Very Small Embryonic/epiblast-like Stem Cells Research Articles

HISTOLOGICAL STRUCTURE AND FUNCTIONAL PROPERTIES OF THE TUNICA ALBUGINEA OF THE OVARY

Just below the surface epithelium of the ovary is the tunica albuginea, which is a tight irregular connective tissue structure that gives the tissue its white color and contains fibroblast cells. Tunica albuginea, which is more resistant to environmental factors, contains fewer cells and is rich in collagen fibers, is observed as the niche of preantral follicles. It has been observed that fibroblasts forming collagen fibers provide the development of follicles with the paracrine factors and cytokines they secrete and function as a nourishing cell layer. After the graaf follicle forms the corpus luteum, fibroblasts in the adjacent tunica albuginea proliferate and the thickness of this structure increases. The tunica albuginea undergoes frequent renewal due to the corpus luteum structures formed in rats. When the corpus luteum is formed, new capillaries and venules are formed in the adjacent tunica albuginea. This structure may be a suitable model for investigating the migration of cells from the bone marrow to the ovary via vessels. It can be suggested that mesenchymal cells and very small embryonic/epiblast-like stem cells (VSELs), which show pluripotent stem cell characteristics, may migrate from the bone marrow to the tunica albuginea through vascular structures and that the bone marrow may be the source of these cells, which have been previously shown to be present in the ovary. The aim of this review is to examine the effects of the tunica albuginea on the development of follicles and the dynamic structure of the ovary.

Effect of colorectal cancer on the number of normal stem cells circulating in peripheral blood.

Bone marrow(BM) residing stem cells are mobilized from their BM niches into peripheral blood(PB) in several pathological situations including tissue organ injury and systemic inflammation. We recently reported that the number of BM-derived stem cells(SCs) increases in patients with pancreatic and stomach cancer. Accordingly, we observed higher numbers of circulating very small embryonic/epiblast‑like stem cells(VSELs) and mesenchymal stem cells(MSCs) that were associated with the activation of pro-mobilizing complement cascade and an elevated level of sphingosine-1 phosphate(S1P) in PB plasma. We wondered if a similar correlation occurs in patients with colorectal cancer(CRC). A total of 46 patients were enrolled in this study: 17 with CRC, 18 with benign colonic adenomas(BCA) and 11 healthy individuals. By employing fluorescence-activated cell sorting(FACS) we evaluated the number of BM-derived SCs circulating in PB: i)CD34+/Lin-/CD45- and CD133-/Lin-/CD45- VSELs; ii)CD45-/CD105+/CD90+/CD29+ MSCs; iii)CD45-/CD34+/CD133+/KDR+ endothelial progenitor cells(EPCs); and iv)CD133+/Lin-/CD45+ or CD34+/Lin-/CD45+ cells enriched for hematopoietic stem/progenitor cells(HSPCs). In parallel, we measured in the PB parameters regulating the egress of SCs from BM into PB. In contrast to pancreatic and gastric cancer patients, CRC subjects presented neither an increase in the number of circulating SCs nor the activation of pro-mobilizing factors such as complement, coagulation and fibrinolytic cascade, circulating stromal derived factor1(SDF‑1), vascular endothelial growth factor(VEGF) and intestinal permeability marker (zonulin). In conclusion, mobilization of SCs in cancer patients depends on the type of malignancy and its ability to activate pro-mobilization cascades.

Peripheral trafficking of bone marrow-derived stem cells in patients with different types of gastric neoplasms: Significance of complement and interleukins.

