Phenotypic characterization of outgrowth endothelial cells from cultures of peripheral blood and feasibility studies of using these cells in biomedical applications.

摘要

Normal adults have a small number of circulating endothelial cells (CEC) in peripheral blood. We established a protocol for expanding endothelial cells (EC) from cultures of peripheral blood. Blood outgrowth endothelial cells (BOEC) cultured using this method also survived cryopreservation and continued to expand thereafter. BOEC were quiescent and microvascular in phenotype by both immunofluorescent and FACS studies. Examination of the genotype of BOEC and CEC from peripheral blood of patients who received gender-mismatched bone-marrow transplants revealed that majority of the outgrowth BOEC are from a transplantable bone marrow derived cells while the majority of the CEC are from vessel wall.; A protocol was developed for stable transfection of normal human BOEC with human factor VIII (hFVIII). Stably transfected cells expressed hFVIII in vitro at both RNA and protein levels as detected by RT-PCR and ELISA respectively. These cells were subsequently injected into NOD/SCID mice for detection of hFVIII expression in vivo. ELISA analysis of mouse plasma indicated that stably transfected BOEC expressed hFVIII in mouse for at least 156 days, and that the level of hFVIII in mouse plasma increased with both cell dosage and time after administration. All animals acquired hFVIII levels that would be therapeutic for human hemophilia A.; The adhesion behavior of outgrowth endothelial cells was studied and compared to that of dermal microvascular endothelial cells (MVEC) and human umbilical vein endothelial cells (HUVEC). Static adhesion assay revealed that optimal substrate condition for adhesion is different for the three cell types. All three types of EC maintained coverage of plastic coverslips under the highest physiologic shear stress. BOEC attached and spread on expanded polytetrafluoroethylene (ePTFE) graft 3 hours after seeding. Complete coverage of ePTFE graft by BOEC was observed after 5 days.; In conclusion, peripheral blood can be a useful source for primary autologous endothelial cell culture. These proof-of-principle studies indicate that the outgrowth BOEC from blood can be used for biomedical applications such as gene therapy and biomaterials.