- Open Access
Expression of the erythropoietin receptor by germline-derived cells - further support for a potential developmental link between the germline and hematopoiesis
© Suszynska et al.; licensee BioMed Central Ltd. 2014
Received: 3 May 2014
Accepted: 11 June 2014
Published: 17 June 2014
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). These two properties of VSELs support the possibility of a developmental origin of HSPCs from migrating PGCs.
To address a potential link between VSELs and germ line cells we analyzed by RT-PCR and FACS expression of erythropoietin receptor (EpoR) on murine bone marrow- and human umbilical cord blood-derived VSELs, murine and human teratocarcinoma cell lines and human ovarian cancer cells. A proper gating strategy and immunostaining excluded from FACS analysis potential contamination by erythroblasts. Furthermore, the transwell chemotaxis assays as well as adhesion and signaling studies were performed to demonstrate functionality of erythropoietin - EpoR axes on these cells.
We report here that murine and human VSELs as well as murine and human teratocarcinoma cell lines and ovarian cancer cell lines share a functional EpoR.
Our data provide more evidence of a potential developmental link between germline cells, VSELs, and HSCs and sheds more light on the developmental hierarchy of the stem cell compartment in adult tissues.
The recent hot debate over the existence of developmentally primitive stem cells in bone marrow (BM) with broad differentiation potential has been fueled by the challenge these cells pose to the accepted hierarchy within the stem cell compartment of hematopoietic tissues . Also surprising is the accumulating evidence that hematopoietic stem cells (HSCs) are specified from a population of migrating primordial germ cells (PGCs) during embryogenesis [2–4] and that stem cells with germ cell potential reside in adult BM [5, 6].
In support of this intriguing linkage, HSCs and PGCs are highly migratory cells, and specification of the first primitive HSCs in the yolk sac blood islands as well as the origin of definitive HSCs in the aorta–gonado–mesonephros (AGM) region are chronologically and anatomically correlated with the developmental migration of PGCs in extra- and intra-embryonic tissues . In addition, several papers have described the sharing of chromosomal aberrations between germline tumors and leukemias or lymphomas, which suggests their clonal origin [8–10]. Furthermore, it has been demonstrated in in vitro cultures that murine PGCs isolated from embryos, stem cells isolated from murine testes , and teratocarcinoma cell lines can be specified into hematopoietic stem/progenitor cells (HSPCs) .
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 blood (UCB) [12–16]. These cells, under appropriate co-culture conditions with OP-9 stromal cells, can be specified into HSPCs , 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 Stella, Prdm14, Fragilis, Blimp1, Nanos3, and Dnd1, 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 , 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) . 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 germline and sheds more light on 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 4–6-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 αβ (PE, clone H57-5970), anti-T cell receptor ɣδ (PE, clone GL3), anti-CD11b (PE, clone M1/70), anti-Ter119 (PE, clone TER-119), anti-CD45 (allophycocyanin–Cy7, clone 30 F11) and anti-Ly-6A/E (also known as Sca-1, PE–Cy5 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. 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 HI30), and anti-CD34 (APC or PE, clone 581) or anti-CD133/1 (APC or PE, clone AC133). Cells were washed, resuspended, and sorted using a MoFlo XDP cell sorter (Beckman Coulter) to obtain populations enriched in VSELs (Lin-/CD45-/CD133+ or Lin-/CD45-/CD34+) as well as hematopoietic stem/progenitor cells (HSPCs, Lin-/CD45+/CD133+ or Lin-/CD45+/CD34+).
In vivo BrdU labelling studies with murine VSELs
C57BL/6 mice (4 to 6 weeks old) were injected subcutaneously with human recombinant erythropoietin (Epogen, 25 U/day, 10 times, every third day) and injected intra-peritoneally with BrdU solution (1 mg/animal, BD Pharmingen) every working day. Control mice were injected with saline and BrdU solution. After 30 days, mice were sacrificed, and a single-cell suspension was obtained and stained for the VSEL phenotype as previously described. After cell-surface staining of cells, the FITC BrdU Flow kit (BD Pharmingen) staining protocol was used, which includes fixation and permeabilization of cells, treatment of cells with DNase to expose incorporated BrdU, and staining with anti-BrdU–FITC antibody. After washing, samples were analyzed using a BD LSR II flow cytometer (BD Biosciences). At least 106 events were acquired and analyzed using BD FACSDiva software.
Analysis of VSELs for the presence of erythroid markers
A single-cell suspension was obtained from 4- to 6-week-old C57BL/6 mice and stained for the VSEL phenotype as previously described. Additionally, cells ware stained for markers characteristic of erythroid cells using antibodies: anti-Ter119 (V450, clone TER-119) and anti-CD71 (FITC, clone C2) for 30 minutes on ice. After washing, cells were fixed and permeabilized with BD Cytofix/Cytoperm™ solution for 15 minutes on ice. The fluorescent DNA intercalator 7AAD was added to samples to visualize only flow cytometer events with DNA content. At least 2×106 nucleated events were acquired using a BD LSR II flow cytometer (BD Biosciences, San Jose, CA).