49 Background: Increasing evidence suggests that gastric malignancy may be a stem cell disorder. Methods: Using FACS analysis supported by ImageStream technique verification, various populations of bone marrow-derived stem cells (BMSCs), such as Lin-CD45-CD133+CXCR4+ very small embryonic/epiblast-like stem cells (VSELs), CD45-STRO-1+CD105+ mesenchymal stem cells (MSCs), Lin-CD45+CD133+ hematopoietic stem/progenitor cells (HSPCs), and CD34 +/ KDR +/ CD31 +/ CD45- endothelial progenitor cells (EPCs) were enumerated and sorted from peripheral blood samples of patients with gastric cancer (n = 23) and other gastric tumors (GISTs, NENs, lymphomas; n = 15) and control patients (n = 15). RT-qPCR analysis for the expression of cancerous, embryonic, and tissue trans-differentiation markers (Oct-4, Nanog, TFF-1, TFF-2, Lgr-5, BMI-1) in circulating VSELs was employed. The plasma levels of stromal-derived factor-1 (SDF-1), complement-derived anaphylatoxins/molecules (C3a, C5a, C5b-9/MAC), and interleukins (IL-6, IL-8, IL-17 and IL-23) were measured. Results: Significantly lower numbers of circulating HSPCs, and intensified peripheral trafficking of both VSELs and MSCs was observed in patients with gastric cancer, but not in those with other types of gastric tumors. In gastric cancer patients, the circulating VSELs presented higher gene expression of Oct-4, Nanog, Lgr-5, BMI-1, and TFF-1/2. The abnormal balance in the peripheral trafficking of BMSCs in patients with gastric cancer was not associated with the clinical stage of the disease or with the systemic levels of stromal-derived factor-1 (SDF-1), but correlated with the levels of C5b-9/MAC, IL-6, IL-8 and IL-23. Conclusions: Abnormal peripheral trafficking of BMSCs occurs in patients with gastric cancer, but not in those with other types of gastric neoplasms. Selected interleukins and complement cascade-derived molecules, but not SDF-1, seem to be significant players associated with this phenomenon (POIG.01.01.02-00-109/09 and IP2014003273).

Peripheral trafficking of bone-marrow-derived stem cells in patients with different types of gastric neoplasms

ABSTRACTRecently, there has been a growing interest in the importance of stem cells (SCs) in the development/progression of gastric neoplasms. In this study, we performed a comprehensive analysis of different populations of bone-marrow-derived stem cells (BMSCs) in patients with various types of gastric malignancies, including gastric cancer, gastrointestinal stromal tumors (GISTs), neuroendocrine neoplasms (NENs), and lymphomas. We found significantly lower numbers of circulating Lin-/CD45 +/ CD133 + hematopoietic stem/progenitor cells (HSPCs), and intensified peripheral trafficking of both Lin−/CD45−/CXCR4+/CD34+/CD133+ very small embryonic/epiblast-like stem cells (VSELs) and CD105 + /STRO-1 +/ CD45- mesenchymal SCs (MSCs) in patients with gastric cancer, but not in those with other types of gastric neoplasms. No significant differences in the absolute numbers of circulating CD34 +/ KDR +/ CD31 +/ CD45- endothelial progenitor cells (EPCs) were observed between the groups. This abnormal balance in the peripheral trafficking of BMSCs in patients with gastric cancer was neither associated with clinical stage of the disease nor with systemic levels of stromal-derived factor-1 (SDF-1), as these were comparable to the values observed in control individuals. Interestingly, the absolute numbers of circulating BMSCs correlated with the concentrations of complement cascade-derived anaphylatoxins/molecules (mainly C5b-9/membrane attack complex-MAC) and sphingosine-1-phosphate (S1P). In summary, our translational study revealed that abnormal peripheral trafficking of BMSCs occurs in patients with gastric cancer, but not in those with other types of gastric neoplasms. Further, our findings indicate that highlighted complement cascade-derived molecules and S1P, but not SDF-1, are significant players associated with this phenomenon.

Evidence that the population of quiescent bone marrow‐residing very small embryonic/epiblast‐like stem cells (VSELs) expands in response to neurotoxic treatment

The concept that bone marrow (BM)-derived cells may participate in neural regeneration remains controversial, and the identity of the specific cell type(s) involved remains unknown. We recently reported that the adult murine BM contains a highly mobile population of Sca-1+Lin−CD45− cells known as very small embryonic/epiblast-like stem cells (VSELs) that express several markers of pluripotency such as Oct-4. In the BM microenvironment, these cells are kept quiescent because of epigenetic modification of certain paternally imprinted genes. However, as reported, these cells can be mobilized in mice in an experimental model of stroke and express several genes involved in neurogenesis while circulating in peripheral blood (PB). In the current work, we employed a model of toxic brain damage, which is induced by administration of kainic acid, to see not only whether VSELs can be mobilized into PB in response to this neurotoxin, but, more importantly, whether they proliferate and expand in BM tissue. We report here for the first time that brain damage leads to activation and expansion of the BM pool of quiescent VSELs, which precedes their subsequent egress into PB. Harnessing these cells in neural tissue regeneration is currently one of the challenges in regenerative medicine.

