells (r\u0026thinsp;=\u0026thinsp;0.759, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Furthermore, a noticeable amount of VSELs were observed as actively dividing stages or closely contacting two cell stages (around 8%) in severe oligozoospermic samples. But, this occurrence is rare in normozoosperic samples. Multiple cell stages were rarely observed in few samples.\u003c/p\u003e \u003cp\u003eIsolated VSELs were stained with eosin, and 97% of the cells did not take up the stain indicating the majority are viable. A clear color difference was observed among cells of similar size stained with Giemsa. Based on the color intensity, two distinct populations were identified: one appearing dark purple (53%) and the other light purple (47%) respectively. Minor subpopulations of VSELs expressing GPR125, with the mean percentage recorded as 5.56%. and positive for alkaline phosphatase activity (6.06%) were also observed.\u003c/p\u003e \u003cp\u003eOur protocol developed for the isolation of VSELs was effective with minimal contamination of sperm (15%) and immature germ cells (1%). However, efficacy of separation was poor in samples with high sperm counts. This was partly overcome by reducing the active motile spermatozoa count using swim-up sperm separation at the first step.\u003c/p\u003e \u003cp\u003eFigure.1\u003c/p\u003e \u003cp\u003eUpon transfer of hanging drops into 24-well culture plates, loosely assembled cell clumps resembling embryoid bodies were noticed. However, they were not long lasting and gradually dissociated into individual cells with time. Nevertheless, the formation of small embryoid bodies was observed with continuation of culture for five weeks. Some embryoid bodies were positive for ALP activity.\u003c/p\u003e \u003cp\u003eThe VSELs exhibited a limited growth rate in culture and the cell count was increased only by around 3% within five weeks. Cellular divisions were confirmed with the presence of both asymmetric and symmetric cellular arrangements. Furthermore, 4 and 8 cell stages and clumps with different sizes of cells were noted, indicating active proliferation and differentiation within the VSEL population.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe existence of VSELs and their capacity to fulfill all the criteria utilized to define pluripotency have been an ongoing debate over two decades. However, several authors have shown the existence of very small cell population in both adult and embryonic tissues, expressing specific pattern of pluripotency (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The regenerative potential of damaged organs such as spermatogenesis in chemo ablated testes (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), and restoration of cisplatin induced acute kidney injury by VSELs have also been reported (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In addition to the deposition of VSELs in different organs during embryonic development, they are assumed to be mobilized from the bone marrow to peripheral blood during tissue injury and stress. Those cells can facilitate tissue regeneration by giving rise to tissue committed progenitors and stimulating mesenchymal stem cells to divide and differentiate (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVSELs are reported to be located in the basal layer of seminiferous epithelium and are remains viable in cancer survivors even after chemo/radio therapy (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). As suggested by different authors VSELs are indeed the backup pool of stem cells which give rise to progenitor spermatogonial stem cells (SSCs) by underg oing asymmetric cell divisions (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Successful differentiation of mouse VSELs into sperm has also been reported (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to our observations VSELs were found in all semen samples with the shedding of a high count of cells into the semen of oligozoospermic men as expected. We assume all the VSELs found in semen are endogenous to the testes, and contamination with peripheral blood VSELs is unlikely due to the blood testes barrier. The other significant observation is presenting a high percentage of dividing or already divided multi cell stages in oligozoospermic samples and a very rare presentation of those stages in normozoospermic samples. Amongst dividing cells majority are symmetrical divisions and occasional asymmetrical divisions. This observation provides a strong support for VSELs as an endogenous dormant stem cells population residing in seminiferous tubules, and activate with stress situations in their biological niche. However, a very low percentage of GPR125 expression and ALP activity suggest that the presence of heterogeneous cell population with different maturation stages.