Molecular and ultrastructural characterization of endothelial cells differentiated from human bone marrow mesenchymal stem cells (original) (raw)
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2008
Human bone marrow derived mesenchymal stem cells (HBMSCs) have the potential to differentiate into cells such as adipocyte, osteocyte, hepatocyte and endothelial cells. In this study, the differentiation of hBMSCs into endothelial like-cells was induced in presence of vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF-1). The differentiated endothelial cells were examined for their ability to express VEGF receptor-2 (VEGFR2) and von willebrand factor (vWF). Then the cells were adopted to grow and develop capillary network in a semisolid gel matrix in vitro. The capillary network formation in a well of 24-well plate was found to be 85% in presence of VEGF (50ng/ml) and IGF-1 (20ng/ml) of the culture media. These data may suggest that the expression of endothelial markers in endothelial like-cells derived from hBMSCs is associated with their ability to form capillaries.
International Journal of Stem Cells, 2016
Understanding the mechanisms of vascular remodeling could lead to more effective treatments for ischemic conditions. We aimed to compare between the abilities of both human Wharton jelly derived mesenchymal stem cells (hMSCs) and human cord blood endothelial progenitor cells (hEPCs) and CD34 ＋ to induce angiogenesis in vitro. hMSCs, hEPCs, and CD34 ＋ were isolated from human umbilical cord blood using microbead (MiniMacs). The cells characterization was assessed by flow cytometry following culture and real-time PCR for vascular endothelial growth factor receptor 2 (VEGFR2) and von Willebrand factor (vWF) to prove stem cells differentiation. The study revealed successful isolation of hEPCs, CD34 ＋ , and hMSCs. The hMSCs were identified by gaining CD29 ＋ and CD44 ＋ using FACS analysis. The hEPCs were identified by having CD133 ＋ , CD34 ＋ , and KDR. The potential ability of hEPCs and CD34 ＋ to differentiate into endothelial-like cells was more than hMSCs. This finding was assessed morphologically in culture and by higher significant VEGFR2 and vWF genes expression (p＜0.05) in differentiated hEPCs and CD34 ＋ compared to differentiated hMSCs. hEPCs and CD34 ＋ differentiation into endothelial-like cells were much better than that of hMSCs.
Differentiation, 2010
A murine embryonic mesenchymal cell line C3H/10T1/2 possesses the potential to differentiate into multiple cell phenotypes and has been recognized as multipotent mesenchymal stem cells, but no in vitro model of its endothelial differentiation has been established and the effect of angiogenic factors on the differentiation is unknown. The aim of the present study was to evaluate the role of angiogenic factors in inducing endothelial differentiation of C3H/10T1/2 cells in vitro. C3H/10T1/2 cells were treated with angiogenic factors, VEGF (10 ng/mL) and bFGF (5 ng/mL). At specified time points, cells were subjected to morphological study, immunofluorescence staining, RT-PCR, LDL-uptake tests and 3-D culture for the examination of the structural and functional characteristics of endothelial cells. Classic cobblestone-like growth pattern appeared at 6 day of the induced differentiation. Immunofluorescence staining and RT-PCR analyses revealed that the induced cells exhibited endothelial cellspecific markers such as CD31, von Willebrand factor, Flk1, Flt1, VE-cadherin, Tie2, EphrinB2 and Vezf1 at 9 day. The induced C3H/10T1/2 cells exhibited functional characteristics of the mature endothelial phenotype, such as uptake of acetylated low-density lipoproteins (Ac-LDL) and formation of capillarylike structures in three-dimensional culture. At 9 day, Weibel-Palade bodies were observed under a transmission electron microscope. This study demonstrates, for the first time, endothelial differentiation of C3H/10T1/2 cells induced by angiogenic factors, VEGF and bFGF, and confirms the multipotential differentiation ability. This in vitro model is useful for investigating the molecular events in endothelial differentiation of mesenchymal stem cells.
PubMed, 2016
Objective: In vivo and in vitro stem cell differentiation into endothelial cells is a promising area of research for tissue engineering and cell therapy. Materials and methods: We induced human mesenchymal stem cells (MSCs) to differentiate to endothelial cells that had the ability to form capillaries on an extracellular matrix (ECM) gel. Thereafter, the differentiated endothelial cells at early stage were characterized by expression of specific markers such as von Willebrand factor (vWF), vascular endothelial growth factor (VEGF) receptor 2, and CD31. In this experimental model, the endothelial cells were transplanted into the groins of severe combined immunodeficiency (SCID) mice. After 30 days, we obtained tissue biopsies from the transplantation sites. Biopsies were processed for histopathological and double immunohistochemistry (DIHC) staining. Results: Endothelial cells at the early stage of differentiation expressed endothelial markers. Hematoxylin and eosin (H&E) staining, in addition to DIHC demonstrated homing of the endothelial cells that underwent vascularization in the injected site. Conclusion: The data clearly showed that endothelial cells at the early stage of differentiation underwent neovascularization in vivo in SCID mice. Endothelial cells at their early stage of differentiation have been proven to be efficient for treatment of diseases with impaired vasculogenesis.
