Abstract
BackgroundTransplantation of endothelial progenitor cells (EPCs)/endothelial cells (ECs) has been used for the treatment of ischemic diseases and hemophilia A, due to their great capacity for producing factor VIII and for repairing vascular damage. We established an effective approach to stimulate the expansion and differentiation of EPCs for potential therapeutic applications.MethodsCD34+ cells isolated from human cord blood were cultured in a two-step system for 21 days. The generated adherent cells were characterized via flow cytometry and immunofluorescent staining. Moreover, single-cell clonogenic and tube-forming assays were carried out to evaluate their potential to proliferate and form vessel networks. Furthermore, these cells were transplanted into a mouse model of hepatic sinusoidal endothelium injury by hepatic portal vein injection to investigate their in-vivo behavior.ResultsThe two-step culture protocol promoted the expansion and differentiation of human cord blood CD34+ cells efficiently, resulting in a large number of adherent cells within 3 weeks. The generated adherent cells were identified as EPCs/ECs based on the expression of CD31, CD144, vWF, and FVIII, and cell numbers showed a 1400-fold increase compared with the initial number. Moreover, these EPCs/ECs were capable of proliferating and establishing colonies as individual cells, and forming tube-like structures. More significantly, tissue examination of mice after transplantation revealed that the injected EPCs/ECs migrated and integrated into the liver, reconstituting the sinusoidal endothelial compartment.ConclusionsWe developed an approach for the generation of cord blood-derived EPCs/ECs on a large scale, characterized them phenotypically, and demonstrated their in-vivo functional capacity. Our approach provides an excellent source of healthy EPCs/ECs for use in cell therapy in a clinical setting.
Highlights
Transplantation of endothelial progenitor cells (EPCs)/endothelial cells (ECs) has been used for the treatment of ischemic diseases and hemophilia A, due to their great capacity for producing factor VIII and for repairing vascular damage
On day 6, CD34+ cells were maintained at the relatively high level of 58.6 ± 2.7%, and CD133+ and VEGFR2+ cells decreased to 27.9 ± 1.8% and 0.14 ± 0.02%, respectively
These results indicated that CD34+ cells and early EPCs both undergo extensive proliferation in step I of the culture protocol
Summary
Transplantation of endothelial progenitor cells (EPCs)/endothelial cells (ECs) has been used for the treatment of ischemic diseases and hemophilia A, due to their great capacity for producing factor VIII and for repairing vascular damage. The generated adherent cells were identified as EPCs/ECs based on the expression of CD31, CD144, vWF, and FVIII, and cell numbers showed a 1400-fold increase compared with the initial number. These EPCs/ECs were capable of proliferating and establishing colonies as individual cells, and forming tube-like structures. EPCs represent a very small proportion of the cells in the circulation, ranging from 0.002 to 0.01% in peripheral blood and from 0.2 to 1% in umbilical cord blood They can expand exponentially, migrate efficiently to sites of neovascularization, and differentiate into ECs in ex-vivo studies [16, 17], the number of cells is limited in the systemic infusion of allogenic EPCs in patients. Difficulties in obtaining adequate numbers of cells for transplantation currently restrict their use in clinical treatments [18]
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