\u003c/p\u003e \u003cp\u003eIt is well known that ALP activity is excessively presence in migratory and early colonization periods of PGCs, and putative germ cells obtained from induced stem cells (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Furthermore, alkaline phosphatase is considered one of the key identification markers of pluripotent embryonic and related cells (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Some authors suggest that the VSELs are a population of pluripotent primitive stem cells and serve as a backup pool for tissue committed stem cells (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). According to our observations majority of cells were non-reactive with ALP stain and only 6% of cells were reactive. In a separate \u003cem\u003ein vitro\u003c/em\u003e study, we observed that ALP activity is high up to the primary SSCs and declines towards secondary SSCs levels. From those evidence we suggest that only active state VSELs shows ALP activity and others may be dormant. The presence of both reactive and non-reactive two cell stages in symmetrical cell divisions provided the supporting evidence for this hypothesis.\u003c/p\u003e \u003cp\u003eGPR 125 is expressed only in spermatogonial stem cells within the seminiferous tubules in rodents and humans (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). ID4 and GPR 125 are accepted as reliable markers for the identification of early spermatogonial subpopulations in the testes (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Similar to ALP activity, GPR 125 was expressed only in a small subpopulation of cells. This heterogeneity was also reported with SSCs and suggested that expression of such markers may vary in a dynamic fashion according to the status of the surrounding microenvironment (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). However, identification of both ALP\u003csup\u003e+\u003c/sup\u003e and GPR 125\u003csup\u003e+\u003c/sup\u003e cells strongly support for the presence of a sub population of VSELs with primitive or early germ cells properties. Whether the negative cells for both markers are in a dormant state or subjected to dynamic changes are questions to be answered.\u003c/p\u003e \u003cp\u003eSimilar to dark and pale cells available among SSCs we observed dark and pale color VSELs with Giemsa stain. The role of those subtypes is an avenue for further studies. Continuation of culture showed cell aggregates and formation of small embryoid bodies which are positive for ALP enzyme activity. This observation is a supportive evidence for continuous proliferation of cells in \u003cem\u003ein vitro\u003c/em\u003e culture conditions. The major drawback in our study was to use of limited markers for characterizing the VSELs. As this is an ongoing study we will rectify all those shortcoming in the next step of the study.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo the best of our knowledge this is the first report on the presence of VSELs in human semen samples. Sub-population of cells with primitive germ cell properties would be a promising source of stem cells for future studies on \u003cem\u003ein vitro\u003c/em\u003e spermatogenesis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval for the research project was obtained the institutional ethics review committee (Ref. No. P/34/04/2021)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinancial support for this research was provided by the national research council (NRC), Sri Lanka under the grant number 20-066.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: DMABD, Investigation: HMD and NRYW, Supervision: PSW\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRatajczak MZ, Zuba-Surma EK, Shin DM, et al. Very small embryonic-like stem cells (VSELs) represent a real challenge in stem cell biology: recent pros and cons in the midst of a lively debate. Stem Cells Transl Med 2014; 3(2): 140\u0026ndash;150.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRatajczak MZ, Zuba-Surma EK, Ratajczak J, et.al. Very Small Embryonic Like (VSEL) stem cells \u0026ndash; characterization, developmental origin and biological significance. Exp Hematol 2008; 36(6): 742\u0026ndash;751\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaichman RS, Wang Z, Shiozawa Y, Jung Y, Song J, Balduino A, Wang J, Patel LR, Havens AM, Kucia M, Ratajczak MZ, Krebsbach PH. Prospective identification and skeletal localization of cells capable of multilineage differentiation in vivo. Stem Cells Dev 2010;19 (10): 1557\u0026ndash;70. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1089/scd.2009.0445\u003c/span\u003e\u003cspan address=\"10.1089/scd.2009.0445\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRatajczak MZ, Kucia M, Shin DM, et.al. A unique population of mobile very small embryonic/epiblast like (VSEL) stem cells resides in adult tissues: physiological and pathological consequences. Cellular Therapy and Transplant 2008; l (2): 36\u0026ndash;43. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3205/ctt-2008-en-000023.01\u003c/span\u003e\u003cspan address=\"10.3205/ctt-2008-en-000023.01\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZuba-Surma EK, Wojakowski W, Ratajczak MZ, Dawn B. Very small embryonic-like stem cells: biology and therapeutic potential for heart repair. Antioxid Redox Signal 2011; 15(7): 1821\u0026ndash;34. