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

Transcriptional landscape of bone marrow-derived very small embryonic-like stem cells during hypoxia

<p>Abstract</p> <p>Background</p> <p>Hypoxia is a ubiquitous feature of many lung diseases and elicits cell-specific responses. While the effects of hypoxia on stem cells have been examined under <it>in vitro </it>conditions, the consequences of <it>in vivo </it>oxygen deprivation have not been studied.</p> <p>Methods</p> <p>We investigated the effects of <it>in vivo </it>hypoxia on a recently characterized population of pluripotent stem cells known as very small embryonic-like stem cells (VSELs) by whole-genome expression profiling and measuring peripheral blood stem cell chemokine levels.</p> <p>Results</p> <p>We found that exposure to hypoxia in mice mobilized VSELs from the bone marrow to peripheral blood, and induced a distinct genome-wide transcriptional signature. Applying a computationally-intensive methodology, we identified a hypoxia-induced gene interaction network that was functionally enriched in a diverse array of programs including organ-specific development, stress response, and wound repair. Topographic analysis of the network highlighted a number of densely connected hubs that may represent key controllers of stem cell response during hypoxia and, therefore, serve as putative targets for altering the pathophysiologic consequences of hypoxic burden.</p> <p>Conclusions</p> <p>A brief exposure to hypoxia recruits pluripotent stem cells to the peripheral circulation and actives diverse transcriptional programs that are orchestrated by a selective number of key genes.</p

Spinal Cord Injury Repair by Intrathecal Infusion of Stromal Cell-Derived Factor-1/CXC Chemokine Receptor 4 in a Rat Model

Background: Stromal cell-derived factor-1 (SDF-1)/CXC Chemokine receptor 4 (CXCR4) is an important cytokine, with multiple functions, which plays a crucial role in the recruitment of multiple stem cell types in the defect sites of central nervous system (CNS). Various strategies have been managed to improve functional recovery after spinal cord injury (SCI). One of these strategies is the use of factors to limit damage and increase recovery. Objectives: In this study we investigated the effect of SDF-1 in spinal cord injury repair in a rat model. Materials andMethods: Adult male Wistar rats were randomly divided to four groups (n = 5) as follows: Sham, SCI, SDF-1 and Vehicle. Spinal cord injury model was created by contusion of T8-T9 by clips and SDF-1 infusion pump implanted in the neck region. One week after injury, 5-Bromo-20-Deoxyuridine (BrdU) was injected to trace the proliferative cells. Basso-Beattie-Bresnahan (BBB) test was performed to evaluate locomotor activity following SCI. Immunohistochemistry test was performed to determine proliferating cells, and real time polymerase chain reaction (PCR) was performed to detect the CXCR4 cells in tissue. Results: Significant improvements in locomotor function were detected in the SDF-1 group compared with the SCI and vehicle groups (P < 0.05). The results showed that SDF-1 treatment increased proliferative cells at the spinal cord injury site. Real time PCR revealed that these proliferative cells are CXCR4 positive that intake Bromodeoxyuridine (Brdu). Conclusions: These results showed that the administration of SDF-1a increases the number of proliferating cells in the injured area in the spinal cord and improves functional recovery

Validation of a Thin-Layer Chromatography for the Determination of Hydrocortisone Acetate and Lidocaine in a Pharmaceutical Preparation

A new specific, precise, accurate, and robust TLC-densitometry has been developed for the simultaneous determination of hydrocortisone acetate and lidocaine hydrochloride in combined pharmaceutical formulation. The chromatographic analysis was carried out using a mobile phase consisting of chloroform + acetone + ammonia (25%) in volume composition 8 : 2 : 0.1 and silica gel 60F 254 plates. Densitometric detection was performed in UV at wavelengths 200 nm and 250 nm, respectively, for lidocaine hydrochloride and hydrocortisone acetate. The validation of the proposed method was performed in terms of specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and robustness. The applied TLC procedure is linear in hydrocortisone acetate concentration range of 3.75 ÷ 12.50 g⋅spot −1 , and from 1.00 ÷ 2.50 g⋅spot −1 for lidocaine hydrochloride. The developed method was found to be accurate (the value of the coefficient of variation CV [%] is less than 3%), precise (CV [%] is less than 2%), specific, and robust. LOQ of hydrocortisone acetate is 0.198 g⋅spot −1 and LOD is 0.066 g⋅spot −1 . LOQ and LOD values for lidocaine hydrochloride are 0.270 and 0.090 g⋅spot −1 , respectively. The assay value of both bioactive substances is consistent with the limits recommended by Pharmacopoeia

Very small embryonic-like stem cells (VSELs) represent a real challenge in stem cell biology : recent pros and cons in the midst of a lively debate

