Very Small Embryonic-like Cells Research Articles

Stem cells mobilization by cardiopulmonary bypass after coronary artery bypass grafting.

Expression of haematopoietic stem and progenitor cells occurs as a result of an inflammatory reaction caused by a damaged organ. Patients undergoing coronary artery bypass surgery (CABG) using cardiopulmonary bypass (CPB) are exposed to an inflammatory reaction, which may be a trigger in the mobilization of regenerative cells. To assess the impact of CPB on stem and progenitor cells mobilization in patients scheduled for CABG. Twenty patients with stable coronary disease, who were scheduled for CABG, were included in the study. Peripheral blood samples were collected perioperatively: 2 h before surgery, before CPB, at CPB weaning, 24 h postoperatively, and on the 6th postoperative day. Analyses of immune-labelled cells were carried out on an ImageStream (IS) system. The following cell populations were identified during cardiac surgery: haematopoietic stem cells (HSC), mesenchymal cells (MSC), endothelial progenitors (EPC), and very small embryonic-like cells (VSEL). The profile of cell mobilization coincides with the perioperative inflammatory response. Mobilization of stem and progenitor cells induced by CPB did not occur in any of the isolated cell lines (p > 0.05). The expression profile of stem and progenitor cells was related with the inflammatory reaction associated with traumatic stress in all cell lines. Type 2 diabetes is a disease that is hampering the trend of MSC mobilization. Mobilization of haematopoietic stem and progenitor cells was not related with CPB. The inflammatory reaction was associated with perioperative trauma. Cell release was inhibited in patients with diabetes.

The Treasury of Wharton's Jelly

BackgroundPostnatal umbilical cord tissue contains valuable mesenchymal progenitor cells of various differentiation stages. While mesenchymal stem cells are plastic-adherent and tend to differentiate into myofibroblastic phenotypes, some round cells detach, float above the adherent cells, and build up cell aggregates, or form spheroids spontaneously. Very small luminescent cells are always involved as single cells or within collective forms and resemble the common well-known very small embryonic-like cells (VSELs). In this study, we investigated these VSELs-like cells in terms of their pluripotency phenotype and tri-lineage differentiation potential.MethodsVSELs-like cells were isolated from cell-culture supernatants by a process that combines filtering, up concentration, and centrifugation. To determine their pluripotency character, we measured the expression of Nanog, Sox-2, Oct-4, SSEA-1, CXCR4, SSEA-4 on gene and protein level. In addition, the cultured cells derived from UC tissue were examined regarding their potential to differentiate into three germ layers.ResultThe VSELs-like cells express all of the pluripotency-associated markers we investigated and are able to differentiate into meso- endo- and ectodermal precursor cells.ConclusionsUmbilical cord tissue hosts highly potent VSELs-like stem cells.Graphical

Markers of Regenerative Processes in Patients with Bipolar Disorder: A Case-control Study.

Progress in medical science has allowed the discovery of many factors affecting the pathogenesis of bipolar disorder, and among the most recent research directions are found regenerative and inflammatory processes. The role of regenerative processes remains particularly poorly explored, but available data encourage further research, which may explain the pathogenesis of bipolar disorder (BD). The aim of this study was to evaluate the mobilization of stem cells into peripheral blood, in patients with bipolar disorder during stable phase, not treated with lithium salts. The study included 30 unrelated individuals with the diagnosis of bipolar disorder, with disease duration of at least 10 years, not treated with lithium salts for at least five years prior to the study. The control group consisted of 30 healthy subjects, matched for age, sex, body mass index (BMI), origin, socio-demographic factors and nicotine use. Blood samples underwent cytometric analyses to assess concentrations of: Very Small Embryonic Like (VSEL) CD34+, VSEL AC133+, HSC CD34+, HSC AC133+. There were no significant differences in stem cell levels between patients with BD and healthy controls. However, the level of VSEL cells AC133 + was significantly higher in type I BD patients compared to healthy controls. Our results indicate a disturbance in regenerative processes in patients with bipolar disorder.