Detection of EpoR expression by FACS
Single-cell suspensions of mononuclear cells were obtained from UCB research units and stained for the VSEL phenotype as previously described. Additionally, cells ware stained for markers characteristic of erythroid cells using antibodies: anti-CD235a (PE–Cy7, clone GA-R2 [HIR2]) and anti-EpoR (APC, clone 38421) for 30 minutes on ice. After washing, cells were fixed and permeabilized with BD Cytofix/Cytoperm™ solution for 15 minutes on ice. 7AAD was added to samples to visualize only events exhibiting DNA content. At least 2×106 nucleated events were acquired using a BD LSR II flow cytometer (BD Biosciences, San Jose, CA).
Immunohistochemical staining of FACS-sorted mBM-derived VSELs
Lin-/CD45-/Sca-1+ murine VSELs as well as HSCs were sorted, plated on 22-mm diameter plates coated with poly-L-lysine (P9155, Sigma, MO, USA), and incubated for 24 h. Subsequently, cells were fixed in 3.5% paraformaldehyde for 15 min, permeabilized by 0.1% Triton X100, washed in PBS, and pre-blocked with 2.5% BSA for 2.5 h at RT to avoid nonspecific binding of antibodies. Immunocytochemistry for POU5F1 (clone 7 F9.2, mouse monoclonal IgG1κ, Millipore, MA, USA), and EpoR (rabbit polyclonal IgG, Abcam, Cambridge, MA, USA) proteins was performed with appropriate secondary Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-rabbit IgG antibodies. Nuclei were stained with DAPI (Invitrogen) for 20 min at 37°C. All images were captured with an Olympus FV1000 confocal microscope.
Detection of EpoR expression by real-time quantitative PCR (RQ-PCR)
Total RNA from FACS-sorted or cultured cells was isolated using the RNeasy Mini kit (Qiagen Inc, Valencia, CA, USA), with removal of genomic DNA using the DNA-free™ kit (Life Technologies, Grand Island, NY, USA). cDNA was prepared with Taqman Reverse Transcription Reagents (Applied Biosystems, Grand Island, NY, USA ), according to the manufacturer’s instructions. Quantitative assessment of mRNA levels of target genes was performed by RQ-PCR using an ABI Prism 7500 sequence detection system (Applied Biosystems). The cDNA templates from each cell were amplified using SYBR Green PCR master mix (Applied Biosystems), and specific primers (mEPO R: forward, 5′-cattctggtcctcatctcgctg-3′, reverse, 5′-tgaagagaccctcaaactcgct-3′; hEPO R: forward, 5′-ccatggacactgtgccctg-3′, reverse, 5′- ccatcggataagccccctt-3′) were designed with Primer Express software (Applied Biosystems). The threshold cycle (Ct), the cycle number at which the fluorescence of the amplified gene reached a fixed threshold, was subsequently determined, and the relative quantification of the expression level of target genes was performed with the 2–ΔΔCt method.
Total RNA from various cells was isolated using the RNeasy Mini kit (Qiagen Inc.), including treatment with DNase I (Qiagen Inc.). The mRNA (200 ng) was reverse-transcribed with Taqman Reverse Transcription Reagents (Applied Biosystems) according to the manufacturer’s instructions. The resulting cDNA fragments were amplified using Amplitaq Gold at 1 cycle of 8 min at 95°C, 2 cycles of 2 min at 95°C, 1 min at 60°C, 1 min at 72°C, and subsequently by 37 cycles of 30 s at 95°C, 1 min at 60°C, 1 min at 72°C, and 1 cycle of 10 min at 72°C by using sequence-specific primers (mEpoR: F5′-AGGGCTGCATCATGGACAAA-3′, R5′-GGTGATAGCGAGGAGAACCG-3′; hEpoR: F5′-CTCCCTTTGTCTCCTGCTCG-3′, R5′-TAGGCAGCGAACACCAGAAG-3′). All primers were designed using the NCBI/Primer-Blast program, as at least one primer included an exon–intron boundary.
Chemotaxis assays were performed in a modified Boyden’s chamber with 8-μm pore polycarbonate membrane inserts (Costar Transwell; Corning Costar, Lowell, MA, USA) as described previously. In brief, cells detached with 0.25% trypsin were seeded into the upper chamber of an insert at a density of 6 × 104 in 100 μl. The lower chamber was filled with pre-warmed culture medium containing test reagents. Medium supplemented with 0.5% BSA was used as a negative control. After 24 hours, the inserts were removed from the Transwell supports. The cells that had not migrated were scraped off with cotton swap from the upper membrane, and the cells that had transmigrated to the lower side of the membrane were fixed and stained with HEMA 3 (protocol, Fisher Scientific, Pittsburgh, PA) and counted on the lower side of the membrane using an inverted microscope.
