Very Small Embryonic-like Research Articles

Peripheral Blood Stem Cells: Phenotypic Diversity and Potential Clinical Applications

A small proportion of cells in peripheral blood are actually pluripotent stem cells. These peripheral blood stem cells (PBSCs) are thought to be heterogeneous and could be exploited for a variety of clinical applications. The exact number of distinct populations is unknown. It is likely that individual PBSC populations detected by different experimental strategies are similar or overlapping but have been assigned different names. In this mini review, we divide PBSCs into seven groups: hematopoietic stem cells (HSCs), CD34- stem cells, CD14+ stem cells, mesenchymal stem cells (MSCs), very small embryonic-like (VSEL) stem cells, endothelial progenitor cells (EPCs), and other pluripotent stem cells. We review the major characteristics of these stem/progenitor cell populations and their potential applications in ophthalmology.

Optimal Recovery of SSEA-4+/OCT-4+/CD133+/CXCR4+/Lin-/CD45- Very Small Embryonic-Like (VSEL) Stem Cells From Umbilical Cord Blood (CB) Using Plasma Depletion/Reduction (PDR) Compared to Red Cell Reduction (RCR)

Optimal Recovery of SSEA-4+/OCT-4+/CD133+/CXCR4+/Lin-/CD45- Very Small Embryonic-Like (VSEL) Stem Cells From Umbilical Cord Blood (CB) Using Plasma Depletion/Reduction (PDR) Compared to Red Cell Reduction (RCR)

Identification and isolation from either adult human bone marrow or G-CSF−mobilized peripheral blood of CD34 +/CD133 +/CXCR4 +/ Lin −CD45 − cells, featuring morphological, molecular, and phenotypic characteristics of very small embryonic-like (VSEL) stem cells

Recently, we demonstrated that normal human bone marrow (hBM)-derived CD34(+) cells, released into the peripheral blood after granulocyte colony-stimulating factor mobilization, contain cell subpopulations committed along endothelial and cardiac differentiation pathways. These subpopulations could play a key role in the regeneration of post-ischemic myocardial lesion after their direct intracardiac delivery. We hypothesized that these relevant cells might be issued from very small embryonic-like stem cells deposited in the BM during ontogenesis and reside lifelong in the adult BM, and that they could be mobilized into peripheral blood by granulocyte colony-stimulating factor. Samples of normal hBM and leukapheresis products harvested from cancer patients after granulocyte colony-stimulating factor mobilization were analyzed and sorted by multiparameter flow cytometry strategy. Immunofluorescence and reverse transcription quantitative polymerase chain reaction assays were performed to analyze the expression of typical pluripotent stem cells markers. A population of CD34(+)/CD133(+)/CXCR4(+)/Lin(-) CD45(-) immature cells was first isolated from the hBM or from leukapheresis products. Among this population, very small (2-5 μm) cells expressing Oct-4, Nanog, and stage-specific embryonic antigen-4 at protein and messenger RNA levels were identified. Our study supports the hypothesis that very small embryonic-like stem cells constitute a "mobile" pool of primitive/pluripotent stem cells that could be released from the BM into the peripheral blood under the influence of various physiological or pathological stimuli. In order to fully support that hBM- and leukapheresis product-derived very small embryonic-like stem cells are actually pluripotent, we are currently testing their ability to differentiate in vitro into cells from all three germ layers.

Polycomb Group Protein-Mediated Bivalent Domains Regulate Pluripotency of Oct4+ Very Small Embryonic-Like (VSEL) Stem Cells In Adult Bone Marrow

