Directed Endothelial Progenitor Differentiation from Human Pluripotent Stem Cells Via Wnt Activation Under Defined Conditions

Abstract

Efficient derivation of endothelial cells and their progenitors from human pluripotent stem cells (hPSCs) can facilitate studies of human vascular development, disease modeling, drug discovery, and cell-based therapy. Here we provide a detailed protocol for directing hPSCs to functional endothelial cells and their progenitors in a completely defined, growth factor- and serum-free system by temporal modulation of Wnt/β-catenin signaling via small molecules. We demonstrate a 10-day, two-stage process that recapitulates endothelial cell development, in which hPSCs first differentiate to endothelial progenitors that then generate functional endothelial cells and smooth muscle cells. Methods to characterize endothelial cell identity and function are also described.

Keywords: Chemically defined; Endothelial cells; Endothelial progenitors; Growth factor-free; Human pluripotent stem cells; Serum-free; Small molecules; Wnt signaling.

Figure 1

Schematic of the protocol for the differentiation of endothelial progenitors from hPSCs with small-molecule modulators of canonical Wnt signaling. Bright-field images of the typical morphology of day −2, day 0, day 2 and day 5 cells from 19-9-11 are shown at ×4 magnifications along with flow cytometry analysis of indicated markers. Scale bar, 200 μm.

Figure 2

Schematic of the protocol for the purification of hPSC-derived endothelial progenitors.

Figure 3

Characterization of endothelial cells and their progenitors derived from hPSCs. (AC) 19-9-11 iPSCs were differentiated as illustrated in Fig. 1. At day 5, pre-sort CD34+ cells were immunostained for CD31/CD34 (A) and VE-cadherin/vWF (B) and quantitatively analyzed for CD31/CD34 (C). (D–F) the post-sort CD34+ endothelial progenitors were quantitatively analyzed for CD31 (D) and immunostained for CD31 (E) and VE-cadherin/vWF (F).