Cell adhesion assay
P19 murine teratocarcinoma cells were made quiescent by incubation for 3 h at 37°C in αMEM medium supplemented with 0.5% BSA. The cells were then stimulated with EpO 0.1, 1, and 100 U/ml for 5 min at 37°C, then added to plates treated with fibronectin (10 μg/ml) and incubated for 5 min at 37°C. After the non-adherent cells had been discarded, cells that adhered to the stromal cells were stained and counted under an inverted fluorescence microscope.
Human and murine teratocarcinoma or ovarian cancer cells were kept overnight in medium containing low levels of bovine serum albumin (BSA, 0.5%) to render the cells quiescent. After the cells were stimulated with EpO (1 or 100 U/ml for the P19 cell line; 0.5 or 10 U/ml for the NTERA2 cell line; 50, 100, or 150 U/ml for the A2780 cell line) for 5 min, cells were lysed for 20 min on ice in RIPA lysis buffer containing protease and phosphatase inhibitors (Santa Cruz Biotech, Santa Cruz, CA). The extracted proteins were separated on a 4–12% SDS-PAGE gel and transferred to a PVDF membrane. The phosphorylation of the p44/42 mitogen-activated kinase (phospho-p44/42 MAPK) was detected by phospho-specific p44/42 MAPK mouse and rabbit polyclonal antibodies (Cell Signaling, Danvers, MA, USA) with HRP-conjugated goat anti-mouse and anti-rabbit secondary antibodies (Santa Cruz Biotech). Equal loading of protein in all the lanes was evaluated by stripping the blots and reprobing with anti-p42/44 MAPK monoclonal antibody (clone no. 9102, Cell Signaling). The membranes were developed with an enhanced chemiluminescence (ECL) reagent (Amersham Life Sciences, Arlington Heights, IL), dried, and subsequently exposed to film (Hyperfilm, Amersham Life Sciences).
All results were presented as mean ± SD. Statistical analysis of the data was performed using Student’s t-test for unpaired samples, with p < 0.05 considered significant.
Effect of EpO on proliferation of VSELs in murine BM
Murine and human VSELs express EpoR
Functional EpoR is expressed by murine and human germline-derived cell lines
Our current understanding of stem cells in adult tissues suffers somewhat from the lack of a broader view of the stem cell compartment, which is a continuum between early stages of development and adult postnatal tissues. The recent hot debate on the existence in BM of early-development stem cells, such as spore-like stem cells, multipotent adult stem cells (MASCs) , multilineage-differentiating, stress-enduring (Muse) cells [31–33], multipotent adult progenitor cells (MAPCs) [34, 35], marrow-isolated adult multilineage-inducible (MIAMI) cells , multipotent progenitor cells (MPCs) [30, 37], and very small embryonic-like stem cells (VSELs) [12–14, 38] with broader specification, resulted from the implicit challenge of these cells to the accepted hierarchy within the stem cell compartment in adult tissues.
We have proposed that VSELs initially identified by us [12, 13, 38] and confirmed by others [39–43] in murine and human adult tissues are a link between early stages of development and the compartment of adult stem cells that reside in adult tissues. The epigenetic mechanism identified in VSELs, which changes expression of certain genes involved in insulin/insulin-like growth factor signaling (IIS), keeps these cells in a highly quiescent state in adult tissues [44–47]. However, we have demonstrated that both murine and human VSELs may be specified into the hematopoietic lineage. This finding was indirectly corroborated by a recent report that the most primitive HSCs in murine BM, corresponding to long-term repopulating hematopoietic stem cells (LT-HSCs), like VSELs, are kept quiescent by changes in certain paternally imprinted genes involved in IIS [24, 47]. In addition, in an old report [17, 48] small murine BM-derived cells that share many characteristics with VSELs have been demonstrated to be a population of LT-HSCs.
The existence in BM of developmentally primitive stem cells with broader specification challenged the accepted hierarchy within the stem cell compartment in murine BM and the developmental position in this hierarchy of HSCs. Evidence has accumulated that HSCs can become specified from a population of migrating primordial germ cells (PGCs) during embryogenesis [3–5]. In support of this intriguing possibility, HSCs and PGCs are highly migratory cells, and specification of the first primitive HSCs in yolk sac blood islands as well as the origin of definitive HSCs in the aorta–gonado–mesonephros (AGM) region are chronologically and anatomically correlated with the developmental migration of PGCs in extra- and intra-embryonic tissues . Moreover, murine VSELs express several markers, such as Stella, Prdm14, Fragilis, Blimp1, Nanos3, and Dnd1, that are shared with migratory primordial germ cells PGCs [18, 19]. Based on these data, we have postulated a potential developmental link between PGCs, VSELs, and HSCs [3, 5, 19], and, as reported here in this study, the presence of EpoR on VSELs purified from murine BM and human UCB lends further support to this hypothesis.