AbstractAbstract 4788Recently, we identified a population of very small embryonic-like (VSEL) stem cells (SCs) in adult bone marrow (BM; Leukemia 2006;20:857). These Oct4+CXCR4+SSEA-1+Sca-1+CD45-Lin- VSELs are capable of differentiation in vitro into cells of all three germ lineages and are able to form spheres (VSEL-derived spheres[VSEL-DS]) composed of primitive stem cells in co-cultures with C2C12 myoblasts. Cells isolated from VSEL-DS are also able to differentiate into all three germ layers (cardiomyocytes, neural cells, and insulin-producing cells). Our previous epigenetic study proved that VSELs exhibit open chromatin structures in the promoters of pluripotent stem cell markers, such as Oct4 and Nanog (Leukemia 2009;23:2042). However, it still remains unclear whether the pluripotency of VSELs could be orchestrated by a mechanism similar to that seen in embryonic stem cells (ESCs). To address this issue, we evaluated whether adult BM-derived VSELs, in a similar way as ESCs, display bivalent-domain–marked histone modifications in the promoter region of homeodomain-containing developmental master transcription factors (TFs), such as Dlx-, Irx-, Lhx-, Pou-, Pax-, and Six-family proteins. To overcome the problem that VSELs sorted by FACS are heterogeneous, we constructed a cDNA library from a small number of cells (~25) by employing the protocols used for single-cell analysis (Nat Protoc 2007;2:739). Analysis of this cDNA library indicated that Oct4+ VSELs highly express several epigenetic regulators, especially polycomb repressive complex 2 (PRC2) proteins, including Ezh2, Suz12, and Eed. It is known that PRC2, due to its histone methyltransferase activity on lysine27 of histone3 (H3K27), is responsible for transcriptionally repressive trimethylation of H3K27 (HKK27me3) in promoters for several homeobox domain-containing developmental TFs, such as Sox21, Nkx2.2, Dlx1, Zfpm2, Irx2, Lbx1h, Hlxb9, and Pax5. Moreover, we observed that most of these promoters are “bivalent” and marked with the transcriptionally active histone code that includes trimethylated H3K4 together with H3K27me3, similarly to ESCs. Such bivalent epigenetic marks prevent the premature expression of these TFs in freshly isolated quiescent VSELs. Furthermore, we noticed that during formation of VSEL-DS, Ezh2 expression was decreased and most of the bivalent domains in VSELs were transformed into unmodified histone marks, resulting in the expression of some bivalent-targeted genes, including Hlxb9, HoxA3, and Evx1. Moreover, most of the pluripotent SC markers were also reduced during VSEL-DS progression, which was paralleled by inactivation of the Oct4 promoter. In conclusion, our results suggest that the pluripotency of adult tissue-derived VSELs is modulated, in a similar manner as in ESCs, by transient repression of developmental master TFs through bivalent epigenetic marks. Although PRC2 proteins stimulate cellular proliferation and are highly expressed in various tumors, unique epigenetic reprogramming of genomic imprinting maintains VSEL quiescence under steady-state conditions and also protects these cells from teratoma formation (Leukemia 2009;23:2042). Thus, we suggest that attenuation of this protective mechanism in VSELs, for example during chronic tissue injury and ageing, may lead to tumor formation. Disclosures:No relevant conflicts of interest to declare.

A Novel Paradigm In Stem Cell Trafficking: The Ratio of Peripheral Blood Sphingosine-1 Phosphate (S1P) to Bone Marrow Ceramide-1 Phosphate (C1P) Regulates Mobilization and Homing of Hematopoietic Stem Cells

AbstractAbstract 554The stromal derived factor-1 (SDF-1)–CXCR4 axis plays an unquestioned role in developmental migration of hematopoietic stem cells (HSPCs) and their retention in the bone marrow (BM). However, changes in the SDF-1 gradient between BM and peripheral blood (PB) do not always support its having a crucial role as chemoattractant for mobilization or homing of HSPCs. As demonstrated by others (e.g., Bone Marrow Transplantation 2003; 31:651–654, and Transfus Apher Sci 2009;40:159) and us (Leukemia 2010;24:976–985) the plasma SDF-1 level does not correlate with mobilization of HSPCs. On the other hand, there is increasing doubt about an exclusive role for SDF-1 in homing of HSPCs in BM. This is based on evidence that i) CXCR4−/− fetal liver HSPCs may home to BM in an SDF-1–independent manner (Immunity 1999;10:463-471), ii) homing of murine HSPCs made refractory to SDF-1 by incubation and co-injection with a CXCR4 receptor antagonist is normal or only mildly reduced (Science 2004;305:1000), and finally iii) HSPCs in which CXCR4 has been knocked down by means of an SDF-1 intrakine strategy also engraft in lethally irradiated recipients (Blood 2000;96:2074–,2080). All this strongly suggests the existence of other factors involved in the mobilization and homing of HSPCs. Moreover, while SDF-1 is a potent chemoattractant for HSPCs when employed at supraphysiological concentrations in vitro, as a peptide it is highly susceptible to degradation by proteases that are elevated, for example, in PB during stem cell mobilization or in the BM microenvironment after myeloablative conditioning for transplantation. Employing ELISA for detection in the present study, we observed insignificant changes in SDF-1 level both in PB during mobilization and in BM after myeloablative conditioning. We also found that mobilized PB (mPB) plasma as well as conditioned media (CM) from lethally irradiated mice chemoattract HSPCs in an SDF-1–independent manner as demonstrated by i) normal chemotaxis of AMD3100 pre-treated cells and ii) preservation of chemotactic activity of plasma and BM-derived CM following heat inactivation. However, the chemotactic activity of mPB plasma and BM CM was inhibited after stripping by activated charcoal. This suggested the involvement of small molecule bioactive lipids. It is known that sphingolipids, which are important components of cell membranes, give rise to two bioactive derivatives, sphingosine-1 phosphate (S1P) and ceramide-1 phosphate (C1P), with S1P already identified as a chemoattractant for HSPCs (Ann N Y Acad Sci. 200;1044:84–89). To our surprise, we found that C1P is also a strong chemoattractant for human and murine HSPCs. In addition, we observed that at physiological concentrations both these bioactive lipids i) activate phosphorylation of MAPKp42/44 and AKT in HSPCs, ii) induce expression of matrix metalloproteinases (MMPs), and iii) modulate adhesion to stroma and endothelium. Interestingly, by employing ELISA and/or mass spectophotometry we found that, while the S1P level increases in PB during mobilization, the C1P level increases in BM after myeloablative conditioning for transplantation. Based on these findings, we propose a new paradigm in which the S1P:C1P ratio plays a role in mobilization and homing of HSPCs. While S1P is a major chemoattractant that directs egress of HSPCs from BM into PB, C1P released from damaged cells in BM after myeloablative conditioning creates a homing gradient for circulating HSPCs. We also postulate that the S1P:C1P ratio plays a more universal role and is involved in regulating migration of other types of stem cells, such as circulating mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like (VSEL) stem cells. Accordingly, while S1P plays a role in egress of stem cells into PB, C1P released from damaged cells (e.g., in infarcted myocardium or brain tissue after stroke) chemoattracts circulating stem cells for potential repair. Disclosures:No relevant conflicts of interest to declare.

