Background Expressing several markers of migrating primordial germ cells (PGCs), the rare population of quiescent, bone marrow (BM)-residing very small embryonic-like stem cells (VSELs) can be specified like PGCs into hematopoietic stem/progenitor cells (HSPCs). erythropoietin – EpoR axes on these cells. Results We report here that murine and human VSELs as well as murine and human Nestoron teratocarcinoma cell lines and ovarian cancer cell lines share a functional EpoR. Conclusions Our data provide more evidence of a potential developmental link between germline cells, VSELs, and HSCs and sheds more light around the developmental hierarchy of the stem cell Nestoron compartment in adult tissues. cultures that murine PGCs isolated from embryos, stem cells isolated from murine testes [4], and teratocarcinoma cell lines can be specified Nestoron into hematopoietic stem/progenitor cells (HSPCs) [11]. Our recent work demonstrated the presence of small, quiescent, Sca-1+Lin-CD45- stem cells in adult murine BM and small CD133+Lin-CD45- cells in human BM and umbilical cord Nestoron blood (UCB) [12-16]. These cells, under appropriate co-culture conditions with OP-9 stromal cells, can be specified into HSPCs [17], and, based on the presence of a primitive type of chromatin in their nuclei as well as expression of embryonic stem cell markers such as Oct-4 and Nanog, these small cells were named very small embryonic-like stem cells (VSELs). Interestingly, murine VSELs express several markers, such as and that are shared with migratory primordial germ cells PGCs [18,19]. Based on these observations, we have proposed a developmental link between PGCs, VSELs, and HSCs [17,19]. We have also hypothesized that these cells, if mutated, may give rise to certain types of malignancies [20], which may reconcile the presence of these cells in adult tissues with the more than 150-year-old embryonic rest hypothesis of cancer development. The erythropoietin receptor (EpoR) is well known to be essential for production of red blood cells. Recently, however, evidence has accumulated that it is also expressed in non-hematopoietic tissues (e.g., some neuronal cells) [21]. Accordingly, EpoR has also been reported to be expressed by several types of malignant cells, including ovarian cancer cell lines [22,23]. These findings prompted us to evaluate the expression of EpoR on murine and human VSELs as well as murine and human germline-derived cell lines. The presence of EpoR on VSELs and germline cell lines provides new evidence for a developmental link between VSELs, HSCs, and the Nestoron germline and sheds more light around the developmental hierarchy within the adult stem cell compartment. Materials and methods Murine BM and human umbilical cord blood cells and cell lines Mononuclear cells were isolated from BM of 4C6-week-old C57Bl/6 mice and human umbilical cord blood in agreement with approval by the Animal Care and Use Committee (IACUC) and Institutional Review Board (IRB) of the University of Louisville (Louisville, KY). All cell lines employed in our studies (P19, NTERA2, A2780) were purchased from ATCC (Manassas, VA, USA). C57Bl/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Clinical-grade UCB research units were shipped from the Cleveland Cord Blood Center. Isolation of murine VSELs Mice were sacrificed and bone marrow was flushed from the femur and tibia. A single-cell suspension was obtained by agitation through the syringe. Cells were lysed in BD lysing buffer (BD Pharmingen) for 10?min at room temperature and washed twice in RPMI medium with 2% FBS. The cell suspension was stained for the VSEL phenotype using antibodies: anti-CD45R/B220 (PE, clone RA-6B2), anti-Gr-1 (PE, clone RB6-8 C5), anti-T cell receptor (PE, clone H57-5970), anti-T cell receptor ? (PE, clone GL3), anti-CD11b (PE, clone M1/70), anti-Ter119 (PE, clone TER-119), anti-CD45 (allophycocyaninCCy7, clone 30?F11) and anti-Ly-6A/E (also known as Sca-1, PECCy5 or Alexa Fluor 647, clone E13-161.7) for 30?minutes on ice. Cells were washed and resuspended in RPMI medium with 2% fetal bovine serum. Populations of Lin-/CD45-/Sca-1+ (VSELs) and Lin-/CD45+/Sca-1+ (HSCs) were sorted with a Moflo XDP cell sorter (Beckman Coulter). Isolation of human VSELs Clinical-grade UCB research units were shipped from Cleveland Cord Blood Center. Total nucleated cells (TNCs) were isolated by hypotonic lysing of red blood cells (RBCs) employing BD PharmLyse lysing buffer (2) for 10?minutes at room temperature. RAB7A After a washing step, magnetic labeling of TNCs for the depletion of mature hematopoietic lineage-positive cells (Lineage Cell Depletion kit, Miltenyi Biotec) was performed. The population of lineage-negative cells was isolated by using an autoMACS separator (Miltenyi Biotec GMBH, Germany) and incubated with antibodies: anti-CD2 (FITC, clone RPA-2.10), anti-CD3 (FITC, clone UCHT1), anti-CD14 (FITC, clone M5E2), anti-CD16 (FITC, clone 3G8), anti-CD19 (FITC, clone HIB19), anti-CD24 (FITC, clone ML5), anti-CD56 (also known as N-CAM, FITC, clone NCAM16.2), anti-CD66b (FITC, clone G10F5), anti-CD235a (FITC, clone GA-R2 [HIR2]), anti-CD45 (PE or V450, clone.