The embryonic rest hypothesis of cancer development – an old XIX century theory revisited

In the middle of XIX century, two German pathologists Rudolf Virchow and Julius Cohnheim proposed the intriguing hypothesis that cancer may develop from cell remnants that remain in the developing organs following embryo- genesis. This hypothesis is known as the embryonic rest hypothesis of cancer development and was popular among pathologist in the 19th and 20th centuries and later on somehow abandoned. In fact the morphology of most primitive tumors often mimics developmentally early tissues, and such tumors may express markers that are characteristic for epiblast and/or germ line cells. This observation could reflect the phenomenon of dedifferentiation of somatic cells in which cancer develops to moreprimitivestateorincontrastapossibilitythatcanceroriginatesinprimitivestemcellscloselyrelatedtotheepiblast/germline. The identification of primitive epiblast/germline-derived so called very small embryonic/epiblast-like stem cells (VSELs) in adult organs raised the possibility that these cells may give rise to some malignancies. In addition VSELs may also support tumor growthbyprovidingstromaandareinvolvedinneovasculogenesis.Inthisreview,wewillpresentahypothesizethatVSELscould be a missing link that supports the rest- or germline-origin hypothesis of cancer development; however, further experimental evidence is needed to support this intriguing concept.

Circulating bone marrow-derived stem cells in patients with pancreatic cancer.

200 Background: Various experimental studies suggest that bone marrow-derived stem cells (BMSCs) may be involved in the development and progression of pancreatic cancer. However, these observations require further studies, and delineation of exact mechanisms responsible for mobilization of BMSCs from the bone marrow to peripheral blood. Methods: A total of 23 patients with newly diagnosed pancreatic cancer (17 with distal metastases) and 15 healthy individuals were recruited to this study. Using fluorescence-activated cell sorter circulating hematopoietic (HSPCs), mesenchymal (MSCs), endothelial (EPCs), and very small embryonic/epiblast-like stem cells (VSELs) were enumerated and sorted. Plasma levels of various growth/inhibitory factors (hepatocyte [HGF], vascular-endothelial [VEGF], leukemia [LIF]), stem cells’ chemoattractants (stromal-derived factor-1 [SDF-1], sphingosine-1-phosphate [S1P]), and complement molecules (anaphylatoxins [C3a, C5a] and complexes [C5b-9/MAC]) were measured. Results: Significantly higher numbers of circulating VSELs and MSCs were detected in pancreatic cancer patients (in both p<0.05), while the numbers of circulating HSPCs and EPCs were comparable between the analyzed groups. Among analyzed biochemical and immunomodulatory factors, levels of C5a, C5b-9/MAC, HGF and S1P, but not of SDF-1, LIF nor VEGF were associated with observed mobilization of BMSCs. Conclusions: In patients with pancreatic cancer selective mobilization of BMSCs from the bone marrow to peripheral blood is observed. This seems to be strongly associated with increased levels of immunomodulatory substances. Our study also highlights that it is S1P and HGF but not SDF-1 that seem to be associated with BMSCs mobilization from the bone marrow to peripheral blood in patients with pancreatic cancer. (MNiSW grant 402 423038).