Intermittent Hypoxia Mobilizes Bone Marrow-Derived Very Small Embryonic-Like Stem Cells and Activates Developmental Transcriptional Programs in Mice

obstructive sleep apnea is a prevalent disorder associated with cognitive dysfunction and cardiovascular and metabolic morbidity and is characterized by recurrent episodes of hypoxia during sleep. Bone marrow-derived very small embryonic-like (VSEL) pluripotent stem cells represent a recruitable pool that may play an important role in organ repair after injury. We hypothesized that exposure to intermittent hypoxia (IH) can mobilize VSELs from the bone marrow (BM) to peripheral blood (PB) in mice and can activate distinct transcriptional programs. adult mice were exposed to IH or normoxia for 48 hours. VSELs were sorted from BM and PB using flow cytometry. Plasma levels of stem cell chemokines, stromal cell derived factor-1 (SDF-1), hepatocyte growth factor (HGF), and leukemia inhibitory factor (LIF) were measured. Transcriptional profiling of VSELs was performed, and differentially expressed genes were mapped to enriched functional categories and genetic networks. exposure to IH elicited migration of VSELs from BM to PB and elevations in plasma levels of chemokines. More than 1100 unique genes were differentially expressed in VSELs in response to IH. Gene Ontology and network analysis revealed the activation of organ-specific developmental programs among these genes. exposure to IH mobilizes VSELs from the BM to PB and activates distinct transcriptional programs in VSELs that are enriched in developmental pathways, including central nervous system development and angiogenesis. Thus, VSELs may serve as a reserve mobile pool of pluripotent stem cells that can be recruited into PB and may play an important role in promoting end-organ repair during IH.

Bone Marrow Transplantation Temporarily Improves Pancreatic Function in Streptozotocin-Induced Diabetes: Potential Involvement of Very Small Embryonic-Like Cells

The role of bone marrow (BM)-derived cells in pancreatic beta-cell regeneration remains unresolved. We examined whether BM-derived cells are recruited to the site of moderate pancreatic injury and contribute to beta-cell regeneration. Low-dose streptozotocin (STZ) treatment was used to induce moderate pancreatic damage and hyperglycemia. Enhanced green fluorescent protein-positive (EGFP) BM chimeras were evaluated for beta-cell regeneration after STZ treatment. To test the hypothesis that pancreatic tissue injury induces a stromal cell-derived factor (SDF)-1 gradient to chemoattract the stem cells, we evaluated the expression of mRNA for SDF-1 in damaged pancreatic tissue. SDF-1 was significantly increased in the pancreas after damage, peaking at day 10. The majority of BM cells expressing mRNA for pancreatic development markers were detected in the subpopulation of CD45/Sca-1/Lin very small embryonic-like (VSEL) cells. VSEL cells mobilized from BM to peripheral blood in response to pancreatic damage, peaking in peripheral blood at day 5, and were enriched in the pancreas 10 to 15 days after STZ treatment. To confirm a role for BM-derived cells in pancreatic beta-cell regeneration, we prepared EGFP-->B6 chimeras. In the EGFP chimeras, EGFP cells were detected around duct and islets and were positive for insulin after STZ treatment. However, STZ-induced hyperglycemia was reduced only transiently (49-77 days) after pancreatic injury. These data suggest that VSEL cells are mobilized into injured pancreatic tissue and contribute to beta-cell regeneration. Transplantation of BM-derived cells improves the function of injured pancreas, although the response is not sufficient to restore sustained normoglycemia.