Very small embryonic-like cells in the mirror of regenerative medicine.

Regenerative medicine with the promise of complete organ restoration could change the treatment paradigm for various degenerative disorders. In order to develop successful, safe and efficient regenerative therapies, appropriate amount of pluripotent autologous cells are desired. Pluripotent stem cell lines, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), offer a unique opportunity to derive various cell types that can be exploited for regenerative medicine. However, their application in therapies is limited due to inherent potential tumorigenesis, problems with immune-rejection, applied reprogramming methods, and ethical considerations. Recent reports on the establishment of very small embryonic-like cells (VSELs) are pointing to a way out of this dilemma, since it is proposed that these cells have similar characteristics as ESCs and could serve as basis for therapeutic applications. A careful scientific comparison of VSELs is now needed in order to make judgments about their capabilities and characteristics. In any case, the development of successful, safe and efficient regenerative therapies based on human pluripotent cells requires additional quality assessments of critical parameters that are also summarized within the review.

Role of Adult Tissue-Derived Pluripotent Stem Cells in Bone Regeneration

BackgroundBone marrow-derived mononuclear cells (BM-MNC) consist of a heterogeneous mix of mesenchymal stem cells (MSC), hematopoietic progenitor cells (HPC), endothelial progenitor cells (EPC), monocytes, lymphocytes and pluripotent stem cells. Whereas the importance of MSC and EPC has been well documented in bone healing and regeneration studies, the role of pluripotent stem cells is still poorly understood. In the present study we evaluated if and how Very Small Embryonic Like cells (VSEL), isolated from rat BM-MNC, contribute to bone healing.MethodsLarge bone defects were made in the femurs of 38 Sprague Dawley female rats and treated with β-TCP scaffold granules seeded with male VSEL; BM-MNC, VSEL-depleted BM-MNC or scaffold alone, and bone healing was evaluated at 8 weeks post-surgery.ResultsBone healing was significantly increased in defects treated with VSEL and BM-MNC, compared to defects treated with VSEL-depleted BM-MNC. Donor cells were detected in new bone tissue, in all the defects treated with cells, and in fibrous tissue only in defects treated with VSEL-depleted BM-MNC. The number of CD68+ cells was the highest in the VSEL-depleted group, whereas the number of TRAP positive cells was the lowest in this group.ConclusionsBased on the results, we can conclude that VSEL play a role in BM-MNC induced bone formation. In our rat femur defect model, in defects treated with VSEL-depleted BM-MNC, osteoclastogenesis and bone formation were decreased, and foreign body reaction was increased.

İNSAN PERİFERİK KANINDAN ÇOK KÜÇÜK EMBRİYONİK (VSEL) KÖK HÜCRELERİN ELDE EDİLMESİ VE TANIMLANMASI

<div><!--block-->Objective: Stem cell transplantation is considered to be one of the available treatments for malign or hereditary blood diseases and bone marrow failure. Peripheral blood progenitor cells (PBPC) are widely used for this technique. Recently a new type of stem cell with a pluripotent potential has been identified. These cells, called very small embryonic-like (VSEL) stem cells, are thought to be found in peripheral blood (PB) in adult individuals. The aim of this study was to obtain and identify VSEL stem cells using a peripheral blood source.&nbsp;<br><br>Material and Method: VSEL cells were isolated from mononuclear and erythrocyte layers obtained by using lysis and ficoll gradient methods from the materials taken from donors. The presence of NANOG, OCT4, SSEA-4 and CXCR-4 embryonal stem cell markers by flow cytometry and immunofluorescence staining was investigated. The presence of NANOG and OCT4 proteins was investigated using the Western blot method.&nbsp;<br><br>Results: According to the flow cytometry results, the number of cells carrying the VSEL marker was higher in the debris layer than in the CD45-population. Western and immunofluorescence results showed high levels of OCT4 and NANOG expression. It was also determined that these proteins are present in both the cytoplasm and the nucleus.&nbsp;<br><br>Conclusions: High expression of these proteins, known as pluripotent stem cell markers, in adult peripheral blood brings to mind the question of how they work in differentiated tissues. These findings strengthen the thesis of a new stem cell population that have pluripotent markers in peripheral blood and provide the basis for future clinical studies about this point.</div>

A Novel Method for Isolation of Pluripotent Stem Cells from Human Umbilical Cord Blood.

