Efficient Differentiation of Human Pluripotent Stem Cells to Endothelial Progenitors via Small-Molecule Activation of WNT Signaling

Abstract

SummaryHuman pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors may provide the means for vascularization of tissue-engineered constructs and can serve as models to study vascular development and disease. Here, we report a method to efficiently produce endothelial cells from hPSCs via GSK3 inhibition and culture in defined media to direct hPSC differentiation to CD34+CD31+ endothelial progenitors. Exogenous vascular endothelial growth factor (VEGF) treatment was dispensable, and endothelial progenitor differentiation was β-catenin dependent. Furthermore, by clonal analysis, we showed that CD34+CD31+CD117+TIE-2+ endothelial progenitors were multipotent, capable of differentiating into calponin-expressing smooth muscle cells and CD31+CD144+vWF+I-CAM1+ endothelial cells. These endothelial cells were capable of 20 population doublings, formed tube-like structures, imported acetylated low-density lipoprotein, and maintained a dynamic barrier function. This study provides a rapid and efficient method for production of hPSC-derived endothelial progenitors and endothelial cells and identifies WNT/β-catenin signaling as a primary regulator for generating vascular cells from hPSCs.

Chemically-defined albumin-free differentiation of human pluripotent stem cells to endothelial progenitor cells

“…We previously demonstrated that activation of canonical Wnt signaling in hPSCs in LaSR basal medium generates functional CD34+/CD31+ endothelial progenitors in numerous hPSC lines (Lian et al, 2014). Figures 1A and S1 show schematics of the endothelial differentiation and purification protocols.…”

“…Recently, we reported a rapid and robust endothelial progenitor differentiation protocol under serum-free conditions, which only employs a Gsk-3β inhibitor in LaSR basal medium (Advanced DMEM/F12, 2.5 mM GlutaMAX and 60 μg/mL ascorbic acid) (Lian et al, 2014). The presence of bovine serum albumin (BSA) in this medium increases the cost, adds xenogenic components, and heightens lot-to-lot variability.…”

Generation, Characterization, and Multilineage Potency of Mesenchymal-Like Progenitors Derived from Equine Induced Pluripotent Stem Cells

“…This is because of the nature of the serial plating method, where some iPSCs were lost during media replacement due to lack of adherent properties. Efficiency of MSC generation is difficult to establish and reports on iPSC-MSC or embryonic cell conversion often do not include efficiency data [52][53][54][55][56]. However, the flow cytometry results show that the cells that remained attached throughout iPSC-MSC conversion are homogenous and phenotypically similar to CB-MSCs.…”

The microRNA-dependent cell fate of multipotent stromal cells differentiating to endothelial cells

“…To search for specific miRNAs that affect EC differentiation via glycogen synthase kinase (GSK)-3β inhibition [15], we used a systematic bioinformatics database, TargetScan 6.2 (www.targets can.org). Based on the total context score (4 À 0.1) and the aggregated P CT score (4 0.5), we selected miR-26a, thus conferring stronger regulation than miRNAs with only one binding site ( Fig.…”

“…The inhibition of GSK-3β in ECFCs leads to increase in ECFC adhesion, survival, proliferation, differentiation, and angiogenic function, which results in accelerating re-endothelialization after vascular injury [12,13]. In addition, GSK-3β inhibition and VEGF exposure in human pluripotent cells induces differentiation of EC [14,15]. However, little is known about the mechanism through which these cells commit to becoming ECs.…”