A model of hematopoietic development from hPSCs. (A) The most critical factors involved in specification of hematovascular precursors from PSCs and regulation of blood formation from HE. (B) Stages of hematopoietic development from hPSCs. Mesodermal stage of development is defined as expression of the mesodermal markers, APLNR and KDR.^62,77,80  The lack of expression of typical endothelial (CD31, VE-cadherin), endothelial/mesenchymal (CD73, CD105), and hematopoietic (CD43, CD45) markers, ie, ^EMHlin⁻ phenotype, separates mesoderm from lineage-committed cells.^86,90  The most primitive mesodermal precursors with hematopoietic potential arise in coculture with OP9 or an embryoid body system on day 3 of differentiation. These cells have features of a posterior PS, coexpress KDR, APLNR, and PDGFRα and capable of forming BL (hemangioblast) colonies in the presence of FGF2 and VEGF.^62,77,80,86  The formation of BL colonies in clonogenic medium proceeds through VE-cadherin⁺ endothelial intermediates, which generate primitive hematopoietic cells with erythroid, megakaryocytic, and macrophage potentials.^77,86  Progressive mesodermal commitment to endothelial and hematopoietic cells is associated with downregulation of PDGFRα^71,86  and PS genes, and upregulation of KDR, TAL1, and GATA2 genes associated with angiohematopoietic development leading to formation of ^EMHlin⁻KDR^brightAPLNR⁺PDGFRα^low/− hematovascular mesodermal precursors (HVMPs).⁸⁶  HVMPs lack BL-CFC potential, but are highly enriched in cells that form hematoendothelial clusters on OP9.⁸⁶  The endothelial stage of development was defined as expression of the typical endothelial markers VE-cadherin, CD31, and CD34 and the absence of the panhematopoietic marker CD43 (supplemental Table 1; see the Blood Web site).^{66,86,90,101,102}  Within the VE-cadherin⁺CD43⁻ population, HE cells (ie, cells lacking hematopoietic CFC potential but capable of forming blood cells after culture with stromal cells) were discriminated from non-HE cells based on lack of CD73 expression.^86,102  The first hematopoietic progenitors emerging from the VE-cadherin⁺ population express CD235a, low levels of CD43, and lack CD41a expression. These cells have a unique potential to form hematopoietic colonies in the presence of FGF2 and hematopoietic cytokines, but also retain endothelial potential and therefore were designated as angiogenic hematopoietic progenitors (AHPs).⁸⁶  Advanced hematopoietic development is associated with upregulation of CD43 expression; segregation of all hematopoietic CFCs to the CD43⁺ fraction^66,100,128 ; and establishment of distinct subsets of CD43⁺ hematopoietic cells, including CD41a⁺CD235a⁺ erythro-megakaryocytic progenitors^100,115,117  and lin⁻CD34⁺CD43⁺CD45^+/− multipotent myelolymphoid progenitors.^66,100,128  Progressive acquisition of the angiogenic and hematopoietic program by differentiated cells is emphasized by green and red colors, respectively.