Very Small Embryonic Like (VSEL) Stem Cells: Characterization, Biological Significance, and Potential Applications

Bone marrow (BM) was for many years primarily envisioned as the "home organ" of hematopoietic stem cells (HSC). Augmenting evidence demonstrates, however that BM in addition to HSC also contains a heterogeneous population of non‐hematopoietic stem cells (non‐HSC). Recently our group identified in BM and other adult tissues including brain, kidney, pancreas, skeletal muscles, liver, spleen and lungs a population of very small embryonic like stem cells (VSELs) which express several markers characteristic for pluripotent stem cells (PSC) that are characteristic for epiblast/germ line‐ derived stem cells (Leukemia 2006;20:857‐869 & Leukemia 2007;21:297‐303). We hypothesize that VSELs are a population of epiblast‐derived cells that are deposited during early gastrulation in developing tissues/organs and play an important role in turnover of tissue specific/committed stem cells. In this context for example VSELs deposited in BM may give rise to long term repopulating HSC. We noticed that VSELs could be also mobilized into peripheral blood (PB) and their number of these cells circulating in PB increases during stress and tissue/organ injuries (e.g., G‐CSF administration, heart infarct and stroke). However, the number of these cells decreases with the age. Our recent data indicate that VSELs could be harnessed as a source of pluripotent stem cells for regenerative medicine. Finally, we envision that in pathological situations VSELs are involved in development of some malignancies (e.g., teratomas, germinal tumors, pediatric "small round blue cell" sarcomas).

Clinical Evidence That Very Small Embryonic-Like Stem Cells Are Mobilized Into Peripheral Blood in Patients After Stroke

In a murine model of stroke, we identified a population of very small embryonic-like (VSEL) stem cells (SCs) in adult murine bone marrow that could be mobilized into peripheral blood (PB). This raised the question of whether a similar population of cells is mobilized in human stroke patients. We evaluated a number of cells that corresponded to VSEL SCs in the PB of 44 stroke patients and 22 age-matched controls. After each patient's stroke, PB samples were harvested during the first 24 hours, on day +3, and on day +7 and then compared with normal controls. The circulating human cells with the phenotype of VSEL SCs were evaluated in PB by real-time quantitative polymerase chain reaction, fluorescence-activated cell sorting analysis, and direct immunofluorescence staining. In parallel, we also measured the serum concentration of stromal derived factor-1 by ELISA. In stroke patients, we found an increase in the number of circulating cells expressing SC-associated antigens, such as CD133, CD34, and CXCR4. More important, we found an increase in the number of circulating primitive cells expressing the VSEL phenotype (CXCR4(+)lin(-)CD45(-) small cells), mRNA for Octamer-4 and Nanog, and Octamer-4 protein. All changes were accompanied by an increased serum concentration of stromal derived factor-1. Additionally, we found a positive correlation between stroke extensiveness, stromal derived factor-1 concentration in serum, and the number of CXCR4(+) VSEL SCs circulating in the PB. We conclude that stroke triggers the mobilization of CXCR4(+) VSEL SCs that have potential prognostic value in stroke patients. However, the potential role of these mobilized cells in brain regeneration requires further study.

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?

Recently, we identified a population of very small embryonic like (VSEL) SCs in adult bone marrow (BM) (Leukemia 2006: 20;857). These VSELs are:very small in size (~3.6 um);Oct-4+CXCR4+SSEA-1+Sca-1+CD45−lin−;possessing large nuclei containing unorganized chromatin (euchromatin); andwe learned that in co-cultures with C2C12 cells, VSELs form embryoid body-like spheres (VSEL-DSs) that contain primitive SCs capable to differentiate into all three germ layers (e.g., myocardium, neural tissue, and pancreas).To better characterize this intriguing population of SCs, we employed bisulfite sequencing, combined bisulfite restriction analysis (COBRA), and methylation specific PCR (MSP) to analyze the methylation status of i) Oct-4 promoter and ii) differentially methylated regions (DMRs) of imprinted genes that are known to be crucial for maintaining the pluripotency of embryonic (E)SCs [e.g., insulin-like growth factor 2 (Igf2)-H19 locus, Igf2 receptor (Igf2R), paternally expressed gene 1(Peg1/Mest), small nuclear ribonucleoprotein polypeptide N (SNRPN), and KCNQ1 locus that includes cyclin-dependent kinase inhibitor 1c (CDKN1C/p57Kip2)]. The methylation status of these genes was evaluated in adult bone marrow (BM)-derived Sca+lin−CD45− VSELs, Sca+lin− CD45+ hematopoietic (H)SCs, mesenchymal stem cells (MSCs), and the murine ESC line ESC-D3 (Table 1). We noticed that Oct-4 promoter is hypomethylated in VSELs similarly to ESCs. In contrast, Oct-4 promoter was hypermethylated in HSCs and MSCs. This observation combined with our mRNA and protein expression data provides evidence that Oct-4 gene is transcribed in VSELs residing in adult tissues. We also found that the DMR of the Igf2-H19 locus, which is crucial for controlling SC pluripotency, was significantly hypomethylated in VSELs. Of note methylation of this locus is known to be erased in migrating primordial germ cells (PGC), and is envisioned as a crucial mechanism that controls “unleashed proliferation” of PGC and prevents them from forming teratomas. On other hand, Igf2-H19 locus was hypermethylated in ESCs and the proper somatic pattern of methylation (~50%) was observed in HSCs and MSCs (Table 1). Furthermore, VSEL similarly to ESCs showed hypermethylation of DMRs of Igf2R, KCNQ1, and Peg1/Mest – loci, which show proper somatic imprint (~50%) in HSCs and MSCs. Of note, SNRPN DMR methylation was normally maintained in all cells tested. Our methylation data were subsequently confirmed by mRNA expression studies. Accordingly, as predicted VSELs showed increased expression levels of mRNA for H19, Igf2R, and CDKN1C/p57Kip2, but reduced expression level of Igf2 as compared to HSCs. Finally, when VSELs were cultured in our “expansion” model over a C2C12 feeder layer, the methylation pattern of VSELs (hypomethylation of Igf2-H19, hypermethylation of Igf2R and Peg1/Mest) had been properly restored in cells inside VSEL-DSs to levels observed in normal somatic cells. At the same time, however, the Oct-4 promoter became hypermethylated and Oct-4 mRNA was downregulated. In conclusion, our methylation studies at Oct-4 promoter and Igf2-H19 locus provide additional evidence that VSELs show a similar methylation pattern to PGC, what supports their developmental origin directly form epiblast/germ line. We believe that they are deposited during embryogenesis in the adult tissues as a backup for tissue committed SCs and that their proliferative potential is tightly regulated/controlled by the status of Oct-4 promoter and Igf2-H19 DMR locus methylation. Erasure of methylation at Igf2-H19 locus on one hand prevents them from “unleashed proliferation” and formation of teratomas. Thus, identification of mechanisms that control and modify genomic imprinting in VSELs will be crucial for developing more powerful strategies to “unleash a power” of these cells and employ them in regenerative medicine.Table 1. Methylation status of Oct-4 promoter and crucial somatic imprinted genes. [Display omitted]