Very small embryonic-like cells (VSELs) are a population of very rare pluripotent stem cells isolated in adult murine bone marrow and many other tissues and organs, including umbilical cord blood (UCB). VSEL existence is still not universally accepted by the scientific community, so for this purpose, we sought to investigate whether presumptive VSELs (pVSELs) could be isolated from human UCB with an improved protocol based on the isolation of enriched progenitor cells by depletion of nonprogenitor cells with magnetic separation. Progenitor cells, likely including VSELs, cultured with retinoic acid were able to form dense colonies and cystic embryoid bodies and to differentiate toward the ecto-meso-endoderm lineages as shown by the positivity to specific markers. VSEL differentiative potential toward mesodermal lineage was further demonstrated in vitro upon exposure to an established inductive protocol, which induced the acquisition of renal progenitor cell phenotype. VSEL-derived renal progenitors showed regenerative potential in a cisplatin model of acute kidney injury by restoring renal function and tubular structure through induction of proliferation of endogenous renal cells. The data presented here foster the great debate that surrounds VSELs and, more in general, the existence of cells endowed with pluripotent features in adult tissues. In fact, the possibility to find and isolate subpopulations of cells that fully fit all the criteria utilized to define pluripotency remains, nowadays, almost unproven. Thus, efforts to better characterize the phenotype of these intriguing cells are crucial to understand their possible applications for regenerative and precision medicine purposes.

Levels of Stromal Derived Factor-1 (SDF-1) and Brain Derived Neurotropic Factor (BDNF) and Very Small Embryonic-Like Cells (VSEL) in Ischemic Stroke Patients

Levels of Stromal Derived Factor-1 (SDF-1) and Brain Derived Neurotropic Factor (BDNF) and Very Small Embryonic-Like Cells (VSEL) in Ischemic Stroke Patients

Confirmation and Characterization of Lin‐/Sca‐1+/CD45‐ Very Small Embryonic‐Like Cells (VSELs) Purified from Bone Marrow

Previously published studies have identified the presence of a rare population of very small cells (&lt; 5.0 microns in diameter) in the bone marrow of mice (Leukemia 2006 May;20(5):857‐69). In addition to size, these cells are characterized by a gene expression profile consistent with pluripotent embryonic stem cells and can differentiate into multiple cell lineages. While the discovery of these cells, termed 'very small embryonic like' (VSEL) cells, generated much enthusiasm in the field, it was not without controversy. Most recently, a failed attempt at repeating the published protocol that was used to isolate VSEL cells from murine bone marrow concluded that “the existence of adult mouse VSELs in the bone marrow remains dubious.” (Stem Cell Reports 2013 Jul 24;1(2):198‐208). However, close inspection of the methodology employed in this attempt reveals key missteps that may have led to this conclusion. Accordingly, we repeated the protocol as published to determine whether cells with features consistent with VSEL cells could be isolated (Stem Cells Dev. 2014 Apr 1;23(7):702‐13).Bone marrow was flushed from the tibiae and femurs of 10 5‐week female C57BL/6 mice. Following lysis of red blood cells, the bone marrow preparation was incubated with antibodies targeting the following cell surface markers: stem cell antigen 1 (Sca‐1), protein tyrosine phosphatase receptor type C (CD45), and lineage (Lin). The labeled sample was subjected to fluorescence activated cell sorting (FACS), using a gating strategy that employs both size (2–10µm) and fluorescence to determine the appropriate gates. Putative VSEL cells (Lin‐/Sca‐1+/CD45‐) and hematopoietic cells (Lin‐/Sca‐1+/CD45+) were collected for downstream analysis.Using the FACS gating strategy described above, Lin‐/Sca‐1+/CD45‐ cells approximately 3‐5 µm in diameter were successfully identified, lending further support to the existence of VSEL cells in bone marrow of mice.Source of Research Support: Schafer Co‐op Research Scholarship

