OBJECTIVE: Vascular Stem Cells (VSCs) are recently defined as cells reside within the vessel wall, which show the typical mesenchymal characteristics and can differentiate into smooth muscle cells (SMCs) and endothelial cells (ECs), the cell types that constitute a functional blood vessel. Recently, we described the isolation of vascular precursor cells from the tunica media of porcine thoracic aortic (porcine Aortic Vascular Precursor cells, pAVPCs). These cells have mesenchymal phenotypic characteristics, pericytes typical functional properties and are able to differentiate spontaneously into smooth muscle cells. The aim of this study was to assess the differentiation potential of pAVPCs toward the endothelial lineage. MATERIALS AND METHODS: pAVPCs were seeded in a 24 well plate (5000 cells/cm2) and cultured in Endothelial Differentiation Medium (EDM) (human Endothelial Serum Free Medium supplemented with 5% FBS and 50 ng/mL of human recombinant Vascular Endothelial Growth Factor [hVEGF]) or in Perycite Growth Medium (PGM) as control. After 21 days of culture, the expression level of differentiated endothelial cells markers, CD31, VE-Cadherin, von Willebrand Factor (vWF) and endothelial Nitric Oxide Synthase (eNOS) was analyzed by qPCR. Expression of CD31 was also tested by immunofluorescence. RESULTS: The gene expression analysis revealed a significant increase of expression levels of all the endothelial markers in cells cultured in EDM respect to the control (PGM), with VE-Cadherin and CD31 showing an increase of 33 and 22 times, respectively. Immunofluorescence analysis of CD31 revealed that only cells cultured in EDM expressed the protein with the classical membrane localization. CONCLUSIONS: We demonstrated that pAVPCs, grown in specific endothelial medium and stimulated with VEGF, are able to differentiate toward the endothelial cells phenotype. Therefore, according to the recent definition of VSC, the pAVPCs could be considered a population of VSC-like cells.
Martina Bertocchi, Andrea Zaniboni, Chiara Bernardini, Augusta Zannoni, Marco Alessandri, Laura Calza', et al. (2014). Endothelial differentiation of porcine Aortic Vascular Precursor Cells.
Endothelial differentiation of porcine Aortic Vascular Precursor Cells
BERTOCCHI, MARTINA;ZANIBONI, ANDREA;BERNARDINI, CHIARA;ZANNONI, AUGUSTA;ALESSANDRI, MARCO;CALZA', LAURA;BACCI, MARIA LAURA;FORNI, MONICA
2014
Abstract
OBJECTIVE: Vascular Stem Cells (VSCs) are recently defined as cells reside within the vessel wall, which show the typical mesenchymal characteristics and can differentiate into smooth muscle cells (SMCs) and endothelial cells (ECs), the cell types that constitute a functional blood vessel. Recently, we described the isolation of vascular precursor cells from the tunica media of porcine thoracic aortic (porcine Aortic Vascular Precursor cells, pAVPCs). These cells have mesenchymal phenotypic characteristics, pericytes typical functional properties and are able to differentiate spontaneously into smooth muscle cells. The aim of this study was to assess the differentiation potential of pAVPCs toward the endothelial lineage. MATERIALS AND METHODS: pAVPCs were seeded in a 24 well plate (5000 cells/cm2) and cultured in Endothelial Differentiation Medium (EDM) (human Endothelial Serum Free Medium supplemented with 5% FBS and 50 ng/mL of human recombinant Vascular Endothelial Growth Factor [hVEGF]) or in Perycite Growth Medium (PGM) as control. After 21 days of culture, the expression level of differentiated endothelial cells markers, CD31, VE-Cadherin, von Willebrand Factor (vWF) and endothelial Nitric Oxide Synthase (eNOS) was analyzed by qPCR. Expression of CD31 was also tested by immunofluorescence. RESULTS: The gene expression analysis revealed a significant increase of expression levels of all the endothelial markers in cells cultured in EDM respect to the control (PGM), with VE-Cadherin and CD31 showing an increase of 33 and 22 times, respectively. Immunofluorescence analysis of CD31 revealed that only cells cultured in EDM expressed the protein with the classical membrane localization. CONCLUSIONS: We demonstrated that pAVPCs, grown in specific endothelial medium and stimulated with VEGF, are able to differentiate toward the endothelial cells phenotype. Therefore, according to the recent definition of VSC, the pAVPCs could be considered a population of VSC-like cells.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
