Directed differentiation of human induced pluripotent stem cells into endothelial cells
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Endothelial cells (ECs) are part of almost every human organ, and disturbance in their function can cause several human diseases such as pulmonary arterial hypertension. We sought to develop an in vitro model to derive ECs from human induced pluripotent stem cells (hiPSCs) in an effort to develop future regenerative therapies or disease models. By stimulating signaling pathways gleaned from prior studies of the early embryonic development of endothelial cells and their precursor germlayer, mesoderm, hiPSCs were differentiated in vitro using a media containing 10ng/ml of Activin, FGF2, VEGF and BMP4 to induce mesoderm over 4 days, followed by VEGF and FGF2 to subsequently specify ECs. By day 12, putative endothelial cells identified by the co-expression of CD31, CD144 and KDR, emerged and could be sorted and re-plated in endothelial maintenance media for expansion over several passages. Time series characterization of mRNA and protein gene expression indicates that to form endothelial cells, hiPSCs developmentally differentiate first into a posterior primitive streak-like stage (indicated by high T and low FOXA2 expression), then into a heterogeneous population of mesodermal subsets (day 4). Subsequently, lateral plate mesoderm cells predominate over intermediate or paraxial mesoderm. To understand the role of BMP4 in this process, each factor was withheld from various stages of the protocol. Results indicate that before day 4, BMP4 alone is necessary and sufficient to direct cells through posterior primitive streak into mesodermal progenitors that upon subsequent VEGF/FGF2 exposure can give rise to CD31+CD144+KDR+ endothelial-like cells. In contrast, BMP4 is dispensable after day4, whereas VEGF and FGF2 are dispensable in the first 4 days of the protocol. To develop a disease model hiPSCs from patients with BMPR2 mutations associated with pulmonary hypertension were generated by reprogramming their archived fibroblasts. By differentiating these patient-specific iPSCs into mesodermal progenitors and ECs using our protocol, preliminary results indicate that the induction of mesodermal cells by day 4 was diminished in cells carrying BMPR2 mutations,, but whether their efficiency of differentiation into ECs is affected by these mutations remains in question. It can be concluded from this project, that we develop an in vitro differentiation model that replicates the developmental pathways of mesodermal and EC derivation in embryos, and suggests that BMP4 is necessary and sufficient to derive mesodermal subsets with EC competence but is dispensable for subsequent endothelial lineage specification.