Secondary blast colonies can arise directly from adherent MHE colonies and differentiate directly to primitive erythroblasts, macrophages, or mixed erythromyeloid colonies. During their initial differentiation, adherent MHE clusters (A; magnification × 100) often produced secondary budding, blast colonies (B, magnification × 200) with a Wright-Giemsa stain hematopoeitic blast morphology (C; magnification × 1000, oil) and that differentiated rapidly into hemoglobinizing cells. To further evaluate the clonogenic potential of MHE-derived budding hematopoietic blast colonies derived from day 7 to 9 hEB cells, they were individually picked prior to full differentiation (about 1 week after hEB plating) and recultured in H4436 supplemented with 50 ng/mL bone morphogenetic protein 4 (BMP4) (which greatly improved their replating efficiency). Single, picked, MHE-derived primary blast colonies (B) were demonstrated to be multipotential by their ability to give rise to multiple secondary mixed, macrophage, or erythroid colonies (D,E) in secondary replating experiments ([E] with 5 of 7 primary blast colonies successfully replated in a representative experiment repeated 3 times). After 4 to 6 weeks of continuous culture, prolific, mature MHE clusters (F, left panel; magnification × 40) are composed of nonadherent hematopoietic cells arising from an adherent bed of mesenchymal-endothelial cells. When nonadherent cells are extensively washed away and adherent cells were incubated overnight with acetylated Dil-LDL, approximately 20% to 30% of adherent cells were positive (F, right panel). To further evaluate the endothelial component of MHE colonies, adherent cells from individual colonies (Gi) were picked, disaggregated, analyzed by FACS, or replated in endothelial medium (EGM2) on Matrigel-coated plates. Replated adherent cells from 6 of 6 individual MHE colonies gave rise to cells with endothelial morphology (ii) and ability to take up acetylated Dil-LDL after several days of EGM2 endothelial culture. (iv) These cells were further evaluated for expression of endothelial genes including von Willebrand factor (VWF) and VE-cadherin by qRT-PCR (HU indicates human umbilical vein endothelial cell [HUVEC] RNA control; C, cord blood RNA control; E, endothelial cells from replated MHE adherent layers). More than 50% to 70% of elongated, adherent cells expressed CD34 and/or CD31 (iii, left); EGM2 replated adherent cells continued to express CD31/CD34 and were devoid of CD45 expression (iii, right).