2016
Neo vessel formation can be initiated by co-transplantation of mesenchymal stem cells (MSC) with endothelial colony-forming cells (ECFC). The two adult stem cell types can be isolated and expanded from a variety of tissues to be used for regenerative applications pro-angiogenesis.Here we performed a systematic study to evaluate the neo-vasculogenesis potential of MSC and ECFC isolated from various human tissues. MSC were isolated, purified and expanded in vitro from umbilical cord (UC) and umbilical cord blood (UCB), white adipose tissue (WAT), bone marrow (BM), and amniotic membrane of placenta (AMN).ECFC were isolated from UC and UCB, WAT and peripheral blood (PB). ECFC and MSC and were co-transplanted admixed with extracellular matrix (Matrigel®) at a ratio of 5:1 to immune-deficient NSG mice, subcutaneously. The transplants were harvested after two weeks and the state of vessel formation and stability in the explants were investigated using immune-histochemical methods. The numb...
Isolation and culture of endothelial cells from human bone marrow
British Journal of Haematology, 1994
Adhesive interactions between haemopoietic progenitor cells and bone marrow sinusoidal endothelium are potentially important in the homing of these cells back to the extravascular compartment of the marrow to reestablish haemopoiesis following stem cell transplantation. A simple method for the isolation and culture of human bone marrow endothelial cells is described using bone marrow aspirates obtained from patients undergoing bone marrow harvests for autologous or syngeneic bone marrow transplantation. The method is based on the selective binding of the lectin Ulex europaeus agglutinin-1 (LEA-1) to endothelial cells. Magnetic Dynabeads coupled with UEA-1 were incubated with single cell suspensions of bone marrow following red cell lysis, and bound cells were isolated with a magnet. The isolated cells demonstrated positive immunofluorescence staining for von Willebrand factor. Cells were plated onto tissue culture flasks coated with extracellular matrix derived from human umbilical vein endothelial cells in an endothelial serum-free medium together with 5% fetal calf serum for 24 h. Cells were then cultured in endothelial serum-free growth medium supplemented with 5% fetal calf serum, endothelial cell growth supplement and heparin. After 2-4 weeks in culture. two morphologically different cell populations can be identified. One has a polygonal spindle-shaped morphology with a rapid growth rate, the other a rounded morphology and a slow growth rate. Both populations have a vesiculated cytoplasm. Positive immunostaining of the cells was demonstrated with a number of endothelial cell markers including von Willebrand factor, and antibodies to ICAM-1, VCAM-1, E-selectin, CD31 and BMAl20. Weibel-Palade bodies were observed by electron microscopy. Culture of these ceIls will allow detailed in vitro studies of adhesion mechanisms in the homing of haemopoietic progenitor cells.
Biological Procedures Online, 2010
The aim of this study was to compare different cell sources and culture conditions to obtain endothelial progenitor cells (EPCs) with predictable antigen pattern, proliferation potential and in vitro vasculogenesis. Pig mononuclear cells were isolated from blood (PBMCs) and bone marrow (BMMCs). Mesenchymal stem cells (MSCs) were also derived from pig bone marrow. Cells were cultured on fibronectin in the presence of a high concentration of VEGF and low IGF-1 and FGF-2 levels, or on gelatin with a lower amount of VEGF and higher IGF-1 and FGF-2 concentrations. Endothelial commitment was relieved in almost all PBMCs and BMMCs irrespective of the protocol used, whilst MSCs did not express a reliable pattern of EPC markers under these conditions. BMMCs were more prone to expand on gelatin and showed a better viability than PBMCs. Moreover, about 90% of the BMMCs pre-cultured on gelatin could adhere to a hyaluronan-based scaffold and proliferate on it up to 3 days. Pre-treatment of BMMCs on fibronectin generated wellshaped tubular structures on Matrigel, whilst BMMCs exposed to the gelatin culture condition were less prone to form vessel-like structures. MSCs formed rough tubule-like structures, irrespective of the differentiating condition used. In a relative short time, pig BMMCs could be expanded on gelatin better than PBMCs, in the presence of a low amount of VEGF. BMMCs could better specialize for capillary formation in the presence of fibronectin and an elevated concentration of VEGF, whilst pig MSCs anyway showed a limited capability to differentiate into the endothelial cell lineage.
In vitrodifferentiation of bone marrow derived porcine mesenchymal stem cells to endothelial cells
Journal of Tissue Engineering and Regenerative Medicine, 2012
Mesenchymal stem cells (MSCs) hold potential for the regeneration of damaged tissues in cardiovascular diseases. In this study, we investigated the potential of porcine MSCs to differentiate into endothelial cells (ECs) in-vitro. The cultured bone marrow derived cells were CD11b − CD34 − CD44 + CD45 − CD90 + and showed mesodermal lineage differentiation which are characteristic of MSCs. The MSCs were induced to differentiate into ECs using endothelial growth media (EGM) with and without high concentrations of VEGF (EGM+VEGF; 50ng/ml). The endothelial basal medium (EBM) without growth factors served as the control. The EC differentiation was assessed by the presence of vWF, ability to take up acetylated LDL, in vitro angiogenesis assay, flow cytometry and qPCR of EC markers vWF, VE-cadherin, PECAM-1, CD34, VEGF-R1 and VEGF-R2 after 10days of stimulation. The cells cultured in EGM+VEGF media demonstrated higher amount of DiI-AcLDL positive cells, and enhanced presence of vWF (90%), VE-Cadherin (60%) and PECAM-1 (48%) positive cells, than in EBM. These cells showed profuse sprouting of capillary tubes and closed polygon formation in the angiogenesis assay. There was 1.5-2 fold increase in the mRNA expression of endothelial markers in the cells stimulated with EGM+VEGF media when compared to control. The results demonstrate the ability of porcine MSCs to differentiate into ECs under in-vitro inducing conditions. The differentiated cells would provide new options for re-endothelialization following interventional procedures and tissue engineering.