Objective: Previous studies have demonstrated development of endothelial cells (ECs) and smooth muscle cells (SMCs) as separate cell lineages derived from human embryonic stem cells (hESCs). We demonstrate CD34+ cells isolated from differentiated hESCs function as vascular progenitor cells capable of producing both ECs and SMCs. These studies better define the developmental origin and reveal the relationship between these two cell types, as well as provide a more complete biological characterization. Materials and Methods: hESCs are cocultured on M2-10B4 stromal cells or Wnt1-expressing M2-10B4 for 13 to 15 days to generate a CD34+ cell population. These cells are isolated using a magnetic antibody separation kit and cultured on fibronectin-coated dishes in EC medium. To induce SMC differentiation, culture medium is changed and a morphological and phenotypic change occurs within 24 to 48 hours. Results: CD34+ vascular progenitor cells give rise to ECs and SMCs. The two populations express respective cell-specific transcripts and proteins, exhibit intracellular calcium in response to various agonists, and form robust tube-like structures when cocultured in Matrigel. Human umbilical vein endothelial cells cultured under SMC conditions do not exhibit a change in phenotype or genotype. Wnt1-overexpressing stromal cells produced an increased number of progenitor cells. Conclusions: The ability to generate large numbers of ECs and SMCs from a single vascular progenitor cell population is promising for therapeutic use to treat a variety of diseased and ischemic conditions. The stepwise differentiation outlined here is an efficient, reproducible method with potential for large-scale cultures suitable for clinical applications.
Bibliographical noteFunding Information:
We appreciate additional assistance from Melinda Hexum and Julie Morris, and helpful discussions with Dr. Xinghui Tian. We thank Drs. Randall Moon and Ken Dorshkind for supplying cells used in these studies. The authors would also like to express gratitude to Dr. Wei Shen and Andrew Lewis for advice and assistance while collecting tube-formation images. These studies were supported by an National Institutes of Health (Bethesda, MD, USA) R01 (HL077923) (D.S.K.), a scholarship from the Fulbright Foundation (P.O.), MSM 0021620808 (Prague, Czech Republic) (P.O.), and funding from the Engdahl Foundation (Minneapolis, MN, USA).