We are aware that expression of EpoR (detected by FACS) on human VSELs was low (~15%). This result, however, depends on the affinity of antibodies against EpoR as well as the number of copies of the receptor on the cell surface. Moreover, we have demonstrated that VSELs residing in adult BM are somewhat heterogeneous, being most likely (more or less) pre-committed to various lineages [17, 39–43, 49]. We cannot exclude the possibility that the cells that highly express EpoR as detected by FACS staining are committed to HSCs. On the other hand, as we have demonstrated, VSELs do not express classical erythroid lineage markers such as Terr119 and CD71, and if this hematopoietic pre-commitment occurs in BM-residing VSELs, it occurs at the level of LT- HSCs.
In further support of a link between hematopoiesis and the germline, it has been demonstrated that PGCs isolated from murine embryos are able to grow hematopoietic colonies in vitro . Furthermore, the presence of early-development stem cells with germline markers in BM that are able to differentiate along the germline lineage has already been demonstrated by several investigators. For example, murine BM SSEA-1+ cells stimulated by bone morphogenic factor 4 may differentiate Oct-4+Stella+Mvh+ cells into gamete precursors . Similarly, a population of Oct-4+Mvh+Dazl+Stella+ cells present in murine BM has been reported to affect the recurrence of oogenesis in mice sterilized by chemotherapy [51, 52], and Oct-4+Mvh+Stella+ cells isolated as Stra8–GFP cells from BM of Stra8–GFP transgenic mice express several molecular markers of spermatogonial stem cells and spermatogonia . Furthermore, Oct-4+SSEA-1/3/4+ cells isolated from chicken BM are able to give rise to functional sperm after injection into testes . In addition to BM, stem cells with germline potential have also been isolated from newborn mouse skin and porcine skin as well as porcine adipose tissue .
The identification of early-development stem cells in adult tissues raises several questions, such as: i) Are these cells functional and do they play a role in tissue/organ rejuvenation? ii) Are they involved in regulating life span of the individual? iii) Are they involved in regeneration of damaged tissues? iv) If regulatory mechanisms fail, could these cells give rise to malignancies?
To address this last question, the concept that adult tissues contain developmentally primitive cells with embryonic features that can lead to tumors is, surprisingly, not so novel. During the 19th and early 20th centuries, it was proposed that cancer may develop in populations of cells that are left in a dormant state in developing organs during embryogenesis. This so-called “embryonic rest hypothesis of cancer origin” was proposed by Virchow (1855), Durante (1874), and Conheim (1875) [55–57]. According to these pathologists, adult tissues contain embryonic remnants that normally lie dormant but that can be activated to become cancerous. In agreement with these theories, Wright (1910) proposed a germinal cell origin of nephroblastoma, and Beard (1911) proposed that tumors arise from displaced and activated trophoblasts or germ cells [58, 59]. However, the putative cells responsible for these effects were at that time neither clearly identified nor purified from adult tissues. As mentioned above, we propose that mutated VSELs could be the missing link in this hypothesis by being the origin of these malignancies.
There are several pieces of evidence supporting the embryonic rest hypothesis of cancer development and the potential involvement of VSELs. First, there is the existence of classical germline tumors seen in patients with seminomas, ovarian tumors, yolk sac tumors, mediastinal or brain germ cell tumors, teratomas, and teratocarcinomas [60, 61]. Second, several types of cancer cells express cancer testis (C/T) antigens (~40 identified), which are encoded by genes that are normally expressed only in the human germline but are also often expressed unexpectedly in various non-gonadal tumor types (e.g., gastric, lung, liver, and bladder carcinomas) [62, 63]. Third, in several types of malignancies, embryonic markers, such as chorionic gonadotropin (hCG) and/or carcinoembryonic antigen (CEA), are detected in patient plasma . Finally, it is known that several solid tumors (e.g., gastric, lung, bladder, and oral mucosa carcinomas as well as germinal tumors) express, like VSELs, the embryonic/germline transcription factor Oct-4 [65, 66]. However, we are aware that more studies are needed to shed light on the potential role of VSELs in tumorigenesis.
In conclusion, we postulate that some support for the embryonic rest hypothesis of cancer development is lent by described by us herein presence of EpoR in both VSELs and germline-derived cell lines. On the other hand, since EpoR has been described in established cell lines of several malignancies [8, 11, 22, 23, 25], though it may be barely detectable on cells isolated from primary tumors [67, 68], we envision that it could be a novel marker of cancer stem cells, for example, in gonadal malignancies (e.g., ovarian cancer stem cells). This, however, requires further study. Finally, since it is known that EPO signals in erythroid cells via EpoR-EpoR homodimers and in nonerythroid cells may signal via EpoR-CD131 heterodimers , thus while this paper was in review we evaluated by FACS expression of CD131 on human germ-line derived cells (VSELs, NTERA2, Ovarian Cancer) and did not find expression of CD131 (not shown).
This work was supported by NIH grants 2R01 DK074720 and R01HL112788, the Stella and Henry Endowment, and Maestro grant 2011/02/A/NZ4/00035 to MZR.
