Project description:Human induced pluripotent stem (hiPS) cells and human embryonic stem (hES) cells differentiate into cells of the endothelial lineage, but derivation of cells with human umbilical cord blood endothelial colony forming cell (ECFC)-like properties has not been reported. Here we describe a novel serum- and stromal cell-free ECFC differentiation protocol for the derivation of clinically relevant numbers of ECFCs (> 108) from hiPS and hES cells. We identified NRP-1+CD31+ selected cells that displayed a stable endothelial phenotype exhibiting high clonal proliferative potential, extensive replicative capacity, formation of human vessels that inosculated with host vasculature upon transplantation, but lacking in teratoma formation in vivo. We also identified NRP-1-VEGF165-KDR-mediated activation of KDR as a critical mechanism for the emergence and derivation of ECFCs from hiPS and hES cells. This protocol advances the field by generating highly replicative but stable endothelial cells for use as a potential cell therapy for human clinical disorders. Transcriptome sequencing of undifferentiated day 0 hiPS cells, Day 3 differentiated hiPS-derived mesoderm proginator cells, Day 12 hiPS-derived NRP-1+CD31+ cells, Day 12 H9-hES-derived NRP-1+CD31+ cells and cord blood-derived Endothelial colony forming cells.
Project description:Human induced pluripotent stem (hiPS) cells and human embryonic stem (hES) cells differentiate into cells of the endothelial lineage, but derivation of cells with human umbilical cord blood endothelial colony forming cell (ECFC)-like properties has not been reported. Here we describe a novel serum- and stromal cell-free ECFC differentiation protocol for the derivation of clinically relevant numbers of ECFCs (> 108) from hiPS and hES cells. We identified NRP-1+CD31+ selected cells that displayed a stable endothelial phenotype exhibiting high clonal proliferative potential, extensive replicative capacity, formation of human vessels that inosculated with host vasculature upon transplantation, but lacking in teratoma formation in vivo. We also identified NRP-1-VEGF165-KDR-mediated activation of KDR as a critical mechanism for the emergence and derivation of ECFCs from hiPS and hES cells. This protocol advances the field by generating highly replicative but stable endothelial cells for use as a potential cell therapy for human clinical disorders.
Project description:Derivation and expansion of human umbilical cord blood-derived endothelial colony forming cells under serum-free conditions - a transcriptome analysis. Endothelial colony forming cells (ECFCs) were isolated from term umbilical cord blood units. ECFCs were expanded under standard, fetal bovine serum (FBS) containing endothelial medium, or transferred to chemically defined endothelial media without FBS. Microarray expression profiling was applied to compare the transcriptome profiles in FBS-containing versus FBS-free culture.
Project description:Derivation and expansion of human umbilical cord blood-derived endothelial colony forming cells under serum-free conditions - a transcriptome analysis. Endothelial colony forming cells (ECFCs) were isolated from term umbilical cord blood units. ECFCs were expanded under standard, fetal bovine serum (FBS) containing endothelial medium, or transferred to chemically defined endothelial media without FBS. Microarray expression profiling was applied to compare the transcriptome profiles in FBS-containing versus FBS-free culture. Comparison of the expression patterns of ECFCs that were either cultured in FBS-containing medium or in serum-free medium (five replicates each).