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1089/ars.2010.3817\u003c/span\u003e\u003cspan address=\"10.1089/ars.2010.3817\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMariusz Z. Ratajczak, Rui Liu, Wojciech Marlicz, Wojciech Blogowski, Teresa Starzynska, Wojciech Wojakowski, Ewa Zuba-Surma, Chap. 3 - Identification of Very Small Embryonic/Epiblast-Like Stem cells (VSELs) circulating in peripheral blood during organ/tissue Injuries, Editor(s): Zbigniew Darzynkiewicz, Elena Holden, Alberto Orfao, William Telford, Donald Wlodkowic, Methods in Cell Biology, Academic Press, Volume 103, 2011, p 31\u0026ndash;54\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShin DM, Zuba-Surma EK, Ratajczak MZ, Kucia M. The unique pattern of somatic imprint in Oct-4\u0026thinsp;+\u0026thinsp;Very Small Embryonic Like (VSEL) stem cells isolated from adult tissues further supports both their epiblast/germ line origin and explains quiescent status: Potential modification of somatic imprint as a key to longevity?, Blood 2008; 112 (11): 385. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.1182/blood.V112.11.385.385\u003c/span\u003e\u003cspan address=\"10.1182/blood.V112.11.385.385\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRatajczak MZ, Liu R, Ratajczak J, Kucia M, Shin DM. The role of pluripotent embryonic-like stem cells residing in adult tissues in regeneration and longevity. Differentiation 2011; 81(3): 153\u0026ndash;61. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.diff.2011.01.006\u003c/span\u003e\u003cspan address=\"10.1016/j.diff.2011.01.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhartiya D, Jha N, Tripathi A, Tripathi A. Very small embryonic-like stem cells have the potential to win the three-front war on tissue damage, cancer, and aging. Front Cell Dev Biol 2023; 10: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fcell.2022.1061022\u003c/span\u003e\u003cspan address=\"10.3389/fcell.2022.1061022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhartiya D. Pluripotent stem cells in adult tissues: Struggling to be acknowledged over two decades. Stem Cell Rev and Rep 2017; 13: 713\u0026ndash;724\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVirant-Klun I. 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; 14(5): 715\u0026ndash;721. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12015-018-9832-y\u003c/span\u003e\u003cspan address=\"10.1007/s12015-018-9832-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnand S, Patel H, Bhartiya D. Chemoablated mouse seminiferous tubular cells enriched for very small embryonic-like stem cells undergo spontaneous spermatogenesis in vitro. Reprod Biol Endocrinol 2015; 13: 33\u0026ndash;43. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12958-015-0031-2\u003c/span\u003e\u003cspan address=\"10.1186/s12958-015-0031-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhartiya D, Unni S, Parte S, Anand S. Very small embryonic-like stem cells: implications in reproductive biology. Biomed Res Int 2013; 2013: 682326. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2013/682326\u003c/span\u003e\u003cspan address=\"10.1155/2013/682326\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhartiya D, Anand S, Patel H. et. al. Making gametes from alternate sources of stem cells: past, present and future. Reprod Biol Endocrinol 2017; 15: 89. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12958-017-0308-8\u003c/span\u003e\u003cspan address=\"10.1186/s12958-017-0308-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLinher K, Dyce P, Li J. Primordial germ cell-like cells differentiated in vitro from skin-derived stem cells. PLoS ONE 2009; 4(12): e8263. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0008263\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0008263\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVirant-Klun I. Very small embryonic-like stem cells: A potential developmental link between germinal lineage and hematopoiesis in humans. Stem Cells and Devel 2016; 25(2): 101\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1089/scd.2015.0275\u003c/span\u003e\u003cspan address=\"10.1089/scd.2015.0275\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhatiya D. Stem cells survive oncotherapy can regenerate non-functional gonads: A paradigm shift for oncofertility. Indian J Med Res 2018; 148 (1): S38-49. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4103/ijmr.IJMR\u003c/span\u003e\u003cspan address=\"10.4103/ijmr.IJMR\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e 2065 17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMonti M, Imberti B, Bianchi N, Pezzotta A, Morigi M, Del Fante C, Redi CA and Perotti C. A Novel Method for Isolation of Pluripotent Stem Cells from Human Umbilical Cord Blood. Stem Cells and Development 2017; 26(17): 1258\u0026ndash;69\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanna P, Hadi LA, Jimenez RAR, Hernandez AAB, Aya Z, et al. Very Small Embryonic Like Stem Cells: A Review of Basic Science, Applications, and Potential Use in Orthopedics. Am J Biomed Sci \u0026amp; Res 2022; 17(4): 442\u0026ndash;49. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.34297/AJBSR.2022.17.002377\u003c/span\u003e\u003cspan address=\"10.34297/AJBSR.2022.17.002377\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurkure P, Prasad M, Dhamankar V. et. al. Very small embryonic-like stem cells (VSELs) detected in azoospermic testicular biopsies of adult survivors of childhood cancer. Reprod Biol Endocrinol 2015; 13 (122): \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12958-015-0121-1\u003c/span\u003e\u003cspan address=\"10.1186/s12958-015-0121-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhartiya D, Anand S, Patel H. Making gametes from pluripotent stem cells: embryonic stem cells or very small embryonic-like stem cells?. Stem Cell Investigat 2016; 3: 57\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalaei-khozani T, Zarei fard N, Bahmanpour S, Jaberipour M, Hosseini A, Esmaeilpour T. Romanian Journal of Morphology and Embryology 2014; 55(2): 297\u0026ndash;303\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStefkov\u0026aacute; K, Prochazkova J, Pachernik J. Alkaline phosphatase in stem cells. Stem Cells Internat 2015; doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2015/628368\u003c/span\u003e\u003cspan address=\"10.1155/2015/628368\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhartiya D, Shaikh A, Anand S, Patel H, Kapoor S, Sriraman K, Parte1 S, Unni1 S. Endogenous, very small embryonic-like stem cells: critical review, therapeutic potential and a look ahead. Hum Reprod Update 2017; 23 (1): 41\u0026ndash;76\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe Z, Kokkinaki M, Jiang J, Dobrinski I, Dym M. Isolation, characterization, and culture of human spermatogonia. Biol Reprod 2010; 82(2): 363\u0026ndash;72. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1095/biolreprod.109.078550\u003c/span\u003e\u003cspan address=\"10.1095/biolreprod.109.078550\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeandel M, James D, Shmelkov SV, Falciatori I, Kim J, Chavala S, Scherr DS, Zhang F, Torres R, Gale NW, Yancopoulos GD, Murphy A, Valenzuela DM, Hobbs RM, Pandolfi PP, Rafii S. Generation of functional multipotent adult stem cells from GPR125\u0026thinsp;+\u0026thinsp;germline progenitors. Nature 2007; 449(7160): 346\u0026ndash;50. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature06129\u003c/span\u003e\u003cspan address=\"10.1038/nature06129\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSachs C, Robinson BD, Andres Martin L, Webster T, Gilbert M, Lo HY, Rafii S, Ng CK, Seandel M. Evaluation of candidate spermatogonial markers ID4 and GPR125 in testes of adult human cadaveric organ donors. Andrology 2014; 2(4): 607\u0026ndash;14. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.2047-2927.2014.00226.x\u003c/span\u003e\u003cspan address=\"10.1111/j.2047-2927.2014.00226.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Germ cells, In vitro spermatogenesis, Seminal fluid, VSELs","lastPublishedDoi":"10.21203/rs.3.rs-6677204/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6677204/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePresence of Very Small Embryonic-Like Stem Cells (VSELs) in human semen samples; A novel finding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003eThe presence of a very small, quiescent, and pluripotent population of stem cells termed as “VSELs” was first reported in 2006. Those cells have observed in various organs in the mammalian body including bone marrow, peripheral blood, endometrium, ovary and testis so on. But their existence in seminal plasma has not been reported so far. The aim of the present study was to find out whether VSELs are present in human semen samples and if so, study their characteristics and relationship with semen parameters. Semen samples were collected from sub fertile men and analyzed according to WHO guidelines. VSELs in seminal plasma were detected using Giemsa stain. The presence of cells with primitive germ cell properties was confirmed by GPR125 expression and alkaline phosphatase activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003eA very small and round cell population, notably smaller than spermatids was observed in all semen samples. The mean (SD) size of cells was 5.1 (0.1) ranging from 3.16 - 6.8 µm, and exhibited a large centrally located nucleus, resulting in a high nucleus-to-cytoplasm (N/C) ratio of 1.75 (0.3). The mean (SEM) concentration of VSELs was 17.21 (4.42) m/ml. VSELs count was significantly high in oligozoospermics compared to normozoospermics (22.71 (5.89) vs 6.22 (1.81), p \u0026lt; 0.05) respectively. There was a positive correlation between VSEL and immature germ cells (r = 0.759, p \u0026lt; 0.001). Two distinct populations of cells were identified according to their color intensity; VSEL dark and pale, 53% and 47% respectively. Minor percentages of sub-populations positive for alkaline phosphatase activity (6.06%) and expressing GPR 125 (5.56%) were also observed. Very slow rate of propagation (3%) and the ability to make embryoid bodies were exhibited by the isolated cells cultured for five weeks.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e To the best of our knowledge this is the first report on the presence of VSELs in semen samples. Sub-population of cells with primitive germ cell properties would be a good source of stem cells for future studies on \u003cem\u003ein vitro \u003c/em\u003espermatogenesis.\u003c/p\u003e","manuscriptTitle":"Presence of Very Small Embryonic-Like Stem Cells (VSELs) in human semen samples; A novel finding","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-05 16:19:20","doi":"10.21203/rs.3.rs-6677204/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"459c4a03-d187-4c4a-a28d-9af735dae14a","owner":[],"postedDate":"June 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T13:23:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-05 16:19:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6677204","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6677204","identity":"rs-6677204","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}