Optimization of Isolation and Further Molecular and Functional Characterization of SSEA-4+/Oct-4+/CD133+/CXCR4+/LINneg/CD45neg Very Small Embryonic-Like (VSEL) Stem Cells Isolated from Umbilical Cord Blood.

Several lines of evidence support the hypothesis that pluripotent stem cells (PSCs) reside in human tissues. Recently, we identified a population of very small embryonic-like (VSEL) SCs in umbilical cord blood (CB) (Leukemia 2007; 21:297-303). These VSELs are: i) very small in size (<6 um); ii) SSEA-4+/Oct-4+/CD133+/CD34+/CXCR4+/Linneg/ CD45neg; iii) responsive to a stromal derived factor (SDF)-1 gradient; and iv) possess large nuclei that contain primitive euchromatin. In the current study, we optimized their isolation/purification strategy and employed several imaging and molecular techniques to better analyze these primitive cells. We noticed that because of their small size, CD133+/ Linneg/CD45neg VSELs are lost (42.5±12.6%) during routine CB unit processing by volume depletion before storage/freezing. Interestingly, these cells are more resistant to changes following freezing and thawing as compared to normal hematopoietic (H)SCs. Interestingly, 82.7±17.3% of the initially frozen CD133+/Linneg/CD45neg VSELs are preserved in frozen CB units, while only 65.0±6.1% CD133+/Linneg/CD45neg HSCs are recovered. Furthermore, when we employed Ficoll centrifugation to purify CB mononuclear cells (CB MNCs), we found that while 59.8±7.2% of CD133+/Linneg/CD45neg VSELs were lost, their hematopoietic counterparts (CD133+/Linneg/CD45+) were almost fully recovered (Fig. 1A). These data indicate that other more “VSEL-saving” strategies of erythrocyte depletion should be developed because of the unusual size and density of these cells. We also established that the most the optimal “VSEL-saving” strategy to deplete erythrocytes from CB was hypotonic lysis. However, we noticed that during this procedure, lyzed erythrocytes release phosphatidyloserine positive (PS+) membrane-derived microvesicles (MVs) and these PS+ MVs preferentially bind to VSELs. Because of this phenomenon, VSELs become PS+ and may be falsely recognized as apoptotic cells in the Annexin-V-binding assay. The unique morphological features of VSELs were confirmed by several complementary imaging methods. ImageStream analysis revealed that VSELs are smaller than erythrocytes, are larger than platelets, and posses a high nuclear/cytoplasmic ratio (Fig. 1B). The fraction of CD133+/Linneg/CD45neg) VSELs with the smallest size (<6 um) exhibit a high cytoplasmic nuclear ratio and highly express Oct-4 in the nucleus and SSEA-4 and CD133 antigens on the surface. Finally, we found 2 to 3 times higher numbers of VSELs in CB samples from vaginal deliveries as compared to scheduled C-sections. This supports the idea that VSEL are released into CB due to delivery-related stress/hypoxia. In conclusion, CB contains a population of VSELs but ~50% of these cells are not recovered by currently employed volume-reduction strategies because of their unique morphology. Taking into consideration that VSELs may be employed in regenerative medicine, novel volume reduction/erythrocyte depletion strategies require development in CB banking to avoid loss of these rare, primitive, and important cells. [Display omitted]