- Suszynska M, Zuba-Surma EK, Maj M, Mierzejewska K, Ratajczak J, Kucia M, Ratajczak MZ: The proper criteria for identification and sorting of very small embryonic-like stem cells, and some nomenclature issues. Stem Cells Dev 2014, 23: 702–713.PubMed CentralView ArticlePubMedGoogle Scholar
- Ohtaka T, Matsui Y, Obinata M: Hematopoietic development of primordial germ cell-derived mouse embryonic germ cells in culture. Biochem Biophys Res Com 1999, 260: 475–482.View ArticlePubMedGoogle Scholar
- Rich IN: Primordial germ cells are capable of producing cells of the hematopoietic system in vitro. Blood 1995, 86: 463–472.PubMedGoogle Scholar
- Yoshimoto M, Heike T, Chang H, Kanatsu-Shinohara M, Baba S, Varnau JT, Shinohara T, Yoder MC, Nakahata T: Bone marrow engraftment but limited expansion of hematopoietic cells from multipotent germline stem cells derived from neonatal mouse testis. Exp Hematol 2009, 37: 1400–1410.PubMed CentralView ArticlePubMedGoogle Scholar
- Kritzenberger M, Wrobel KH: Histochemical in situ identification of bovine embryonic blood cells reveals differences to the adult haematopoietic system and suggests a close relationship between haematopoietic stem cells and primordial germ cells. Histochem Cell Biol 2004, 121: 273–289.View ArticlePubMedGoogle Scholar
- Nayernia K, Lee JH, Drusenheimer N, Nolte J, Wulf G, Dressel R, Gromoll J, Engel W: Derivation of male germ cells from bone marrow stem cells. Lab Invest 2006, 86: 654–663.View ArticlePubMedGoogle Scholar
- De Miguel MP, Arnalich Montiel F, Lopez Iglesias P, Blazquez Martinez A, Nistal M: Epiblast-derived stem cells in embryonic and adult tissues. Int J Dev Biol 2009, 53: 1529–1540.View ArticlePubMedGoogle Scholar
- Woodruff K, Wang N, May W, Adrone E, Denny C, Feig SA: The clonal nature of mediastinal germ cell tumors and acute myelogenous leukemia. A case report and review of the literatur. Cancer Genet Cytogenet 1995, 79: 25–31.View ArticlePubMedGoogle Scholar
- Chaganti RS, Ladanyi M, Samaniego F, Offit K, Reuter VE, Jhanwar SC, Bosl GJ: Leukemic differentiation of a mediastinal germ cell tumor. Genes Chromosomes Cancer 1989, 1: 83–87.View ArticlePubMedGoogle Scholar
- Nichols CR, Hoffman R, Einhorn LH, Williams SD, Wheeler LA, Garnick MB: Hematologic malignancies associated with primary mediastinal germ-cell tumors. Ann Intern Med 1985, 102: 603–609.View ArticlePubMedGoogle Scholar
- Miwa Y, Atsumi T, Imai N, Ikawa Y: Primitive erythropoiesis of mouse teratocarcinoma stem cells PCC3/A/1 in serum-free medium. Development 1991, 111: 543–549.PubMedGoogle Scholar
- Kucia M, Reca R, Campbell FR, Zuba-Surma E, Majka M, Ratajczak J, Ratajczak MZ: A population of very small embryonic-like (VSEL) CXCR4(+)SSEA-1(+)Oct-4+ stem cells identified in adult bone marrow. Leukemia 2006, 20: 857–869.View ArticlePubMedGoogle Scholar
- Kucia M, Wu W, Ratajczak MZ: Bone marrow-derived very small embryonic-like stem cells: their developmental origin and biological significance. Develop Dynamics 2007, 236: 3309–3320.View ArticleGoogle Scholar
- Kucia M, Wysoczynski M, Ratajczak J, Ratajczak MZ: Identification of very small embryonic like (VSEL) stem cells in bone marrow. Cell Tissue Res 2008, 331: 125–134.View ArticlePubMedGoogle Scholar
- McGuckin CP, Forraz N, Baradez MO, Navran S, Zhao J, Urban R, Tilton R, Denner L: Production of stem cells with embryonic characteristics from human umbilical cord blood. Cell Prolif 2005, 38: 245–255.View ArticlePubMedGoogle Scholar
- Bhartiya D, Shaikh A, Nagvenkar P, Kasiviswanathan S, Pethe P, Pawani H, Mohanty S, Rao SG, Zaveri K, Hinduja I: 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 Dev 2012, 21: 1–6.View ArticlePubMedGoogle Scholar
- Ratajczak J, Wysoczynski M, Zuba-Surma E, Wan W, Kucia M, Yoder MC, Ratajczak MZ: Adult murine bone marrow-derived very small embryonic-like stem cells differentiate into the hematopoietic lineage after coculture over OP9 stromal cells. Exp Hematol 2011, 39: 225–237.PubMed CentralView ArticlePubMedGoogle Scholar
