Figure 2.

Inhibition of ALDH increases the frequency of phenotypic and functional BM progenitor cells in culture. (A): Representative flow cytometric analyses are shown of day 0 BM 34-KSL cells (top) and the progeny of 34-KSL cells after 7-day culture with TSF alone (left) and TSF + DEAB (right). Note that the progeny of TSF + DEAB remain lineage negative at day +7, with increased KSL content compared to the progeny of TSF alone. (B): The addition of DEAB to cultures of 34-KSL cells caused a significant reduction in total cell expansion compared to TSF alone (upper left, *, p = .001), but yielded a significant increase in the percentage of lineage negative progenitor cells (upper right, *, p < .001) and percentage of KSL cells compared to treatment with TSF alone (lower left, *, p = .01) (n = 3–5, means ± SD). The absolute number of KSL cells was 2.6-fold increased in TSF cultures compared to TSF + DEAB (lower right, *, p = .001) (n = 3–5, means ± SD). (C): Inhibition of ALDH preserves CFC content in cultures of BM 34-KSL cells. CFC assays were performed using day 0 BM 34-KSL cells or their progeny after 7-day culture with TSF alone or TSF + DEAB. The mean number of CFU total was 16-fold increased in the TSF + DEAB cultures compared to TSF alone. *, p = .009 for difference between CFCs in TSF + DEAB versus TSF alone (n = 3, means ± SD). Gray bar indicates CFU-GM, black bar indicates BFU-E, and white bar indicates CFU-Mix. Abbreviations: ALDH, aldehyde dehydrogenase; BFU-E, burst forming unit-erythroid; BM, bone marrow; 34-KSL, CD34−c-kit+Sca-1+lineage−; CFC, colony forming cell; CFU, colony-forming unit; CFU-GM, colony forming unit-granulocyte monocyte; CFU-Mix, colony forming unit-mix; DEAB, diethylaminobenzaldehyde; TSF, thrombopoietin.