Efficient differentiation of XXXX embryonic stem cells to arterial and venous endothelial cells under feeder- and serum-free conditions.

@entity1 @entity455 @entity13062 @entity7 @entity19 @entity2279 @entity540 @entity2280
BACKGROUND: Heterogeneity of endothelial cells (ECs) is a hallmark of the vascular system which may impact the development and management of @entity455 . Despite the tremendous progress in differentiation of @entity1 embryonic stem cells (hESCs) towards endothelial lineage, differentiation into arterial and venous endothelial phenotypes remains elusive. Additionally, current differentiation strategies are hampered by inefficiency, lack of reproducibility, and use of animal-derived products. METHODS: To direct the differentiation of hESCs to endothelial subtypes, H1- and H9-hESCs were seeded on @entity1 plasma fibronectin and differentiated under chemically defined conditions by sequential modulation of glycogen synthase kinase-3 (GSK-3), @entity540 ( @entity540 ), @entity13062 ( @entity13062 ) and @entity7 ( @entity7 ) signaling pathways for 5 days. Following the initial differentiation, the endothelial progenitor cells ( @entity2280 (+) @entity2279 (+) cells) were sorted and terminally differentiated under serum-free conditions to arterial and venous ECs. The transcriptome and secretome profiles of the two distinct populations of hESC-derived arterial and venous ECs were characterized. Furthermore, the safety and functionality of these cells upon in vivo transplantation were characterized. RESULTS: Sequential modulation of hESCs with GSK-3 inhibitor, @entity540 , @entity13062 and @entity7 resulted in stages reminiscent of primitive streak, early mesoderm/lateral plate mesoderm, and endothelial progenitors under feeder- and serum-free conditions. Furthermore, these endothelial progenitors demonstrated differentiation potential to almost pure populations of arterial and venous endothelial phenotypes under serum-free conditions. Specifically, the endothelial progenitors differentiated to venous ECs in the absence of @entity7 , and to arterial phenotype under low concentrations of @entity7 . Additionally, these hESC-derived arterial and venous ECs showed distinct molecular and functional profiles in vitro. Furthermore, these hESC-derived arterial and venous ECs were nontumorigenic and were functional in terms of forming perfused microvascular channels upon subcutaneous implantation in the @entity19 . CONCLUSIONS: We report a simple, rapid, and efficient protocol for directed differentiation of hESCs into endothelial progenitor cells capable of differentiation to arterial and venous ECs under feeder-free and serum-free conditions. This could offer a @entity1 platform to study arterial-venous specification for various applications related to drug discovery, disease modeling and regenerative medicine in the future.