- Shin DM, Liu R, Klich I, Ratajczak J, Kucia M, Ratajczak MZ: Molecular characterization of isolated from murine adult tissues very small embryonic/epiblast like stem cells (VSELs). Mol Cells 2010, 29: 533–538.View ArticlePubMedGoogle Scholar
- Shin DM, Liu R, Klich I, Wu W, Ratajczak J, Kucia M, Ratajczak MZ: Molecular signature of adult bone marrow-purified very small embryonic-like stem cells supports their developmental epiblast/germ line origin. Leukemia 2010, 24: 1450–1461.View ArticlePubMedGoogle Scholar
- Ratajczak MZ, Shin DM, Kucia M: Very small embryonic/epiblast-like stem cells: a missing link to support the germ line hypothesis of cancer development? Am J Pathol 2009, 174: 1985–1992.PubMed CentralView ArticlePubMedGoogle Scholar
- Miljus N, Heibeck S, Jarrar M, Micke M, Ostrowski D, Ehrenreich H, Heinrich R: Erythropoietin-mediated protection of insect brain neurons involves JAK and STAT but not PI3K transduction pathways. Neuroscience 2014, 258: 218–227.View ArticlePubMedGoogle Scholar
- Solar P, Hrckova G, Varinska L, Solarova Z, Kriska J, Uhrinova I, Kello M, Mojzis J, Fedorocko P, Sytkowski AJ: Location and the functionality of erythropoietin receptor(s) in A2780 cells. Oncol Rep 2012, 28: 141–146.PubMedGoogle Scholar
- Jeong JY, Feldman L, Solar P, Szenajch J, Sytkowski AJ: Characterization of erythropoietin receptor and erythropoietin expression and function in human ovarian cancer cells. Int J Cancer 2008, 122: 274–280.View ArticlePubMedGoogle Scholar
- Shin DM, Zuba-Surma EK, Wu W, Ratajczak J, Wysoczynski M, Ratajczak MZ, Kucia M: Novel epigenetic mechanisms that control pluripotency and quiescence of adult bone marrow-derived Oct4(+) very small embryonic-like stem cells. Leukemia 2009, 23: 2042–2051.PubMed CentralView ArticlePubMedGoogle Scholar
- Sytkowski AJ: Does erythropoietin have a dark side? Epo signaling and cancer cells. Sci STKE 2007, 395: pe38.Google Scholar
- Dagnon K, Pacary E, Commo F, Antoine M, Bernaudin M, Bernaudin JF, Callard P: Expression of erythropoietin and erythropoietin receptor in non-small cell lung carcinomas. Clin Cancer Res 2005, 11: 993–999.PubMedGoogle Scholar
- Arcasoy MO, Amin K, Vollmer RT, Jiang X, Demark-Wahnefried W, Haroon ZA: Erythropoietin and erythropoietin receptor expression in human prostate cancer. Mod Pathol 2005, 18: 421–430.View ArticlePubMedGoogle Scholar
- Yasuda Y, Musha T, Tanaka H, Fujita Y, Fujita H, Utsumi H, Matsuo T, Masuda S, Nagao M, Sasaki R, Nakamura Y: Inhibition of erythropoietin signalling destroys xenografts of ovarian and uterine cancers in nude mice. Br J Cancer 2001, 84: 836–843.PubMed CentralView ArticlePubMedGoogle Scholar
- McBroom JW, Acs G, Rose GS, Krivak TC, Mohyeldin A, Verma A: Erythropoietin receptor function and expression in epithelial ovarian carcinoma. Gynecol Oncol 2005, 99: 571–577.View ArticlePubMedGoogle Scholar
- Beltrami AP, Cesselli D, Bergamin N, Marcon P, Rigo S, Puppato E, D’Aurizio F, Verardo R, Piazza S, Pignatelli A, Poz A, Baccarani U, Damiani D, Fanin R, Mariuzzi L, Finato N, Masolini P, Burelli S, Belluzzi O, Schneider C, Beltrami CA: Multipotent cells can be generated in vitro from several adult human organs (heart, liver, and bone marrow). Blood 2007, 110: 3438–3446.View ArticlePubMedGoogle Scholar
- Wakao S, Akashi H, Kushida Y, Dezawa M: Muse cells, newly found non-tumorigenic pluripotent stem cells, reside in human mesenchymal tissues. Pathol Int 2014, 64: 1–9.View ArticlePubMedGoogle Scholar
- Wakao S, Kitada M, Kuroda Y, Shigemoto T, Matsuse D, Akashi H, Tanimura Y, Tsuchiyama K, Kikuchi T, Goda M, Nakahata T, Fujiyoshi Y, Dezawa M: Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts. Proc Natl Acad Sci U S A 2011, 108: 9875–9880.PubMed CentralView ArticlePubMedGoogle Scholar
- Kuroda Y, Wakao S, Kitada M, Murakami T, Nojima M, Dezawa M: Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells. Nat Protoc 2013, 8: 1391–1415.View ArticlePubMedGoogle Scholar
- Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM: Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002, 418: 41–49.View ArticlePubMedGoogle Scholar