Very small embryonic/epiblast-like stem cells (VSELs) and their potential role in aging and organ rejuvenation – an update and comparison to other primitive small stem cells isolated from adult tissues

Very small embryonic-like stem cells (VSELs) are a population of developmentally early stem cells residing in adult tissues. These rare cells, which are slightly smaller than red blood cells, i) become mobilized during stress situations into peripheral blood, ii) are enriched in the Sca1+Lin-CD45- cell fraction in mice and the CD133+ Lin-CD45- cell fraction in humans, iii) express markers of pluripotent stem cells (e.g., Oct4, Nanog, and SSEA), and iv) display a distinct morphology characterized by a high nuclear/cytoplasmic ratio and undifferentiated chromatin. Recent evidence indicates that murine VSELs are kept quiescent in adult tissues and protected from teratoma formation by epigenetic modification of imprinted genes that regulate insulin/insulin like growth factor signaling (IIS). The successful reversal of these epigenetic changes in VSELs that render them quiescent will be crucial for efficient expansion of these cells. The most recent data in vivo from our and other laboratories demonstrated that both murine and human VSELs exhibit some characteristics of long-term repopulating hematopoietic stem cells (LT-HSCs), are at the top of the hierarchy in the mesenchymal lineage, and may differentiate into organ-specific cells (e.g., cardiomyocytes). Moreover, as recently demonstrated the number of these cells positively correlates in several murine models with longevity. Finally, while murine BM-derived VSELs have been extensively characterized more work is needed to better characterize these small cells at the molecular level in humans.

Clinical Evidence for Mobilization of Very Small Embryonic/Epiblast Like Stem Cells (VSEL-SCs) in Patients With Inflammatory Bowel Disease

Clinical Evidence for Mobilization of Very Small Embryonic/Epiblast Like Stem Cells (VSEL-SCs) in Patients With Inflammatory Bowel Disease

Hematopoietic differentiation of umbilical cord blood-derived very small embryonic/epiblast-like stem cells

A population of CD133+lin−CD45− very small embryonic-like stem cells (VSELs) has been purified by multiparameter sorting from umbilical cord blood (UCB). In order to speed up isolation of these cells, we employed anti-CD133-conjugated paramagnetic beads followed by staining with Aldefluor to detect aldehyde dehydrogenase (ALDH) activity; we subsequently sorted CD45−/GlyA−/CD133+/ALDHhigh and CD45−/GlyA−/CD133+/ALDHlow cells, which are enriched for VSELs, and CD45+/GlyA−/CD133+/ALDHhigh and CD45+/GlyA−/CD133+/ALDHlow cells, which are enriched for hematopoietic stem/progenitor cells (HSPCs). While freshly isolated CD45− VSELs did not grow hematopoietic colonies, the same cells, when activated/expanded over OP9 stromal support, acquired hematopoietic potential and grew colonies composed of CD45+ hematopoietic cells in methylcellulose cultures. We also observed that CD45−/GlyA−/CD133+/ALDHhigh VSELs grew colonies earlier than CD45−/GlyA−/CD133+/ALDHlow VSELs, which suggests that the latter cells need more time to acquire hematopoietic commitment. In support of this possibility, real-time PCR analysis confirmed that, while freshly isolated CD45−/GlyA−/CD133+/ALDHhigh VSELs express more hematopoietic transcripts (e.g., c-myb), CD45−/GlyA−/CD133+/ALDHlow VSELs exhibit higher levels of pluripotent stem cell markers (e.g., Oct-4). More importantly, hematopoietic cells derived from VSELs that were co-cultured over OP9 support were able to establish human lympho-hematopoietic chimerism in lethally irradiated NOD/SCID mice 4–6 weeks after transplantation. Overall, our data suggest that UCB-VSELs correspond to the most primitive population of HSPCs in UCB.

Molecular characterization of isolated from murine adult tissues very small embryonic/epiblast like stem cells (VSELs).

Pluripotent very small embryonic/epiblast derived stem cells (VSELs) as we hypothesize are deposited at begin of gastrulation in developing tissues and play an important role as backup population of pluripotent stem cells (PSCs) for tissue committed stem cells (TCSCs). We envision that during steady state conditions these cells may be involved in tissue rejuvenation and in processes of regeneration/repair after organ injuries. Molecular analysis of adult bone marrow (BM)-derived purified VSELs revealed that they i) express pluripotent stem cells markers e.g., Oct4, Nanog, Klf-4, SSEA-1 ii) share several markers characteristic for epiblast as well as migratory primordial germ cells (PGCs), and iii) possess a unique pattern of genomic imprinting (e.g., erasure of differently methylated regions at Igf2-H19 and Rasgrf1 loci and hypermethylation at KCNQ1 and Igf2R loci). This supports that VSELs are related to epiblast-derived migrating PGC-like cells and, despite their pluripotent stem cell character, changes in the epigenetic signature of imprinted genes keep these cells quiescent in adult tissues and prevent them from teratoma formation. In contrast epigenetic changes/mutations that lead to activation of imprinted genes could potentially lead to tumor formation by these cells. Mounting evidence accumulates that perturbation of expression of imprinted genes is a common phenomenon observed in developing tumors.

