Comparative analysis of the proliferation and differentiation potential of HAT^+/+ and HAT^−/− CD34⁺/c-kit⁺ hematopoietic progenitors. (A) Cell morphology. Sorted CD34⁺/c-Kit⁺ hematopoietic populations from HAT^+/+ and HAT^−/− ES cell lines were resuspended in serum-free proliferation media at a concentration of 2 × 10⁵ cells/mL, harvested at indicated time points, and stained by May-Grünwald-Giemsa for morphologic analysis. (B) Relative frequency of undifferentiated/blast cells (□) and macrophages (■) at different stages of maturation in the HAT^+/+ and HAT^−/− cultures. A total of 200 cells was scored for each sample. (C) Analysis of CD34⁺/CD45⁻ cell population in HAT^+/+ and HAT^−/− cell cultures. Days of differentiation are indicated. Numbers represent the percentage of total population. (D) Percentage of apoptotic cells in HAT^+/+ and HAT^−/− asynchronous cultures. Percentage of annexin V–positive cells is indicated for each time point. (E) Expansion of CD34⁺/c-Kit⁺ cells and fetal liver cells. Total number of cells (×10⁴) generated during the culture. CD34⁺/c-Kit⁺ cells isolated from day 6 EBs or total fetal liver cells from embryos were seeded at a density of 2 × 10⁶ or 10⁶ cells/mL, respectively. (F) Cell cycle status of HAT^+/+ and HAT^−/− CD34⁺/c-Kit⁺ hematopoietic progenitors in serum-free proliferation media. Asynchronous cultures of CD34⁺/c-Kit⁺ hematopoietic progenitors were stained with propidium iodide and analyzed by flow cytometry. Percentages of cells in G₁ and S/G₂ in HAT^+/+ and HAT^−/− at day 3 of culture are indicated.