CD45−/ALDHlow/SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin− Very Small Embryonic-Like (VSEL) Stem Cells Isolated from Umbilical Cord Blood as Potential Long Term Repopulating Hematopoietic Stem Cells

Recently, we identified a population of very small embryonic-like (VSEL) stem cells in umbilical cord blood (CB) (Leukemia 2007; 21:297–303) These VSELs are:smaller than erythrocytes;SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin−/CD45−;responsive to SDF-1 gradient; and iv) possessing large nuclei that contain unorganized chromatin (euchromatin).Data obtained in a murine model indicate that a similar cell population isolated from bone marrow (BM) does not reveal hematopoietic activity after isolation. However, in appropriate models (i.e., in vitro co-culture over OP-9 cells or in vivo after intra bone injection), these cells contribute to hematopoiesis and thus possesses potential of long term repopulating hematopoietic stem cells (LT-HSCs). To investigate the hematopoietic activity of CB-derived, CD45 negative VSELs, we employed staining with Aldefluor detecting aldehyde dehydrogenase (ALDH), the enzyme expressed in primitive hematopoietic cells. We sorted CD133+/CD45−/ALDHhigh and CD133+/CD45−/ALDHlow sub-fractions of VSELs from CB samples and established that both freshly sorted CB-derived populations did not grow hematopoietic colonies in vitro. However, when activated/expanded over OP-9 stroma cells, they exhibit hematopoietic potential and initiate hematopoietic colonies composed of CD45+ cells when replated into methylcellulose cultures. Furthermore, while CD133+/CD45−/ALDHhigh VSELs gave raise to hematopoietic colonies after the first replating, the formation of colonies by CD133+/CD45−/ALDHlow VSELs was delayed. The data indicate that both populations of CD45− cells may acquire hematopoietic potential; however hematopoietic specification is delayed for CD133+/CD45−/ALDHlow cells (Fig. 1A). In parallel, real time PCR analysis revealed that freshly isolated CD133+/CD45−/ALDHhigh VSELs express more hematopoietic transcripts (e.g., c-myb, 80.2±27.4 fold difference) while CD133+/CD45−/ALDHlow exhibit higher levels of pluripotent stem cell markers (e.g., Oct-4, 119.5±15.5 fold difference) as compared to total CB mononuclear cells (Fig. B). Furthermore and somewhat unexpectedly, we found that because of their unusually small size, these important cells may be partially depleted (in 42.5±12.6%) during standard preparation strategies of CB units for storage that employ volume reduction. In conclusion, our data suggest very small CB mononuclear cells expressing VSEL markers that are CD133+/CD45−/ALDHlow are highly enriched for the most primitive population of LT-HSCs. These cells may be responsible for long term CB engraftment and be a population of cells from which HSCs should be expanded. We are currently testing this in an in vivo model by performing heterotransplants of CD45− ALDHlow VSELs into immunodeficient mice. It is important to stress that currently employed, routine CB processing strategies may lead up to ~50% loss of these small cells that are endowed with such remarkable hematopoietic activity. [Display omitted]

Very small embryonic-like (VSEL) stem cells in adult organs and their potential role in rejuvenation of tissues and longevity

Recently, we purified rare CXC chemokine receptor 4 expressing (CXCR4 +) small stem cells (SCs) from the murine bone marrow (BM) that express markers characteristic for embryonic (E)SCs, epiblast (EP)SCs, and primordial germ cells (PGCs). We named these primitive cells very small embryonic-like (VSEL) SCs (VSELs). Our data indicate that VSELs are also present in many other organs in mice and that they may differentiate into cells from all three germ layers. Similar SCs were also isolated from human cord blood (CB) and mobilized peripheral blood (mPB). We hypothesize that VSELs are deposited during gastrulation and organogenesis in developing organs/tissues of mammals as a population of pluripotent stem cells (PSCs) that give rise to tissue committed monopotent SCs and that their number decreases with age. Therefore VSELs could play a pivotal role in normal rejuvenation of adult tissues as well as involvement in regeneration of damaged organs. Thus, these cells are potential SCs candidates for regenerative medicine and we envision that the regenerative potential of these cells could be harnessed to decelerate the aging processes.

