Isolation, differentiation and characterization of vascular cells derived from human embryonic stem cells

Abstract

Herein, we describe a protocol for the isolation of human embryonic stem cells (hESCs)-derived vascular cells at various stages of development. The cells are isolated from 10 to 15-d-old human embryoid bodies (EBs) cultured in suspension. After dissociation, cells are labeled with anti-CD34 or anti-CD31 (PECAM1) antibody and separated from the cell mixture by magnetic-activated cell separation (MACS) or fluorescent-activated cell sorting (FACS). Isolated vascular cells are then cultured in media conditions that support specific differentiation and expansion pathways. The resulting vascular cell populations contain > 80% endothelial-like or smooth muscle-like cells. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 1.5 months. Vascular cells isolated and differentiated under the described conditions may constitute a potential cell source for therapeutic application toward repair of ischemic tissues, preparation of tissue-engineered vascular grafts and design of cellular kits for drug screening applications.

Generation of pure lymphatic endothelial cells from human pluripotent stem cells and their therapeutic effects on wound repair

“…As there are no statistical data regarding the efficiency of lymphatic differentiation of hPSCs, we are unable to compare our system to other lymphatic endothelial differentiation systems. However, compared to the current published data regarding blood vascular endothelial lineage differentiation from human or mouse PSCs, which shows at most 10% of a single marker expression before cell sorting3536373839404142, the efficiency of our system for lymphatic endothelial cell lineage differentiation was much higher, demonstrated by a single marker expression at more than 40% except for VEGFR3 (10–15%) and expression of the four LEC markers at ~10%. This high yield of differentiation makes this system appealing for testing in vivo behavior and therapeutic potential of hPSC-derived LECs.…”

Multifactorial Optimizations for Directing Endothelial Fate from Stem Cells

“…Endothelial cells (EC) were first successfully derived from both mouse [6–8] and human [9–14] ESC using first three-dimensional (3D) embryoid body (EB) cultures [10, 11, 15] and then 2D cultures with the aid of OP9 cells [12, 13] or mouse embryonic fibroblasts feeder cells [14]. Vascular induction by EB yields very low percentages of EC (1–3%) [10, 11], but EB-monolayer combination inductions [16] and pure monolayer inductions [6, 17–20] lead to greater efficiencies compared with 3D EB differentiation methods.…”

“…Vascular induction by EB yields very low percentages of EC (1–3%) [10, 11], but EB-monolayer combination inductions [16] and pure monolayer inductions [6, 17–20] lead to greater efficiencies compared with 3D EB differentiation methods. Recently, chemically-defined mediums have been used in feeder-free monolayer cultures for the induction of larger numbers of EC from both mouse [21] and human ESC [9], and allow the development of improved approaches for directed differentiation including a labor intensive method sprouting endothelial progenitor cells (EPC) into 3D fibrin scaffolds [22].…”

Induced pluripotent stem cells: at the heart of cardiovascular precision medicine

“…Platelet endothelial cell adhesion molecule 1-positive (PECAM1+) cells isolated from days 13-15 EBs expressed endothelial markers (e.g., CD34, Flk-1) that were comparable to those of human umbilical vein endothelial cells. 53 Orlova et al, later established a protocol for simultaneously deriving ECs and pericytes from monolayers of hESCs and hiPSCs grown on Matrigel and exposed to timely introduction of differentiation growth factors in B(P)EL media. 54 The differentiation efficiency for either cell type was as high as 30% by day 10 of differentiation.…”