Epiblast/Germ Line Hypothesis of Cancer Development Revisited: Lesson from the Presence of Oct-4+ Cells in Adult Tissues

The morphology of several tumors mimics developmentally early tissues; tumors often express early developmental markers characteristic for the germ line lineage. Recently, our group identified a population of very small stem cells (SCs) in murine bone marrow (BM) and other adult organs that express several markers characteristic for epiblast/germ line-derived SCs. We named these rare cells "Very Small Embryonic/Epiblast-like Stem Cells (VSELs)." We hypothesized that these cells that express both epiblast and germ line markers are deposited during early gastrulation in developing tissues and organs and play an important role in the turnover of tissue-committed (TC) SCs. To support this, we envision that the germ line is not only the origin of SCs, but also remains as a scaffold or back-up for the SC compartment in adult life. Furthermore, we noticed that VSELs are protected from uncontrolled proliferation and teratoma formation by a unique DNA methylation pattern in some developmentally crucial imprinted genes, which show hypomethylation or erasure of imprints in paternally methylated genes and hypermethylation of imprints in the maternally methylated. In pathological situations, however, we hypothesize that VSELs could be involved in the development of several malignancies. Therefore, potential involvement of VSELs in cancerogenesis could support century-old concepts of embryonic rest- or germ line-origin hypotheses of cancer development. However, we are aware that this working hypothesis requires further direct experimental confirmation.

Optimization of isolation and further characterization of umbilical cord blood‐derived very small embryonic/ epiblast‐like stem cells (VSELs)

Because of their small size and density, umbilical cord blood (UCB)-derived very small embryonic/epiblast-like stem cells (VSELs) are usually lost at various steps of UCB preparation. Accordingly, we noticed that a significant number of these cells, which are smaller than erythrocytes, are lost during gradient centrifugation over Ficoll-Paque as well as during routine volume depletion of UCB units before freezing. To preserve these cells in final UCB preparations, we propose a relatively short and economical three-step isolation protocol that allows recovery of approximately 60% of the initial number of Lin(-)/CD45(-)/CD133(+) UCB-VSELs present in freshly harvested UCB units. In this novel approach (i) UCB is lysed in a hypotonic ammonium chloride solution to deplete erythrocytes; (ii) CD133(+) including VSELs cells are enriched by employing immunomagnetic beads; and subsequently (iii) Lin(-)/CD45(-)/CD133(+) cells are sorted by fluorescence-activated cell sorting. The whole isolation procedure takes approximately 2-3 h per UCB unit and isolated cells are highly enriched for an Oct-4(+) and SSEA-4(+) population of small Lin(-)/CD45(-)/CD133(+) cells.

Genetic and Epigenetic Studies Reveal That Murine Oct-4+ Very Small Embryonic/Epiblast-Like Stem Cells (VSELs) Present in Adult Tissues Share Several Similarities/Markers with Epiblast-Derived Migratory Primordial Germ Cells (PGCs).

