CD34⁺CD43⁺CD45^−/low cells expanded on overexpression of Sox17 retain hemogenic potential. (A) Experimental design to evaluate effects of withdrawal of 4-OHT on Sox17-overexpressing cells. ECs from day 6 EBs transduced with a Sox17-ERT retrovirus were cultured in the presence of 20 ng/mL of SCF and TPO and 200nM 4-OHT for 15 days. Then, the cells were subjected to coculture with OP9 cells and colony-forming assays. For coculture with OP9 cells, the cells were replated onto OP9 cells and cultured in the presence of 20 ng/mL of SCF and TPO, 10 ng/mL of IL-3, and 3 units/mL of erythropoietin with and without 4-oht. at day 7 of culture, the cells were analyzed for their immunophenotypes by Flow cytometry. For colony-forming assays, the cells were replated in methylcellulose in the presence of 20 ng/mL of SCF, 10 ng/mL of TPO and IL-3, and 3 units/mL erythropoietin with and without 4-OHT. At day 12 of culture, the colonies were counted. (B) Representative flow cytometric profiles of cells overexpressing Sox17-ERT before and after depletion of 4-OHT. (C) The absolute numbers of CD235⁺ erythroblasts and CD11b⁺ myeloid cells in culture at 7 days after depletion of 4-OHT. Data are shown as the means ± SD for triplicate cultures. (D) Ability of Sox17-ERT–overexpressing cells to form hematopoietic colonies in methylcellulose cultures with or without 4-OHT. The numbers of CFUs in culture are presented (left panel). CFU-GM, CFU-M, CFU-G, BFU-E, and CFU-E indicate CFU-granulocyte-macrophage, CFU-macrophage, CFU-granulocyte, burst-forming unit-erythroid, and colony-forming unit-erythroid, respectively. Compact colonies indicate colonies composed by HE cell–like cells. The appearance of a representative compact colony and an erythroid colony observed under an inverted microscope is depicted (right panel).