Evidence That Very Small Embryonic-Like Stem Cells Are Mobilized into Peripheral Blood

Recently, we identified in murine adult tissues, including bone marrow, a population of very small embryonic-like (VSEL) stem cells. Here, we provide further evidence that under steady-state conditions these cells circulate at very low levels in peripheral blood (PB) ( approximately 100-200 cells/ml) and could be additionally mobilized during pharmacological granulocyte-colony-stimulating factor-induced or stress-related mobilization, as demonstrated in a model of toxic liver or skeletal muscle damage induced by injection of carbon tetrachloride or cardiotoxin, respectively. The number of circulating VSEL stem cells under steady-state conditions in PB of 2-month-old animals was five times higher than that in 1-year-old mice. In conclusion, this study supports a hypothesis that VSEL stem cells are a mobile pool of primitive stem cells that could be released from the stem cell niches into PB. Further studies are needed, however, to see whether the level of these cells circulating in PB could become a prognostic indicator to assess the regenerative potential of an adult organism and/or clinical outcome from an injury. Disclosure of potential conflicts of interest is found at the end of this article.

An efficient two-step method to purify very small embryonic-like (VSEL) stem cells from umbilical cord blood (UCB).

The identification in murine bone marrow (BM) of very small embryonic-like (VSEL) stem cells, possessing several features of pluripotent stem cells, encouraged us to investigate if similar population of cells could be also isolated from the human umbilical cord blood (UCB). Here our approach to purify VSEL from human UCB is described by employing a two step isolation strategy based on i) hypotonic lysis of erythrocytes followed ii) by multi-parameter FACS sorting. Accordingly, first, erythrocytes are removed from the UCB samples by hypotonic ammonium chloride solution and next, the UCB mononuclear cells (UCB MNC) are stained with monoclonal antibodies against all hematopoietic lineages including the common leukocyte antigen CD45. The cells carrying these markers (lin+CD45+) are eliminated from the sort by electronic gating. At the same time the antibodies against CXCR4, CD34 and CD133 are employed as positive markers to enrich the UCB MNC for VSEL. This combined two step approach enables to purify VSEL stem cells, which are small and express mRNA for pluripotent stem cells (PSC) (Oct-4 and Nanog) and tissue-committed stem cells (TCSC) (Nkx2.5/Csx, VE-cadherin and GFAP) similarly to those isolated from the adult BM (3-5 microm cells with large nuclei).

An efficient two-step method to purify very small embryonic-like (VSEL) stem cells from umbilical cord blood (UCB).

An efficient two-step method to purify very small embryonic-like (VSEL) stem cells from umbilical cord blood (UCB).

Very Small Embryonic-Like (VSEL) Stem Cells: Purification from Adult Organs, Characterization, and Biological Significance

In this review, we discuss current views of the bone marrow (BM) stem cell (SC) compartment and present data showing that BM contains heterogeneous populations of hematopoietic (H)SCs and non-HSCs. These cells are variously described in the literature as: endothelial progenitor cells (EPCs); mesenchymal (M)SCs; multipotent adult progenitor cells (MAPCs); marrow-isolated adult multilineage inducible (MIAMI) cells; and multipotent adult (MA)SCs. In some cases, it is likely that similar or overlapping populations of primitive SCs in the BM detected using various experimental strategies were assigned different names. Recently, we purified rare CXC chemokine receptor 4 expressing (CXCR4(+)) small SCs from the murine BM that express markers characteristic for embryonic (E)SCs, epiblast (EP)SCs, and primordial germ cells (PGCs). We named these primitive cells very small embryonic-like (VSEL) SCs. Our data indicate that VSELs may differentiate into cells from all three germ layers.

Very small embryonic-like stem cells in adult tissues—Potential implications for aging

Recently our group identified in murine bone marrow (BM) and human cord blood (CB), a rare population of very small embryonic-like (VSEL) stem cells. We hypothesize that these cells are deposited during embryonic development in BM as a mobile pool of circulating pluripotent stem cells (PSC) that play a pivotal role in postnatal tissue turnover both of non-hematopoietic and hematopoietic tissues. During in vitro co-cultures with murine myoblastic C2C12 cells, VSELs form spheres that contain primitive stem cells. Cells isolated from these spheres may give rise to cells from all three germ layers when plated in tissue specific media. The number of murine VSELs and their ability to form spheres decreases with the age and is reduced in short-living murine strains. Thus, developmental deposition of VSELs in adult tissues may potentially play an underappreciated role in regulating the rejuvenation of senescent organs. We envision that the regenerative potential of these cells could be harnessed to decelerate aging processes.

Pluripotent SSEA-1+ OCT-4+ CXCR4+ Sca-1+ lin− CD45− Very Small Embryonic Like (VSEL) Stem Cells Reside in Multiple Murine Organs - Are VSEL Epiblast-Derived Functional Pluripotent Precursors for Tissue Committed Stem Cells or Merely Quesiscent Remants from Embryonic Development?.