Abstract 2521Poster Board II-498Recently, we identified a population of pluripotent VSELs in murine adult bone marrow (BM; Leukemia 2006:20;857). Compared with other adult stem cells (SCs), VSELs show unique epigenetic features including: i) open chromatin structures in the promoter of Oct-4 and Nanog; and ii) parent-of-origin-specific reprogramming of genomic imprinting. These features explain the pluripotent embryonic-like nature and quiescent status of these cells, respectively (Leukemia 2009:In press), and indicate their relation to an epiblast/germ-line pluripotent (P)SC population. To better understand the developmental origin of VSELs, we examined gene expression profiles and the epigenetic status of epiblast and germ-line related genes in these cells. We employed real time quantitative PCR (RQ-PCR) to evaluate gene expression, a bisulfite sequencing strategy to evaluate DNA methylation, and chromatin immunoprecipitation (ChIP) to elucidate histone codes in genes of interest. VSELs were isolated from murine BM by multiparameter fluorescence-activated cell sorter (FACS) as a population of Sca+lin−CD45− along with Sca+lin−CD45+ hematopoietic (H)SCs and BM mononuclear cells (MNCs). We noticed that VSELs, similarly to epiblast PSCs (EpiSCs), highly express the stemness genes (e.g., Oct-4, Nanog, Sox2, Klf4) and epiblast markers (Gbx2, Fgf5, Nodal). However, the Rex1 gene is expressed at a lower level compared to the murine ESC-D3 line. Moreover, VSELs also highly express the Stella, Blimp1, Dnd1, and Nanos3, which are developmental regulators during specification in the proximal epiblast of PGCs. Accordingly, the Stella promoter in VSELs was partially demethylated and highly enriched for transcriptionally active histones (acetylated H3, trimethylated lysine4 of H3) while being simultaneously less enriched for repressive ones (dimethylated lysine9 and trimethylated lysine27 of H3). In particular, we noticed that VSELs resemble migratory PGCs. To support this notion, VSELs: i) express several markers of migratory PGCs (Dppa2, Dppa4, Mvh); ii) do not express post-migratory PGCs genes (Dazl, Sycp3); and iii) have reprogrammed DNA demethylation in the repetitive sequence (LINE1) and the promoters of Mvh, Dazal, and Sycp3. Finally, VSELs express less the transcripts for cMyc, Stat3, Snai, and Ecat1, which are expressed in early specified PGCs. In conclusion, our previous data showing reprogramming of genomic imprinting in VSELs with the present gene expression profile and epigenetic studies strongly supports VSELs developmentally originating from epiblast-derived germ-line SCs (PGCs), particularly migratory PGCs. We believe VSELs are deposited during embryogenesis in the adult tissues as a backup for tissue-committed SCs and that epigenetic reprogramming tightly controls their proliferative potential. Thus, identification of mechanisms that control and modify the epigenetic marks in VSELs will be crucial for developing more powerful strategies to “unleash the power” of these cells and employ them in regenerative medicine. Disclosures:No relevant conflicts of interest to declare.

In Vitro and In Vivo Evidence That Umbilical Cord Blood (UCB)-Derived CD45-/SSEA-4+/OCT-4+/CD133+/CXCR4+/Lin- Very Small Embryonic/Epiblast Like Stem Cells (VSELs) Do Not Contain Clonogenic Hematopoietic Progenitors but Are Highly Enriched in More Primitive Stem Cells - Novel View On Hierarchy of UCB Stem Cell Compartment.

