Abstract

Differentiation of human embryonic stem cells to endothelial progenitor cells on laminins in defined and xeno-free systems

Mien Nguyen, Research Fellow, Duke-NUS Medical School

A major hurdle for in vitro culturing of primary endothelial cells (ECs) is that they readily de-differentiate and exhibit variability among cell lines. This has hampered the use of primary ECs, e.g. human umbilical vein endothelial cells (HUVECs), for cell therapies and in vitro models to study vascular diseases and drug screening. While human embryonic stem cells (hESCs) may provide an unlimited cell source, most current protocols to derive hESCs to endothelial progenitor cells (EPCs) or mature ECs use embryoid bodies on undefined matrices or involve xeno products, yet the final yields are not sufficient for therapeutic purposes. Our laboratory has successfully developed a method to culture monolayer hESCs on a stem cell niche laminin (LN) LN511 or LN521 matrix. Here, we report a novel chemically defined and xeno-free protocol for differentiation of hESCs to EPCs using biologically relevant LN521 as the main culture substrate. Through a three-phase differentiation protocol, we were able to reproducibly generate ~95% cell population expressing EPC markers defined as VEGFR2+CD34+CD31+VE-Cadherin+ after 15 days. These hESC-derived cells were able to form tube-like structures in vitro and take up acetylated low-density lipoprotein. RNA sequencing analyses of hESCs, hESC-derived EPCs and primary HUVECs provided a new insight into differentiation-related expression signatures of ECs regarding basement membrane composition, specific cell-matrix interactions, and changes in endothelial lineage markers. The results may facilitate production of stable ECs in defined and xeno-free systems for treatment of vascular diseases and for in vitro cell modeling such as the blood-brain and renal filtration barriers.