Several lines of evidence support the hypothesis that pluripotent stem cells (PSC) reside in adult tissues. Recently, we identified in adult bone marrow (BM) a population of very small embryonic like (VSEL) stem cells (Leukemia 2006: 20;857) that arei.very small in size (∼3.6 μm),ii.CXCR4+Oct-4+ SSEA-1+Sca-1+CD45−lin−,iii.respond to SDF-1 gradient andiv.possess large nuclei containing unorganized chromatin (euchormatin).We learned that VSEL in co-cultures with C2C12 cells form embryoid body-like spheres that contain primitive stem cells able to differentiate into cells from all three germ layers (e.g., myocardium, neural tissue and pancreas). In the current studies, we investigated if in addition to BM, VSEL are also present in other murine fetal and adult organs such as liver, heart, brain, kidney, skeletal muscles, pancreas, testes. VSEL were identified and enumerated in adult organs that in order to remove circulating blood were perfused before homogenization and subsequently detected byi.FACS,ii.ImageStream analysis,iii.microscopic immunofluorescence staining,iv.RQ-PCR for expression of pluripotent stem cell markers and finallyv.in functional assays in in vitro based on embryoid body-like spheres formation.We found that murine fetal liver and adult organs contain some rare VSEL-like cells that express several markers expressed by epiblast/germ line stem cells such as SSEA-1, CXCR4, Oct-4, Nanog and MvH. These cells are small, contain unorganized euchromatin and display high telomerase activity. Their size, however may vary depending on the organ from which they are isolated. The number of VSEL-like cells is higher in young animals and decreases with age. We hypothesize that VSEL are direct descendants of the germ lineage which, in order to pass genes on to the next generation, creates soma, and thus is a “mother lineage” for all somatic cell lineages present in the adult body. We envision that VSEL are deposited during embryogenesis in various organs as mother cells for tissue committed stem cells and that they survive in these locations into adulthood. VSEL, however erase their somatic imprint (differential methylation of IGF-2 and H19 on maternal and paternal chromosmes). This mechanism of erasure protects the developing organism from the possibility of teratoma formation. However, at the same time it also affects some of the aspects of the “true pluripotentiality” of these cells (e.g., their ability to complete blastocyst development). We noticed that at least in BM VSEL are a population of PSC that may differentiate along hematopoietic lineage and give rise to hematopoietic stem cells (HSC). Thus VSEL, fulfill criteria of long-term repopulating HSC. Based on this we postulate that, VSEL could play an important role as an organ-residing reserve population of PSC that generates stem cells committed to particular organs and tissues. Decrease in VSEL number that reside in various organs correlates with senescence and exhaustion of the regenerative potential.

Novel Direct Evidence That Adult Bone Marrow-Derived Very Small Embryonic Like (VSEL) Stem Cells Are Mobilized into Peripheral Blood - Leukopheresis as a Potential Tool To Isolate Pluripotent Stem Cells for Therapeutic Pruposes.

Recently, we identified in adult bone marrow (BM) a population of very small embryonic like (VSEL) stem cells (Leukemia 2006: 20;857) that arei.very small in size (∼3.5 μm),ii.CXCR4+Oct-4+ SSEA-1+Sca-1+CD45−lin−,iii.respond to SDF-1 gradient andiv.contain large nuclei with open type of euchromatin.We asked if VSEL are mobilized into peripheral blood (PB) similarly as BM-derived hematopoietic stem cells. To address this question, we evaluated VSEL level in PB afteri.pharmacological (G-CSF) andii.stress/organ damage-induced mobilization (toxic liver and skeletal muscle damage after exposure to CCl4 and cardiotoxin respectively).The number of circulating VSEL in PB was measured byi.FACS,ii.direct histochemical staining of PB-cells for SSEA-1 and Oct-4 expression, andiii.RQ-PCR analysis of pluripotent genes (Oct-4, Nanog).Finally, VSEL sorted from the PB were tested for their pluripotency in our established assay based on formation of embryoid-like spheres by VSEL in co-cultures with C2C12 cells. We report that in steady state conditions, VSEL circulate at very low levels in PB (∼100–200 cells/ml) and that the number of these cells/ml of PB is five times higher in two month old mice as compared to one year old animals. Furthermore, we found that VSEL are mobilized into PB both by G-CSF- and tissue injury and the number of circulating VSEL increases 5–6 and 2–3 times after G-CSF and after liver or skeletal muscle injury, respectively. Furthermore, the mobilization of VSEL could be additionally enhanced by blocking CXCR4 receptor by T140 antagonist. Based on this, we provide the evidence that VSELi.is a mobile pool of primitive CXCR4+SSEA-1+Oct-4+ stem cells,ii.is released into circulation during tissue/organ injury andiii.their trafficking is regulated by SDF-1-CXCR4 axis.Further studies will asses if the level of mobilized VSELi.correlates with overall regenerative potential of an organism andii.could become a helpful prognostic indicator for the clinical outcome from organ damage/injury (e.g., stroke, heart infarct).Finally, mobilized VSEL could also be employed in regenerative medicine and we are testing this possibility in appropriate animal models.