Abstract 35Recently, we identified in umbilical cord blood (UCB) a population of very small embryonic/epiblast-like (VSEL) stem cells (Leukemia 2007;21:297–303) that are i) smaller than erythrocytes, ii) SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin−/CD45−, iii) respond to SDF-1 gradient and iv) possess large nuclei containing primitive euchromatin. We have demonstrated in vitro that UCB-derived VSELs did not reveal hematopoietic activity freshly after isolation, but grow hematopoietic colonies following co-culture/activation over OP-9 cells. To investigate the hierarchy of UCB-derived, CD45 negative VSELs, we employed staining with Aldefluor - detecting aldehyde dehydrogenase (ALDH), the enzyme expressed in primitive hematopoietic cells. Subsequently, we sorted CD45−/CD133+/ALDHhigh and CD45−/CD133+/ALDHlow sub-fractions of VSELs from UCB samples and established that freshly sorted from UCB VSELs in contrast to sorted CD45+/ CD133+/ALDHhigh and CD45+/CD133+/ALDHlow hematopoietic stem cells (HSC) did not grow colonies in vitro. However, when CD45− VSELs were activated/expanded over OP-9 stroma cells, they exhibit hematopoietic potential and grew in routine methylcellulose cultures hematopoietic colonies composed of CD45+ cells. Interestingly, while CD45−/CD133+/ALDHhigh VSELs gave raise to hematopoietic colonies after the first replating, the formation of colonies by CD45−/CD133+/ALDHlow VSELs was somehow delayed, what suggest that they needed more time to acquire hematopoietic commitment. Thus our in vitro data indicate that both populations of CD45− cells may acquire hematopoietic potential; however hematopoietic specification is delayed for CD45−/CD133+/ALDHlow cells, suggesting their more primitive nature. In parallel, real time PCR analysis confirmed that while freshly isolated CD45−/CD133+/ALDHhigh VSELs express more hematopoietic transcripts (e.g., c-myb, 80.2±27.4 fold difference), CD45−/CD133+/ALDHlow exhibit higher levels of pluripotent stem cell markers (e.g., Oct-4, 119.5±15.5 fold difference as compared to total UCB mononuclear cells) (Figure 1 panel A). Next hematopoietic potential of UCB-derived VSELs was tested in vivo after transplantation into NOD/SCID mice (Figure 1 panel B and C). We noticed that both CD45−/CD133+/ALDHhigh and CD45−/CD133+/ALDHlow VSELs, give rise to human lympho-hematopoietic chimerism in lethally irradiated NOD/SCID mice as assayed 4–6 weeks after transplantation. The level of human hematopoietic CD45+ cells in murine peripheral blood (PB), bone marrow (BM) and spleen (SP) were comparable for both transplanted UCB-VSELs fractions - 7.1±2.9% (PB), 23.2±0.2% (SP) and 25.2±1.0% (BM). In conclusion, our data suggest that freshly isolated very small CD45 negative UCB-VSELs are depleted from clonogeneic progenitors, however they are highly enriched for primitive HSC. Based on our in vitro and in vivo data we postulate following hierarchy of hematopoietic stem cells in UCB (from most primitive to more differentiated) i) CD45−/CD133+/ALDHlow, ii) CD45−/CD133+/ALDHhigh , iii) CD45+/CD133+/ALDHlow and iv) CD45−/CD133+/ALDHhigh. We also postulate that as we have already shown for murine BM-derived VSELs, human UCB-derived CD45 negative VSELs correspond to a population of most primitive long term repopulating HSC (LT-HSC). Of note, we also found that currently employed, routine UCB processing strategies may lead up to ∼50% unwanted loss of these small cells that are endowed with such remarkable hematopoietic activity! [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Very Small Embryonic/Epiblast-Like Stem Cells (VSELs) – Novel Supporting Evidence for An Existence of Developmentally Distinct Mobile Pool of Oct-4 + Pluripotent Stem Cells in Embryonic and Adult Tissues: Emerging Concept for a Potential 4th Migratory Germ Layer.

