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Published in final edited form as: Stem Cells. 2010 Mar 31;28(3):523–534. doi: 10.1002/stem.299

Antagonism of ALDH inhibits cell cycle progression of HSCs and increases the frequency of 34+Flt-3-KSL cells in culture compared to cytokines alone. (A): A representative cell cycle analysis of 34-KSL cells and their progeny after 7-day cultures with TSF alone or TSF + DEAB is shown. The majority (mean 83.5%) of day 0 34-KSL cells were in G0/G1. At day +3, the progeny of TSF + DEAB cultures demonstrated a modest increase in cells in G0 and decreased numbers in G2/S/M phase, consistent with overall inhibition of cell cycle progression compared to TSF alone. At day +7, the progeny of TSF cultures demonstrated increased numbers of cells in G0 compared to TSF + DEAB. (B): (i) Representative FACS analyses of 34+Flt-3-KSL cells in culture of BM 34-KSL cells with TSF, TSF + 1 μM ATRA, TSF + DEAB, or TSF + DEAB + ATRA are shown. (ii) Treatment with TSF + DEAB supported an increase in the frequency of 34+Flt-3-KSL cells in culture compared to culture with TSF alone (*, p = .003, n = 3, mean ± SD). The addition of ATRA reversed the effects of DEAB on ST-HSC expansion in culture (∧p = .003, n = 3; mean ± SD). (C): Representative FACS analyses of CMP and MEP content are shown for BM 34-KSL cells after culture with TSF (top) or TSF + DEAB (middle). The progeny of TSF + DEAB cultures contained an increased frequency of CMPs compared to the progeny of TSF alone (mean 36.1% vs. 4.8%, **, p < .001) and a decreased frequency of MEPs (mean 0.1% vs. 0.3%, respectively; *, p = .01) (n = 6; means ± SD) (bottom). Abbreviations: ALDH, aldehyde dehydrogenase; ATRA, All trans retinoic acid; 34-KSL, CD34−c-kit+Sca-1+lineage−; CMP, common myeloid progenitor; DEAB, diethylaminobenzaldehyde; Flt-3, fms-like tyrosine kinase-3; HSCs, hematopoietic stem cells; MEP, megakaryocyte-erythroid progenitor; TSF, thrombopoietin.

Antagonism of ALDH inhibits cell cycle progression of HSCs and increases the frequency of 34+Flt-3-KSL cells in culture compared to cytokines alone. (A): A representative cell cycle analysis of 34-KSL cells and their progeny after 7-day cultures with TSF alone or TSF + DEAB is shown. The majority (mean 83.5%) of day 0 34-KSL cells were in G0/G1. At day +3, the progeny of TSF + DEAB cultures demonstrated a modest increase in cells in G0 and decreased numbers in G2/S/M phase, consistent with overall inhibition of cell cycle progression compared to TSF alone. At day +7, the progeny of TSF cultures demonstrated increased numbers of cells in G0 compared to TSF + DEAB. (B): (i) Representative FACS analyses of 34+Flt-3-KSL cells in culture of BM 34-KSL cells with TSF, TSF + 1 μM ATRA, TSF + DEAB, or TSF + DEAB + ATRA are shown. (ii) Treatment with TSF + DEAB supported an increase in the frequency of 34+Flt-3-KSL cells in culture compared to culture with TSF alone (*, p = .003, n = 3, mean ± SD). The addition of ATRA reversed the effects of DEAB on ST-HSC expansion in culture (∧p = .003, n = 3; mean ± SD). (C): Representative FACS analyses of CMP and MEP content are shown for BM 34-KSL cells after culture with TSF (top) or TSF + DEAB (middle). The progeny of TSF + DEAB cultures contained an increased frequency of CMPs compared to the progeny of TSF alone (mean 36.1% vs. 4.8%, **, p < .001) and a decreased frequency of MEPs (mean 0.1% vs. 0.3%, respectively; *, p = .01) (n = 6; means ± SD) (bottom). Abbreviations: ALDH, aldehyde dehydrogenase; ATRA, All trans retinoic acid; 34-KSL, CD34−c-kit+Sca-1+lineage−; CMP, common myeloid progenitor; DEAB, diethylaminobenzaldehyde; Flt-3, fms-like tyrosine kinase-3; HSCs, hematopoietic stem cells; MEP, megakaryocyte-erythroid progenitor; TSF, thrombopoietin.