Transduction of ECFCs with rhEPO. Human CB-derived ECFCs were transfected using a lentivirus vector encoding for hEPO (epoECFCs) under control of a CMV promoter. laczECFCs, nontransfected ECFCs, and MSCs served as negative controls for EPO expression. (A) Expression of EPO at the mRNA level was only observed in epoECFCs by RT-PCR. (B) At the protein level, expression of EPO was confirmed in cell lysates by immunoblotting analysis. (C) EPO was also detected by immunoblotting analysis in epoECFC-CM, but not in CM from laczECFCs, ECFCs, or MSCs. (D) Immunofluorescent staining of cells in monolayer cultures using Abs against human CD31 (green) and hEPO (red) showed EPO expression only present in epoECFCs, not in nontransfected ECFCs. Cell nuclei were stained with DAPI. Scale bars indicate 50 μm. (E) Secreted EPO was confirmed to be functional in hematopoietic colony-forming assays. CB-derived mononuclear cells cultured in EPO-free methylcellulose medium containing epoECFC-CM developed EPO-dependent erythrocyte-containing colonies such as BFU-E and CFU-GEMM. Cultures containing control ECFC-CM did not generate BFU-E or CFU-GEMM colonies. Scale bars indicate 200 μm.