ROBUST ENDOTHELIAL AND HEMATOPOIETIC DIFFERENTIATION OF HUMAN PLURIPOTENT STEM CELLS

In vitro hematoendothelial differentiation of human pluripotent stem cells (hPSCs) may provide unique opportunities for disease-modeling studies and regenerative medicine by generating transplantable endothelial and blood cells. Endothelial cells have received increased attention for their potential to repair ischemic tissues and assembling a vascular network in organ and tissue engineering. Introducing hematopoietic cells such as erythrocytes, lymphocytes, megakaryocytes, and platelets from hPSCs would have enormous benefits for healthcare. iPSC derived lymphoid cells can facilitate the therapies of patients with immunodeficiency and development of immunotherapies. Such systems could provide tolerance in organ transplant and reversal of autoimmunity by delivering regulatory T cells. In the early vertebrate embryo, canonical Wnt signaling induces a strong mesodermal program, which leads to formation of aggregates of mesodermal cells that form the blood islands in the extraembryonic yolk sac from which both hematopoietic and endothelial lineages derive. This observation led to the hypothesis that both lineages originate from a common precursor named the hemangioblast. By utilizing a combination of enhanced monolayer induction method, which generates large quantities of CD34+ hemangioblast by stimulating Wnt signaling pathway, and various mouse stromal co-culture protocols, we have achieved efficient differentiation of hPSCs along the endothelial, myeloid and lymphoid lineages. Endothelial CD31+CD144+ cells were isolated with positive selection of CD144+ marker and their potential was evaluated using tube formation assay, immunostaining and Acetylated-low density lipoprotein uptake. The cells of myeloid lineages were obtained by co culturing CD34+ cells with OP9 mouse stromal cells followed by their cultivation in semisolid media. Finally, lymphoid differentiation was achieved by co culturing of CD34+ cells with mouse OP9-DLL4 stromal cells in various differentiation cocktails. This work demonstrates a potential of hPSC technology for a robust production of endothelial and blood cells that are suited for cell-fate, disease-modeling studies as well as future hematopoietic and vascular repair and regeneration.