Objective: Endothelial progenitor cells (EPCs) are a promising tool in regenerative medicine. We designed a comparative study of peripheral (adult, PB) versus umbilical cord blood (CB) derived EPCs in order to investigate the angiogenic properties of these two sources. Methods: Mononuclear cells (MNCs) were isolated from PB (n=4) and CB (<24 hours, n=4) and cultured on fibronectin-coated cell culture plates in endothelial cell culture medium until the formation of cobblestone-shaped colonies. Colony-derived cells were expanded for further investigation and their endothelial phenotype was confi rmed evaluating the uptake of Di-Iacetylated-low-density lipoprotein (Ac-LDL) and expression of endothelial cell-surface antigens (CD34, CD31, CD146, vWF, KDR) and CD45. Proliferation rates of CB and PB derived EPCs were determined using the MTT assay. The angiogenic properties of colony-derived EPCs were assessed by in vitro capillary-like network formation assay: early-passage cells were seeded (20,000 cells per well) onto 96-wells plates coated with Matrigel. Capillary-like network formation and maintenance were evaluated with an inverted microscope after 4-18-24-48-72 hours of incubation; images were taken from 3 random fields of Matrigel wells to assess the number of branch points per field to quantify the degree of tubulogenesis. Each experiment was performed in triplicate. Results: The median frequency of colonies obtained was higher in CB than PB (0.49/107MNCs vs 0.05/107MNCs) and CB-derived EPCs had higher proliferative potential than PB-derived EPCs. Both CB and PB colony-derived cells incorporated Ac-LDL, expressed the endothelial cell-surface antigens and were CD45-. Both CB and PB derived EPCs formed capillary-like structures in Matrigel, but CB-derived tubes formed earlier and more complex networks than PB-derived EPCs. Moreover, the number of branch points in CB-derived capillary-like networks was higher than PB-derived networks (16.3±4.3 vs 7.1±-2.6). Finally, CB-derived capillary-like networks were maintained for at least 72 hours, while PB-derived networks started to disassembly in 48 hours. Conclusion: Our study confirms the different clonogenic and proliferative potential of EPCs derived from PB and CB and demonstrates a superior angiogenic potential of CB-derived cells. Our preliminary data indicate that CB-derived EPCs have a better angiogenic potential than PB-derived EPCs and CB is a promising source of EPCs for regenerative medicine purpose.
Cord blood derived endothelial progenitor cells: a superior buiding company in angiogenesis market.
GNECCHI, MASSIMILIANO;
2012-01-01
Abstract
Objective: Endothelial progenitor cells (EPCs) are a promising tool in regenerative medicine. We designed a comparative study of peripheral (adult, PB) versus umbilical cord blood (CB) derived EPCs in order to investigate the angiogenic properties of these two sources. Methods: Mononuclear cells (MNCs) were isolated from PB (n=4) and CB (<24 hours, n=4) and cultured on fibronectin-coated cell culture plates in endothelial cell culture medium until the formation of cobblestone-shaped colonies. Colony-derived cells were expanded for further investigation and their endothelial phenotype was confi rmed evaluating the uptake of Di-Iacetylated-low-density lipoprotein (Ac-LDL) and expression of endothelial cell-surface antigens (CD34, CD31, CD146, vWF, KDR) and CD45. Proliferation rates of CB and PB derived EPCs were determined using the MTT assay. The angiogenic properties of colony-derived EPCs were assessed by in vitro capillary-like network formation assay: early-passage cells were seeded (20,000 cells per well) onto 96-wells plates coated with Matrigel. Capillary-like network formation and maintenance were evaluated with an inverted microscope after 4-18-24-48-72 hours of incubation; images were taken from 3 random fields of Matrigel wells to assess the number of branch points per field to quantify the degree of tubulogenesis. Each experiment was performed in triplicate. Results: The median frequency of colonies obtained was higher in CB than PB (0.49/107MNCs vs 0.05/107MNCs) and CB-derived EPCs had higher proliferative potential than PB-derived EPCs. Both CB and PB colony-derived cells incorporated Ac-LDL, expressed the endothelial cell-surface antigens and were CD45-. Both CB and PB derived EPCs formed capillary-like structures in Matrigel, but CB-derived tubes formed earlier and more complex networks than PB-derived EPCs. Moreover, the number of branch points in CB-derived capillary-like networks was higher than PB-derived networks (16.3±4.3 vs 7.1±-2.6). Finally, CB-derived capillary-like networks were maintained for at least 72 hours, while PB-derived networks started to disassembly in 48 hours. Conclusion: Our study confirms the different clonogenic and proliferative potential of EPCs derived from PB and CB and demonstrates a superior angiogenic potential of CB-derived cells. Our preliminary data indicate that CB-derived EPCs have a better angiogenic potential than PB-derived EPCs and CB is a promising source of EPCs for regenerative medicine purpose.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