Very Small Embryonic-Like Stem Cells from the Murine Bone Marrow Differentiate into Epithelial Cells of the Lung

The view that adult stem cells are lineage restricted has been challenged by numerous reports of bone marrow (BM)-derived cells giving rise to epithelial cells. Previously, we demonstrated that nonhematopoietic BM cells are the primary source of BM-derived lung epithelial cells. Here, we tested the hypothesis that very small embryonic like cells (VSELs) are responsible for this engraftment. We directly compared the level of BM-derived epithelial cells after transplantation of VSELs, hematopoietic stem/progenitor cells, or other nonhematopoietic cells. VSELs clearly had the highest rate of forming epithelial cells in the lung. By transplanting VSELs from donor mice expressing H2B-GFP under a type 2 pneumocyte-specific promoter, we demonstrate that this engraftment occurs by differentiation and not fusion. This is the first report of VSELs differentiating into an endodermal lineage in vivo, thereby potentially crossing germ layer lineages. Our data suggest that Oct4+ VSELs in the adult BM exhibit broad differentiation potential.

Number of circulating pro-angiogenic cells, growth factor and anti-oxidative gene profiles might be altered in type 2 diabetes with and without diabetic foot syndrome.

Type 2 diabetes is often complicated by diabetic foot syndrome (DFS). We analyzed the circulating stem cells, growth factor and anti-oxidant gene expression profiles in type 2 diabetes patients without or with different forms of DFS. Healthy volunteers (n=13) and type 2 diabetes patients: (i) without DFS (n=10); or with (ii) Charcot osteoneuropathy (n=10); (iii) non-infected (n=17); (iv) infected (n=11); and (v) healed ulceration were examined (n=12). Peripheral blood endothelial progenitor cells (EPC), mesenchymal stem cells (MSC), hematopoietic stem cells (HSC) and very small embryonic-like (VSEL) cells were phenotyped using flow cytometry. Plasma cytokine concentrations and gene expressions in blood cells were measured by Luminex and quantitative real-time polymerase chain reaction assays, respectively. Patients with non-complicated type 2 diabetes showed reduced HMOX1 expression, accompanied by HMOX2 upregulation, and had less circulating EPC, MSC or HSC than healthy subjects. In contrast, VSEL cells were elevated in the type 2 diabetes group. However, subjects with DFS, even with healed ulceration, had fewer VSEL cells, more CD45-CD29(+)CD90(+)MSC, and upregulated HMOX1 when compared with the type 2 diabetes group. Patients with Charcot osteopathy had lowered plasma fibroblast growth factor-2. Elevated plasma tumor necrosis factor-α and decreased catalase expression was found in all diabetic patients. Patients with type 2 diabetes and different forms of DFS have an altered number of circulating stem cells. Type 2 diabetes might also be associated with a changed plasma growth factor and anti-oxidant gene expression profile. Altogether, these factors could contribute to the pathogenesis of different forms of DFS.

Very small embryonic‐like cells: Biology and function of these potential endogenous pluripotent stem cells in adult tissues

Very small embryonic-like cells (VSELs), found in murine bone marrow and other adult tissues, are small, non-hematopoietic cells expressing markers of pluripotent embryonic and primordial germ cells. A similar cell type in humans has begun to be characterized, though with a slightly different phenotype and surface markers. Consistent with expression of pluripotency genes, murine VSELs differentiate into cell types from three germ-layer lineages in vitro, though pluripotency has yet to be shown at the single-cell level or in vivo. VSELs appear to be quiescent under steady state conditions, apparently due to partially erased imprinting and overexpression of cell cycle inhibitory genes. In vivo, VSELs can enter the cell cycle under stress conditions, but which factors regulate quiescence versus proliferation and self-renewal versus differentiation are as yet unknown, and in vitro conditions that induce proliferation and self-renewal have yet to be defined. Future experiments are needed to address whether a VSEL niche actively regulates quiescence in vivo or quiescence is cell autonomous under steady state conditions. Insights into these mechanisms may help to address whether or not VSELs could play a role in regenerative medicine in the future.