Abstract 1480Poster Board I-503We demonstrated the presence of very small (smaller than erythrocytes) Oct-4+ SSEA-1+Sca-1+Lin−CD45− VSELs in bone marrow (BM) and several adult organs and tissues (Leukemia 2006:20;857, Cytometry 2009:73;1116). Furthermore, our recent promoter methylation studies and analysis of chromatin's structure revealed that the Oct-4 gene is truly expressed in these cells. The epigenetic changes in selected somatic-imprinted genes govern their quiescent status, thus preventing them from unleashed proliferation and the spontaneous growth of teratomas (Leukemia 2009: in press). In the current study, we addressed the developmental origin of VSELs and significance of their presence in adult tissues. We first evaluated a number of VSELs in developing murine embryos (7-16 dpc). These cells were easily identified at the early stages of embryonic development. Studies in heartbeat-deficient mice (Ncx1−/−) revealed that VSELs are present in the embryo proper before initiation of circulation, which is in contrast to hematopoietic stem cells. Their number rapidly increases to ∼325k/embryo around 10-11 dpc during rapid vascularization of developing tissues/organs. These cells survive into adulthood; however, their number decreases with age. Molecular analysis revealed that VSELs express several primary epiblast- (Gbx2, Fgf5, Nodal) as well as epiblast-derived migratory primordial germ cell markers (Stella, Blimp1, Dnd1, and Nanos3). Furthermore, our recent BrdU staining studies revealed that, e.g., BM-residing VSELs are highly resistant to lethal irradiation and begin to proliferate even after lethal myeloablasia. This suggests that these cells could be a source of tissue-committed stem cells during tissue/organ damage and regeneration. Finally, in direct chemotactic studies we noticed that VSELs are highly mobile and respond robustly to SDF-1 and HGF gradients. This explains why they are mobilized into peripheral blood after heart infarct (Circulation 2004:110;3213, JACC 2009:53;1), stroke (Stroke 2009: 40,1237), and skeletal muscle and toxic liver damage (Stem Cells 2008:26;2083). Based on this, we propose a novel and challenging concept that during embryogenesis, a pool of highly migratory PSCs emerges (VSELs) in proximal epiblasts that contributes to organogenesis, tissue development, and specification of primordial germ cells. Furthermore, these cells survive into adulthood as a pool of PSCs that play some role in: i) steady-state conditions of tissue rejuvenation as a back up for tissue-committed stem cells; and ii) regeneration of damaged organs during emergencies. As such, we postulate that VSELs could somehow be a mammalian counterpart of blastema-like cells present in lower organisms. In mammalian tissues, they are kept in quiescent status due to epigenetic modification of selected imprinted genes. In addition, the age-related decrease of these cells in adult tissues and their primitive nature could explain the aging process. On the other hand, embryonic/epiblast-derived remnants in adult tissues could potentially initiate tumorogenesis (Virchow & Connheim). Finally, identification of VSELs supports the concept of a so-called ‘memoderm', a putative 4th, highly migratory, germ layer (Gaillard & Hernandez). Identification of VSELs in embryonic and adult tissues and our most recent data lend support to all these theories and provide novel view on regeneration, senescence and tumorigenesis. Disclosures:No relevant conflicts of interest to declare.

Fetal Liver Very Small Embryonic/Epiblast Like Stem Cells Follow Developmental Migratory Pathway of Hematopoietic Stem Cells

Fetal liver (FL) has been described as a source of both hematopoietic and nonhematopoietic stem cells. Recently we have purified from murine adult bone marrow (BM) a population of CXCR4(+)Oct-4(+)SSEA-1(+)Sca-1(+)Lin(-)CD45(-) very small embryonic/epiblast-like stem cells (VSELs). By employing several complementary imaging and molecular strategies, we report in this study that VSELs, like hematopoietic stem cells (HSCs), are highly enriched in murine FL during the second trimester of gestation. Subsequently, at the beginning of the third trimester of gestation their number decreases, which corresponds to the time when HSCs egress FL and follow the stromal derived factor-1 (SDF-1) gradient in order to colonize developing BM. Thus, our data support the hypothesis that VSELs are a mobile pool of primitive stem cells that respond to similar chemotactic gradients as HSCs and follow their developmental migratory route.

Identification of small Sca-1 +, Lin −, CD45 − multipotential cells in the neonatal murine retina

Bone marrow contains a subset of stem cells that give rise to nonhematopoietic lineages. These nonhematopoietic stem cells appear heterogeneous and contain cells committed to mesenchymal and endothelial lineages, as well as more primitive multipotential cells resembling progenitors of germ cells and very small embryonic/epiblast-like stem cells (VSELs). Nonhematopoietic stem cells can be mobilized from the bone marrow in response to tissue injury, and cells with similar properties have been found in cord blood and normal adult organs. However, the relationship between bone marrow cells and these adult organ stem cells is still unclear. The differentiation potential of some adult stem cells is organ-restricted, but other populations appear to retain multipotential capacity. A population of small Sca-1(+), lineage-negative (Lin(-)), CD45(-) cells resembling VSELs were isolated from neonatal mouse retina by cell sorting. Differentiation of the cells in culture was achieved by exposure to embryonic stem cell differentiation protocols. VSEL-like cells comprise 1.5% of the neonatal mouse retina. They remain quiescent during retinal differentiation, and thus they do not contribute to normal retinal development. However, they display eye cell differentiation potential in culture and they are also multipotential and can give rise to cells representative of all three embryonic layers. The neonatal retina is an abundant postnatal source of multipotential VSEL-like cells that can differentiate in culture into a variety of lineages.