- Jiang Y, Vaessen B, Lenvik T, Blackstad M, Reyes M, Verfaillie CM: Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp Hematol 2002,30(8):896–904.View ArticlePubMedGoogle Scholar
- D’Ippolito G, Diabira S, Howard GA, Menei P, Roos BA, Schiller PC: Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J Cell Sci 2004, 117: 2971–2981.View ArticlePubMedGoogle Scholar
- Cesselli D, Beltrami AP, Rigo S, Bergamin N, D’Aurizio F, Verardo R, Piazza S, Klaric E, Fanin R, Toffoletto B, Marzinotto S, Mariuzzi L, Finato N, Pandolfi M, Leri A, Schneider C, Beltrami CA, Anversa P: Multipotent progenitor cells are present in human peripheral blood. Circ Res 2009, 104: 1225–1234.View ArticlePubMedGoogle Scholar
- Kucia M, Halasa M, Wysoczynski M, Baskiewicz-Masiuk M, Moldenhawer S, Zuba-Surma E, Czajka R, Wojakowski W, Machalinski B, Ratajczak MZ: Morphological and molecular characterization of novel population of CXCR4+ SSEA-4+ Oct-4+ very small embryonic-like cells purified from human cord blood: preliminary report. Leukemia 2007, 21: 297–303.View ArticlePubMedGoogle Scholar
- Parte S, Bhartiya D, Telang J, Daithankar V, Salvi V, Zaveri K, Hinduja I: Detection, characterization, and spontaneous differentiation in vitro of very small embryonic-like putative stem cells in adult mammalian ovary. Stem Cells Dev 2011, 20: 1451–1464.PubMed CentralView ArticlePubMedGoogle Scholar
- Havens AM, Shiozawa Y, Jung Y, Sun H, Wang J, McGee S, Mishra A, Taichman LS, Danciu T, Jiang Y, Yavanian G, Leary E, Krebsbach PH, Rodgerson D, Taichman RS: Human very small embryonic-like cells generate skeletal structures, in vivo. Stem Cells Dev 2013, 22: 622–630.PubMed CentralView ArticlePubMedGoogle Scholar
- Kassmer SH, Jin H, Zhang PX, Bruscia EM, Heydari K, Lee JH, Kim CF, Krause DS: Very small embryonic-like stem cells from the murine bone marrow differentiate into epithelial cells of the lung. Stem Cells 2013, 31: 2759–2766.PubMed CentralView ArticlePubMedGoogle Scholar
- Virant-Klun I, Zech N, Rozman P, Vogler A, Cvjeticanin B, Klemenc P, Malicev E, Meden-Vrtovec H: Putative stem cells with an embryonic character isolated from the ovarian surface epithelium of women with no naturally present follicles and oocytes. Differentiation 2008, 76: 843–856.View ArticlePubMedGoogle Scholar
- Havens AM, Sun H, Shiozawa Y, Jung Y, Wang J, Mishra A, Jiang Y, O’Neill DW, Krebsbach PH, Rodgerson DO, Taichman RS: Human and murine very small embryonic-like cells represent multipotent tissue progenitors, in vitro and in vivo. Stem Cells Dev 2014, 23: 689–701.PubMed CentralView ArticlePubMedGoogle Scholar
- Kucia M, Masternak M, Liu R, Shin DM, Ratajczak J, Mierzejewska K, Spong A, Kopchick JJ, Bartke A, Ratajczak MZ: The negative effect of prolonged somatotrophic/insulin signaling on an adult bone marrow-residing population of pluripotent very small embryonic-like stem cells (VSELs). Age 2013, 35: 315–330.PubMed CentralView ArticlePubMedGoogle Scholar
- Ratajczak J, Shin DM, Wan W, Liu R, Masternak MM, Piotrowska K, Wiszniewska B, Kucia M, Bartke A, Ratajczak MZ: Higher number of stem cells in the bone marrow of circulating low Igf-1 level Laron dwarf mice–novel view on Igf-1, stem cells and aging. Leukemia 2011, 25: 729–733.PubMed CentralView ArticlePubMedGoogle Scholar
- Ratajczak MZ, Zuba-Surma E, Wojakowski W, Suszynska M, Mierzejewska K, Liu R, Ratajczak J, Shin DM, Kucia M: 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. Leukemia 2014, 28: 473–484.PubMed CentralView ArticlePubMedGoogle Scholar
- Ratajczak MZ, Shin DM, Schneider G, Ratajczak J, Kucia M: Parental imprinting regulates insulin-like growth factor signaling: a Rosetta Stone for understanding the biology of pluripotent stem cells, aging and cancerogenesi. Leukemia 2013, 27: 773–779.View ArticlePubMedGoogle Scholar
- Ratajczak J, Zuba-Surma E, Klich I, Liu R, Wysoczynski M, Greco N, Kucia M, Laughlin MJ, Ratajczak MZ: Hematopoietic differentiation of umbilical cord blood-derived very small embryonic/epiblast-like stem cells. Leukemia 2011, 25: 1278–1285.PubMed CentralView ArticlePubMedGoogle Scholar
