In the last 10 years, several populations of vascular progenitor cells (VPCs) were isolated from vessel wall. In particular, it seems that some of these cells could be considered the in vivo counterpart of mesenchymal stromal cells (MSCs). In this work we describe a new method to isolate VPCs from porcine aorta tunica media. The internal lumen of arteries (n=3) was digested with Collagenase I solution, firstly for 40 minutes, to eliminate endothelial cells, and then for further 4 hours. The cell suspension obtained after 4 hours was cultured overnight in high concentration (10%) antibiotic/antimycotic culture medium (DMEM + 10% FBS). After 3 days cells were overnight serum starved and then recovered with DM medium (10% FBS + 1% antibiotic/antimycotic in DMEM:M199-1:1). Histological analysis revealed that after 4 hours of Collagenase I treatment 1/3 of the tunica media was digested. Progenitor cells derived from media layer, cultured in DM medium, display, when sub-confluent, a polygonal morphology and elongated thin arms at their ends. When confluent, cells showed a spindle shape. Moreover, cells display, an increasing doubling time from P1 to P6 (from 36.5±8.0 to 60.8±2.1 hours). At P3, cells were characterized for immunophenotype, antigen expression and differentiation potential. Immunophenotype analysis showed that VPCs derived from porcine aorta media layer grown in DM were positive for CD105 and CD90 and negative (<2%) for CD34 and CD45. Immunocytochemistry revealed that cells express NG2-, PDGFRα and β-, Nestin-, Vimentin-, Laminin-, Oct4-immunoreactivity (IR), whilst don’t express c-kit-, CD31- and Smooth Muscle Myosin-IR. Cells were also able to differentiate toward adipo-, osteo- and chondrocyte phenotype. In conclusion, we established a new method to isolate a prevalent MSCs phenotype with defined features in terms of differentiation potential and antigen expression.
Vascular progenitor cells derived from porcine aorta tunica media: isolation, characterization and differentiation potential
ZANIBONI, ANDREA;BERNARDINI, CHIARA;ZANNONI, AUGUSTA;SARLI, GIUSEPPE;CALZA', LAURA;BACCI, MARIA LAURA;FORNI, MONICA
2013
Abstract
In the last 10 years, several populations of vascular progenitor cells (VPCs) were isolated from vessel wall. In particular, it seems that some of these cells could be considered the in vivo counterpart of mesenchymal stromal cells (MSCs). In this work we describe a new method to isolate VPCs from porcine aorta tunica media. The internal lumen of arteries (n=3) was digested with Collagenase I solution, firstly for 40 minutes, to eliminate endothelial cells, and then for further 4 hours. The cell suspension obtained after 4 hours was cultured overnight in high concentration (10%) antibiotic/antimycotic culture medium (DMEM + 10% FBS). After 3 days cells were overnight serum starved and then recovered with DM medium (10% FBS + 1% antibiotic/antimycotic in DMEM:M199-1:1). Histological analysis revealed that after 4 hours of Collagenase I treatment 1/3 of the tunica media was digested. Progenitor cells derived from media layer, cultured in DM medium, display, when sub-confluent, a polygonal morphology and elongated thin arms at their ends. When confluent, cells showed a spindle shape. Moreover, cells display, an increasing doubling time from P1 to P6 (from 36.5±8.0 to 60.8±2.1 hours). At P3, cells were characterized for immunophenotype, antigen expression and differentiation potential. Immunophenotype analysis showed that VPCs derived from porcine aorta media layer grown in DM were positive for CD105 and CD90 and negative (<2%) for CD34 and CD45. Immunocytochemistry revealed that cells express NG2-, PDGFRα and β-, Nestin-, Vimentin-, Laminin-, Oct4-immunoreactivity (IR), whilst don’t express c-kit-, CD31- and Smooth Muscle Myosin-IR. Cells were also able to differentiate toward adipo-, osteo- and chondrocyte phenotype. In conclusion, we established a new method to isolate a prevalent MSCs phenotype with defined features in terms of differentiation potential and antigen expression.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
