Generation of definitive hematopoietic progenitors from human pluripotent stem cells

Abstract

The hematopoietic system in the early embryo consists of two distinct programs, primitive hematopoiesis and definitive hematopoiesis that can be distinguished by site and timing of development and the spectrum of lineages generated. Primitive hematopoiesis, the first program to develop, is restricted to the yolk sac and produces primitive erythrocytes, macrophages and megakaryocytes, but does not give rise to lymphoid cells or hematopoietic stem cells. Lymphoid lineage cells and HSCs are generated during the definitive stage of hematopoiesis that emerges following the onset of primitive hematopoiesis. Definitive program develops from a progenitor population known as hemogenic endothelium that is specified at different sites within the vasculature of the embryo. To derive HSCs from human pluripotent stem cells, it is essential to be able to accurately recapitulate these development stages including the formation of HE and its specification to a hematopoietic fate in the differentiation cultures. Using T lymphoid potential and primitive erythropoiesis to monitor the emergence of the two hematopoietic programs from hPSCs, we have recently found that both primitive and definitive hematopoiesis develop from KDR progenitors that are specified within the first three days of differentiation. The primitive KDR progenitors can be distinguished from those of the definitive lineage by the expression of CD235a and by the requirement for Wnt/b-catenin signaling. Development of the primitive KDR+CD235a+ progenitor is Wnt independent, whereas specification of the KDR+CD235a- definitive progenitor is dependent on signaling through this pathway, specifically between days 2 and 3 of differentiation. Following an additional five days of differentiation, the Wnt-induced definitive progenitors generate a CD34+ population that expresses cell surface markers indicative of HE and displays the capacity to give rise to lymphoid, myeloid and erythroid progeny. Based on expression of the vascular markers, CD73 and CD184, this day 8 CD34+ population can be separated into three distinct fractions: CD73+CD184+, CD73+CD184- and CD73-CD184-. Functional analyses revealed that hematopoietic potential reflective of HE segregated to the CD73-CD184- fraction, whereas the CD73+CD184+ and CD73+CD184- fractions were found to contain progenitors of arterial and venous vascular endothelium, respectively. Taken together, these findings demonstrate that it is possible to model definitive hematopoietic development from hPSCs and to identify and isolate a population enriched in HE, the putative progenitor of the human HSC.