- 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: 153–161.View ArticlePubMedGoogle Scholar
- Shirazi R, Zarnani AH, Soleimani M, Abdolvahabi MA, Nayernia K, Ragerdi Kashani I: BMP4 can generate primordial germ cells from bone-marrow-derived pluripotent stem cells. Cell Biol Int 2012, 36: 1185–1193.View ArticlePubMedGoogle Scholar
- Johnson J, Bagley J, Skaznik-Wikiel M, Lee HJ, Adams GB, Niikura Y, Tschudy KS, Tilly JC, Cortes ML, Forkert R, Spitzer T, Iacomini J, Scadden DT, Tilly JL: Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood. Cell 2005, 122: 303–315.View ArticlePubMedGoogle Scholar
- Selesniemi K, Lee HJ, Niikura T, Tilly JL: Young adult donor bone marrow infusions into female mice postpone age-related reproductive failure and improve offspring survival. Aging 2009, 1: 49–57.PubMed CentralGoogle Scholar
- Heo YT, Lee SH, Yang JH, Kim T, Lee HT: Bone marrow cell-mediated production of transgenic chickens. Lab Invest 2011, 91: 1229–1240.PubMed CentralView ArticlePubMedGoogle Scholar
- Dyce PW, Liu J, Tayade C, Kidder GM, Betts DH, Li J: In vitro and in vivo germ line potential of stem cells derived from newborn mouse skin. PLoS One 2011, 6: e20339.PubMed CentralView ArticlePubMedGoogle Scholar
- Durante F: Nesso fisio-pathologico tra la struttura dei nei materni e la genesi di alcuni tumori maligni. 1847, 11: 217–226.Google Scholar
- Conheim J: Congenitales, quergestreiftes Muskelsarkon der Nireren. Virchows Arch 1875, 65: 64.View ArticleGoogle Scholar
- Virchow R: Editorial. Archive fuer pathologische Anatomie und Physiologie fuer klinische Medizin 1855, 8: 23–54.Google Scholar
- Wright JHW: Neurocytoma or Neuroblastoma, a kind of tumor not generally recognized. J Exp Med 1910, 12: 556–561.PubMed CentralView ArticlePubMedGoogle Scholar
- Beard J: The enzyme treatment of cancer. London: Chatto and Windus; 1911.Google Scholar
- Andrews PW, Matin MM, Bahrami AR, Damjanov I, Gokhale P, Draper JS: Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin. Biochem Soc Trans 2005, 33: 1526–1530.View ArticlePubMedGoogle Scholar
- Macchiarini P, Ostertag H: Uncommon primary mediastinal tumours. Lancet Oncol 2004, 5: 107–118.View ArticlePubMedGoogle Scholar
- Sigalotti L, Covre A, Zabierowski S, Himes B, Colizzi F, Natali PG, Herlyn M, Maio M: Cancer testis antigens in human melanoma stem cells: expression, distribution, and methylation status. J Cell Physiol 2008, 215: 287–291.PubMed CentralView ArticlePubMedGoogle Scholar
- Simpson AJ, Caballero OL, Jungbluth A, Chen YT, Old LJ: Cancer/testis antigens, gametogenesis and cancer. Nat Rev Cancer 2005, 5: 615–625.View ArticlePubMedGoogle Scholar
- Hotakainen K, Ljungberg B, Haglund C, Nordling S, Paju A, Stenman UH: Expression of the free beta-subunit of human chorionic gonadotropin in renal cell carcinoma: prognostic study on tissue and serum. Int J Cancer 2003, 104: 631–635.View ArticlePubMedGoogle Scholar
- Cheng L: Establishing a germ cell origin for metastatic tumors using OCT4 immunohistochemistry. Cancer 2004, 101: 2006–2010.View ArticlePubMedGoogle Scholar
- Lee AS, Tang C, Rao MS, Weissman IL, Wu JC: Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies. Nat Med 2013, 19: 998–1004.PubMed CentralView ArticlePubMedGoogle Scholar
- Saito A, Watanabe K, Kusakabe T, Abe M, Suzuki T: Mediastinal mature teratoma with coexistence of angiosarcoma, granulocytic sarcoma and a hematopoietic region in the tumor: a rare case of association between hematological malignancy and mediastinal germ cell tumor. Pathol Int 1998, 48: 749–753.View ArticlePubMedGoogle Scholar
- Elliott S, Swift S, Busse L, Scully S, Van G, Rossi J, Johnson C: Epo receptors are not detectable in primary human tumor tissue samples. PLoS One 2013, 8: e68083.PubMed CentralView ArticlePubMedGoogle Scholar
- Broxmeyer HE: Erythropoietin: multiple targets, actions, and modifying influences for biological and clinical consideration. J Exp Med 2013, 210: 205–208.PubMed CentralView ArticlePubMedGoogle Scholar
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