The concept that adult tissue, including bone marrow (BM), contains early-development cells with broader differentiation potential has again been recently challenged. In response, we would like to review the accumulated evidence from several independent laboratories that adult tissues, including BM, harbor a population of very rare stem cells that may cross germ layers in their differentiation potential. Thus, the BM stem cell compartment hierarchy needs to be revisited. These dormant, early-development cells that our group described as very small embryonic-like stem cells (VSELs) most likely overlap with similar populations of stem cells that have been identified in adult tissues by other investigators as the result of various experimental strategies and have been given various names. As reported, murine VSELs have some pluripotent stem cell characteristics. Moreover, they display several epiblast/germline markers that suggest their embryonic origin and developmental deposition in adult BM. Moreover, at the molecular level, changes in expression of parentally imprinted genes (for example, Igf2–H19) and resistance to insulin/insulin-like growth factor signaling (IIS) regulates their quiescent state in adult tissues. In several emergency situations related to organ damage, VSELs can be activated and mobilized into peripheral blood, and in appropriate animal models they contribute to tissue organ/regeneration. Interestingly, their number correlates with lifespan in mice, and they may also be involved in some malignancies. VSELs have been successfully isolated in several laboratories; however, some investigators experience problems with their isolation

CXCR4 expression on circulating pan-cytokeratin positive cells is associated with survival in patients with advanced non-small cell lung cancer

<p>Abstract</p> <p>Background</p> <p>The CXC chemokine, CXCL12, and its receptor, CXCR4 promote metastases of a variety of solid tumors, including non-small cell lung cancer (NSCLC). The expression of CXCR4 on tumor cells may represent a critical biomarker for their propensity to metastasize. This study was performed to evaluate the hypothesis that co-expression of pan-cytokeratin and CXCR4 may be a prognostic marker for patients with advanced NSCLC.</p> <p>Methods</p> <p>We evaluated CXCR4 levels on circulating pan-cytokeratin positive cells from patients with NSCLC. NSCLC tumor and metastases were also assessed for the presence of CXCR4.</p> <p>Results</p> <p>Pan-cytokeratin positive cells were increased in the circulation of patients with NSCLC, as compared to normal control subjects. Patients with pan-cytokeratin +/CXCR4+ = 2,500 cells/ml had a significant improvement in median survival when compared with patients with pan-cytokeratin +/CXCR4+ >2,500 cells/ml (not achieved versus 14 weeks). CXCR4 expression was found on NSCLC tumors and at sites of tumor metastasis.</p> <p>Conclusion</p> <p>This study suggests that CXCR4 may be a prognostic marker in NSCLC, and provides hypothesis-generating results, which may be important in determining metastatic potential. In future studies, we will prospectively evaluate the prognostic significance of pan-cytokeratin/CXCR4+ cells, and determine the mechanisms involved in the regulation of CXCR4 expression on tumor cells in a larger patient population.</p

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

Adventage of mesenchymal stem cells (MSC) expansion directly from purified bone marrow CD105^+ and CD271^+ cells

Mesenchymal Stem Cells (MSC) are employed in gene and cellular therapies. Routinely MSC are isolated from bone marrow mononuclear cells (MNC) by plastic adherence. Here we compared new isolation strategies of bone marrow MSC including immunodepletion of hematopoietic cells and immunomagnetic isolation of CD105+ and CD271+ populations. Four fractions were obtained: MNC MSC, RosetteSep-isolated MSC, CD105+ and CD271+ sorted MSC. We evaluated i) number of CFU-F colonies, ii) cell phenotype, iii) in vitro differentiation of expanded cells and iv) expression of osteo/adipogenesis related genes. Results: Average number of day 9 CFU-F colonies was the highest for CD271 positive fraction. Real-Time PCR analysis revealed expression of RUNX2, PPARgamma and N-cadherin in isolated cells, particularly high in CD271+ cells. Expression of CD105, CD166, CD44, CD73 antigens was comparable for all expanded populations (over 90%). We observed various levels of hematopoietic contamination with the highest numbers of CD45+ cells in MNC-MSC fraction and the lowest in CD105+ and CD271+ fractions. Cells of all the fractions were CD34 antigen negative. Expanded CD105 and CD271 populations showed higher level of RUNX2, osteocalcin, PTHR, leptin, PPARgamma2 and aggrecan1 genes except for alpha1 collagen. After osteogenic differentiation CD105+ and CD271+ populations showed lower expression of RUNX, PPARgamma2 and also lower expression of osteocalcin and PTHR than MNC, with comparable alpha1-collagen expression. Chondrogenic and adipogenic gene expression was higher in MNC. More clonogenic CD105+ and particularly CD271+ cells, which seem to be the most homogenous fractions based on Real-Time PCR and immunostaining data, are better suited for MSC expansion