Effects of epigenetic treatment on mature hematopoietic populations. (A-D) Murine bone marrow cells were sort-purified into primitive (Lin⁻Sca-1⁺c-Kit⁺; LSK), intermediate (Lin⁻Sca-1⁻c-Kit⁻; L⁻S⁻K⁻), and terminally differentiated B-lymphoid (B220⁺Sca-1⁻) or myeloid (Mac-1⁺/Gr-1⁺Sca-1⁻) cell populations. Each cell population was treated with AZA (25 ng/mL) and TSA (25 ng/mL) (denoted as AT) for 24 hours. (A) The extent of apoptosis after treatment was measured as the percentage of PI⁺ cells. (B) The percentage of L⁻S⁺K⁺ cells was determined by restaining the cells with the indicated antibodies. *Less than 0.1%. (C-D) Effects of epigenetic treatment on B-lymphoid (B220⁺Sca-1⁻) and myeloid (Mac-1⁺/Gr-1⁺Sca-1⁻) cells were examined after restaining cultured cells with the indicated antibodies. Representative FACS profile (C) and mean ± SD% Lin⁻ cells (D) after culture are shown (5 independent experiments). AT indicates treatment with both AZA and TSA. (E) Effects of epigenetic treatment on the repopulating activity of mature hematopoietic cells. B220⁺Sca-1⁻ and Mac-1⁺/Gr-1⁺Sca-1⁻ cells (each 10⁵ cells) were treated with epigenetic modifiers (AT) for 24 hours and transplanted with helper cells (10⁵ cells each) into irradiated recipient mice together. Shown is the percentage of donor-derived cells in the peripheral blood of individual recipient mice 16 weeks after transplantation (n = 5 for control and 8 for treated group). (F-G) Effects of epigenetic treatment on human UCB-derived hematopoietic cells. Sorted CD34⁻ and CD34⁺CD38⁻ cells (10⁶ and 10⁵, respectively) were treated with AZA (25 ng/mL) and TSA (25 ng/mL) for 48 hours and analyzed for apoptosis with PI⁺ cells (F) and percentage CD34⁺ cells after culture (G). Shown are the mean ± SD from 3 experiments. (H) Effects of epigenetic treatment on hematopoietic cells in a long-term culture. CD34⁺38⁻ and CD34⁻ cells (10⁴ and 10⁶ cells, respectively) were treated with AZA and TSA for 48 hours and subjected to a long-term culture as described in “LTC-IC assays” in “Methods.” Shown is the total number of colonies and 12-day colony assays obtained after a 6-week long-term culture from 3 independent experiments. *No colonies were obtained.