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S. Bohler et al.
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Figure 6. MCL-1 is essential for survival of both cord blood- and bone marrow-derived human CD34+ cells. (A) Protein levels of the anti-apoptotic BCL-2 family mem- bers MCL-1, BCL-XL, BCL-2 and BFL1/A1 were determined in CD34+ cells isolated from both cord blood and bone marrow. b-actin served as a loading control. (B) Freshly isolated bone marrow was subjected to density gradient centrifugation and mononuclear cells were divided into CD34+ and CD34- cells using magnetic acti- vated cell sorting technology. Both cell fractions were treated with the indicated concentrations of the MCL-1 inhibitor S63845. After 24 h (upper panel) and 48 h (lower panel), percentages of living cells were determined by flow cytometry using annexin V/7-AAD. Bars represent mean ± standard error of mean (SEM); n=6-7 from seven independent experiments. (C-D) Human CD34+ cells isolated from either cord blood (C) or bone marrow (D) were differentiated in MethoCult medium containing 0.1 or 1 mM S63845. As controls, untreated and dimethylsulfoxide (DMSO)-treated cells were used (n=7 from seven independent experiments). After 11 days, total colony numbers (left) and total cell numbers (middle) were determined using light microscopy and hemocytometry, respectively. Different immature and differentiated cell types were determined by flow cytometry, and their absolute cell numbers were calculated (right). The following cell types were studied; HSC: hematopoietic stem cells (CD34+CD38-CD45RA-CD90+), MPP: multipotent progenitors (CD34+38–CD45RA-CD90-), MLP: mixed lymphoid progenitors (CD34+CD38- CD45RA+CD10+) GM: granulocytic–monocytic progenitors (CD34+CD33+CD115+), M: monocytes (CD34-CD33+CD14+CD115-), immE: immature erythrocytes (CD71hiCD235a-), matE: mature erythrocytes (CD71+CD235a+). Bars represent mean ± SEM. Mann-Whitney test: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
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haematologica | 2021; 106(12)