The influence of cell division history of CD34⁺CD90⁺ cells on frequency and absolute numbers of CFUs-Mix and CAFCs following ex vivo culture. CD34⁺ cells cultured in the presence of cytokines with or without 5azaD/TSA treatment after 9 days were harvested and CD34⁺CD90⁺ cells that have undergone 4 or fewer divisions or 5 or more divisions were reisolated and assayed for CFCs and CAFCs. (A) Frequency of CFUs-Mix generated from populations of day 0 CD34⁺CD90⁺ cells and CD34⁺CD90⁺ cells undergoing 4 or fewer divisions or 5 or more divisions (day 9) was determined. The bar graph indicates the numbers of CFUs-Mix assayed in 500 CD34⁺CD90⁺ cells plated. (B) Numbers of CAFCs/10⁴ cells assayed from day 0 CD34⁺CD90⁺ cells and CD34⁺CD90⁺ cells having undergone 4 or fewer divisions or 5 or more cell divisions (day 9). (C) The absolute numbers of CFUs-Mix/well generated from populations of day 0 CD34⁺CD90⁺ cells and CD34⁺CD90⁺ cells undergoing 4 or fewer divisions or 5 or more divisions (day 9) present in an individual well was determined by the following calculation: (numbers of CFUs-Mix assayed in 500 CD34⁺CD90⁺ cells plated that have undergone ≤ 4 or ≥ 5 cell divisions) × (total number of CD34⁺CD90⁺ cells having undergone ≤ 4 divisions or ≥ 5 cell divisions present in an individual well). (D) The absolute number of CAFCs/well was determined by the following calculation: (number of CAFCs/10⁴ cells assayed from reisolated CD34⁺CD90⁺ cells having undergone ≤ 4 divisions or ≥ 5 cell divisions in an individual well after 9 days of culture) × (total number of CD34⁺CD90⁺ cells having undergone ≤ 4 divisions or ≥ 5 cell divisions present in an individual well). The bar graphs represent mean ± SE of 3 independent experiments. *Previously published data, reprinted with permission.²⁴