In vivo pharmacologic inhibition of the Bcl-2 family members leads to decreased human HSC regenerative and self-renewal function. (A) Experimental strategy used to examine the effect of obatoclax on primitive human hematopoietic cell phenotype and SRC self-renewal capacity. (B) Representative examples of flow cytometric analyses used to assess the effects of obatoclax on human hematopoietic engraftment relative to vehicle control. The inlay plots represent the isotype staining control for each antibody. Percentages represent the frequency of total cells acquired with the indicated cell surface phenotype. (C) Analysis of the effect of obatoclax on human SRC regenerative capacity. The average frequency of human hematopoietic engraftment (CD45⁺ cells) in the BM of primary recipient mice administered obatoclax or vehicle control. Error bars represent the mean ± SEM of 3 independent experiments, each with 3 mice per group. (D) Analysis of the effect of obatoclax on the viability of engrafted human hematopoietic cells. The average frequency of dead cells within the human hematopoietic graft (CD45⁺7AAD⁺) in the BM of primary recipient mice administered obatoclax or vehicle control. Error bars represent the mean ± SEM of 3 independent experiments, each with 3 mice per group. (E) Analysis of the effect of obatoclax on apoptosis in engrafted human hematopoietic cells. Average frequency of viable (7AAD⁻) annexin V⁺ (preapoptotic) cells within the human hematopoietic graft (CD45⁺7AAD⁺) in the BM of primary recipient mice administered obatoclax or vehicle control. Error bars represent the mean ± the SEM (n = 2-3 mice per group). (F) Analysis of the effect of obatoclax on multilineage human hematopoietic engraftment. The average frequency of lymphoid (CD19⁺) and myeloid (CD33⁺) cells within the human hematopoietic graft (gated CD45⁺ cells) in the BM of primary recipient mice administered obatoclax or vehicle control. Error bars represent the mean ± SEM of 3 independent experiments, each with 3 mice per group. (G) Analysis of the effect of obatoclax on primitive (CD34⁺) human hematopoietic engraftment. The average frequency of CD34⁺ cells within the human hematopoietic graft (gated CD45⁺ cells) in primary recipient mice administered obatoclax is expressed relative to mice administered vehicle control. Error bars represent the mean ± SEM of 3 independent experiments, each with 3 mice per group. *P < .05. (H) Analysis of the effect of obatoclax on each population within the human hematopoietic hierarchy. The average frequency of cells in each population within human hematopoietic graft (gated CD45⁺ cells) in primary recipient mice administered obatoclax is expressed relative to mice administered vehicle control. Error bars represent the mean ± SEM of 3 independent experiments, each with 3 mice per group. **P < .01. (I) Analysis of the effect of obatoclax on human SRC self-renewal capacity. The average frequency of human hematopoietic engraftment (CD45⁺ cells) in the BM of secondary recipient mice that received a transplant with human-engrafted BM isolated from primary recipient mice administered obatoclax or vehicle control. Error bars represent the mean ± SEM of 3 independent experiments, each with 3 mice per group. *P < .05.