Efficient Differentiation of Human Pluripotent Stem Cells to Endothelial Cells

Abstract

Endothelial cells (ECs) line the interior surface of blood and lymphatic vessels, and play a key role in a variety of physiological or pathological processes such as thrombosis, inflammation, or vascular wall remodeling. Human-induced pluripotent stem cell (iPSCs)-derived ECs provide a new opportunity for vascular regeneration and serve as a model to study the mechanism and to screen for novel therapies. We use developmental cues in a monolayer differentiation approach to efficiently generate mesoderm cells from iPSCs via small-molecule activation of WNT signaling in chemically defined medium for 4 days, and subsequent EC specification using vascular endothelial growth factor and fibroblast growth factor for another 4 days. After 8 days of differentiation, mature ECs are further purified using magnetic-activated cell sorting for the EC surface marker CD144. These ECs exhibit molecular and cellular characteristics consistent with native ECs, such as expression of specific surface markers, formation of tube-like structures and acetylated low-density lipoprotein uptake. © 2018 by John Wiley & Sons, Inc.

Keywords: disease modeling; endothelial cell; induced pluripotent stem cell; monolayer.

Figure 1

Generation of endothelial cells from iPSCs. (A) Schematic of the differentiation procedure with the medium, extracellular matrices, and small molecules used in each step. (B) Representative images of hiPSC cells at 80% confluency on day 0, followed by mesoderm induction and endothelial cell specification. After MACS sorting against CD144, the iPSC-ECs show homogeneous cobble-stone morphology characteristic of ECs.

Figure 2

Characterization of iPSC-ECs. (A) Flow cytometry of iPSC-ECs before and after purification. The population of mature iPSC-ECs (CD144⁺) is significantly enriched after MACS sorting. (B) iPSC-EC differentiated from skin fibroblast-derived iPSC express the endothelial surface marker CD144 (green) and CD31 (red). Nuclear staining using DAPI is in blue. (C) iPSC-ECs incorporate acetylated low-density lipoprotein (Ac-LDL, red; DAPI, Blue). (D) Angiogenesis assays: representative images of tube formation 6 hours, 12 hours, and 24 hours after seeding cells in Matrigel. iPSC-ECs start forming tubes at 6 hour time point, and will regress after 24 hours.