Cytometry in stem cell research and therapy

recent Nobel Prize in medicine was awarded to two stemcell researchers, John Gurdon and Shinya Yamanaka, for theirachievements in stem cell research and reprogramming ofsomatic cells. Flow cytometry is by nature the ideal tool toidentify, characterize, and isolate stem and progenitor cells forresearch and potential clinical use (1). The major strength offlow cytometry is its ability to rapidly perform highlymultiplexed quantitative measurements on single cells withina heterogeneous cell population. However, when the cell typeof interest is extremely rare, as most stem and progenitor cellsare, several sources of artifact must be addressed. The impor-tance of flow cytometry as a driving force for stem cellresearch was demonstrated in a focused issue of the journalexactly three years ago (1). This current focus issue of Cytome-try A is devoted to the topic of stem cells due to numerouscurrent innovations and discoveries.Applications of stem cells include several disciplines,from embryogenesis, adult tissue maintenance, and repair,and more recently, cancer as well as for toxicity screening anddisease modeling. All of these topics are represented in thisissue, with special emphasis on the role of analytic and pre-parative flow cytometry in the elucidation of stem cell pheno-type and function, and best laboratory practices as they applyto flow cytometry. Image and flow cytometry together withcell sorting have revolutionized the study of stem cell biologyand the implications of these cells and their progeny indevelopmental biology, tissue engineering, and cellulartherapy. The number of parameters and the speed of theirsimultaneous measurements in single cells has continued toincrease with advances in hardware, reagents, and analyticalsoftware (2).

Abstract 19198: Adult Human Heart Contains Two Populations of C-kit-Oct-4+SSEA-4+ Very Small Embryonic-Like Stem Cells (Isl-1- and Isl-1+)

Very small embryonic-like cells (VSELs) express pluripotent stem markers (Oct-4,Nanog, SSEA-4). and in adult mice may be expanded and differentiatiated into three germ layers. In humans VSELs were found in cord blood and peripheral blood (PB). AIM: Comparison of VSELs isolated from human myocardium with cardiac stem cells (CSC) and to measure the mobilization into PB after cardiac surgery. METHODS: 32 patients undergoing CABG were enrolled. Right atrial appendage (RAA), left ventricle (LV) were collected. PB samples were drawn before and immediately after surgery, 24 hrs and 5-7 days after weaning from cardiopulmonary bypass. Tissue samples (0.5-1.0 cm3) were cut, digested in collagenase, passed through 70um cell strainer, centrifugated, resuspended and stained with antibodies CD34 FITC/CD133 APC/SSEA-4PE/CD45 APC-Cy7. Analyses were carried out on Image Stream System which is a combination of FACS and immunofluorescent microscope. RESULTS: VSELs and CSC were identified in LAA. VSELs had markedly smaller diameter (4.7 ± 0.2µm vs. 7.7 ± 0.4µm), were c-kit negative, expressed markers of pluripotent cells (Oct-4, SSEA-4) and CD133. Two populations of VSELs - both Isl-1 negative and positive were identified. Subpopulation of Oct-4+SSEA-4+Isl+ VSELs contained cells of intermediate size (5.7 ± 0.7µm) and might form more mature population of VSELs progenitors. VSELs undergo significant mobilization into PB following the CABG. CONCLUSION: Human myocardium in adults contains two subpopulations of Oct-4+SSEA4+ VSELs which are distinct from CSC. Cardiac surgery with cardiopulmonary bypass induced mobilization of VSELs into peripheral blood.

Murine and human very small embryonic‐like cells: A perspective

In 2006, very small embryonic-like (VSEL) stem cells were described as a pluripotent population of prospectively isolated stem cells in adult murine bone marrow (mBM) and human umbilical cord blood (hUCB). While rigorous proof of pluripotency is still lacking, murine VSEL cells have been shown to overlap with an independently identified population of neural crest derived mesenchymal stem cells (MSC). The presence of primitive mesenchymal precursors within the VSEL cell population may partially explain the findings that have led to the concept of an "embryonic-like" stem cell in mBM. However, our own studies on human VSEL cells revealed very little similarity between murine VSEL cells and their reportedly equivalent population in hUCB. On the contrary, our data strongly suggest that human VSEL cells are an aberrant and inactive population that cannot expand in vitro and has neither embryonic nor adult stem cell like properties. Here we critically re-examine the data supporting stemness and pluripotency of murine and human VSEL cells, respectively.

Very Small Embryonic-Like Stem Cells Purified from Umbilical Cord Blood Lack Stem Cell Characteristics

Very small embryonic-like (VSEL) cells have been described as putatively pluripotent stem cells present in murine bone marrow and human umbilical cord blood (hUCB) and as such are of high potential interest for regenerative medicine. However, there remain some questions concerning the precise identity and properties of VSEL cells, particularly those derived from hUCB. For this reason, we have carried out an extensive characterisation of purified populations of VSEL cells from a large number of UCB samples. Consistent with a previous report, we find that VSEL cells are CXCR4+, have a high density, are indeed significantly smaller than HSC and have an extremely high nuclear/cytoplasmic ratio. Their nucleoplasm is unstructured and stains strongly with Hoechst 33342. A comprehensive FACS screen for surface markers characteristic of embryonic, mesenchymal, neuronal or hematopoietic stem cells revealed negligible expression on VSEL cells. These cells failed to expand in vitro under a wide range of culture conditions known to support embryonic or adult stem cell types and a microarray analysis revealed the transcriptional profile of VSEL cells to be clearly distinct both from well-defined populations of pluripotent and adult stem cells and from the mature hematopoietic lineages. Finally, we detected an aneuploid karyotype in the majority of purified VSEL cells by fluorescence in situ hybridisation. These data support neither an embryonic nor an adult stem cell like phenotype, suggesting rather that hUCB VSEL cells are an aberrant and inactive population that is not comparable to murine VSEL cells.

Circulating Very Small Embryonic-Like Stem Cells in Cardiovascular Disease

Very small embryonic-like cells (VSELs) are a population of stem cells residing in the bone marrow (BM) and several organs, which undergo mobilization into peripheral blood (PB) following acute myocardial infarction and stroke. These cells express markers of pluripotent stem cells (PSCs), such as Oct-4, Nanog, and SSEA-1, as well as early cardiac, endothelial, and neural tissue developmental markers. VSELs can be effectively isolated from the BM, umbilical cord blood, and PB. Peripheral blood and BM-derived VSELs can be expanded in co-culture with C2C12 myoblast feeder layer and undergo differentiation into cells from all three germ layers, including cardiomyocytes and vascular endothelial cells. Isolation of VSLEs using fluorescence-activated cell sorting multiparameter live cell sorting system is dependent on gating strategy based on their small size and expression of PSC and absence of hematopoietic lineage markers. VSELs express early cardiac and endothelial lineages markers (GATA-4, Nkx2.5/Csx, VE-cadherin, and von Willebrand factor), SDF-1 chemokine receptor CXCR4, and undergo rapid mobilization in acute MI and ischemic stroke. Experiments in mice showed differentiation of BM-derived VSELs into cardiac myocytes and effectiveness of expanded and pre-differentiated VSLEs in improvement of left ventricular ejection fraction after myocardial infarction.

Mobilization of very small embryonic-like stem cells in acute coronary syndromes and stroke

The bone marrow (BM) niche contains small heterogenous populations of cells which may contribute to cardiac and endothelial repair, including committed lineages [endothelial progenitor cells (EPCs), multipotent mesenchymal stromal cells (MSCs) and more primitive very small embryonic-like cells (VSELs) expressing pluripotent stem cell (PSC) markers (Oct-4, Nanog, SSEA-1)]. VSELs are present in BM, peripheral blood and some solid organs in mice and were recently identified in peripheral blood in patients with acute coronary syndromes and stroke. VSELs can be expanded in vitro and differentiated into cells from all three germ layers. This population of cells displays the morphology of primitive PSC (small size, open type chromatin, large nucleus, narrow rim of cytoplasm) and express PSC markers. The isolation of human VSELs is based on their size and presence of several surface markers (CXCR4, CD133, CD34) and lack of markers of hematopoietic lineage (lin, CD45). In acute myocardial infarction and ischemic stroke VSELs are rapidly mobilized into peripheral blood, and express increased levels of PSC markers as well as early cardiac (GATA-4, Nkx2.5/Csx), neural (GFAP, nestin, beta-III-tubulin, Olig1, Olig2, Sox2, Musashi) and endothelial lineage markers (VE-cadherin, von Willebrand factor). The number of VSELs mobilized in acute myocardial infarction is inversely correlated with left ventricular ejection fraction and the release of cardiac necrosis markers. Mobilization of these cells is also reduced in patients with diabetes and in the elderly. BM-derived VSELs were expanded and after cardiogenic pre-differentiation injected intramyocardially in mice models of myocardial infarction leading to improved left ventricular contractility. VSELs are probably progeny of epiblast cells which migrated to the BM and developing organs during embryonic development. The cells are present in a quiescent state in the adult BM and solid organs and might serve as a reserve pool of resident stem cells. VSELs are promising candidates for further pre-clinical and clinical studies on cellular cardiovascular therapy.

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.

Abstract 697: Mobilization of Oct-4 + Bone Marrow-Derived Very Small Embryonic-Like Stem Cells in Patients with Acute Myocardial Infarction

Bone marrow-derived CD34 + CXCR4 + progenitor cells are mobilized into peripheral blood early in acute myocardial infarction (MI). Adult murine bone marrow contains population of small CD34 + lin − CD45 − CXCR4 + cells expressing markers of pluripotent stem cells (PSC) SSEA, Oct-4 and Nanog. This population of very small embryonic-like cells (VSEL) has unique morphology (small size 2– 4 μm, large nucleus, euchromatin) and capability to form embrioid bodies (EB). Murine EB-derived cells can in vitro differentiate into cells from all three germ layers including cardiomyocytes. We hypothesized that in patients with acute MI small cells expressing the VSEL immunophenotype and PSC markers are present in bone marrow and mobilized into peripheral blood. Blood samples (20 mL) from 18 patients with acute MI were obtained after 12 hours, 2 and 5 days after symptoms onset. Bone marrow samples (20 mL) were obtained from 2 patients with acute MI and 3 healthy volunteers. Mononuclear cells were isolated using hypotonic lysis and samples were analyzed by FACS. Mobilization of following cell populations was confirmed: hematopoietic lin − CD45 + CXCR4 + , lin − CD45 + CD133 + , lin − CD45 + CD34 + and non-hematopoietic (VSEL) lin − CD45 − CXCR4 + , lin − CD45 − CD133 + , lin − CD45 − CD34 + . Analysis of the cell number using lymphocyte gate showed more significant increase of CD45 + (hematopoietic) populations of lin − CD34 + , lin − CD133 + and lin − CXCR4 + cells. After gating for small events (VSEL size range) we found more significant mobilization of small, non-hematopoietic populations of lin − CD34 + , lin − CD133 + and lin − CXCR4 + cells (Table ). The expression of PSC markers (Oct-4, Nanog, SSEA-1) in VSEL was confirmed using real-time RT-PCR. Conclusion: We report for the first time that acute MI is associated with mobilization of non-hematopoietic VSELs expressing pluripotent stem cells markers.