American Society of Hematology

Figure 5

$Figure 5. A functional dependence on Mcl-1 for self-renewal capacity hierarchically distinguishes human HSCs from HPCs. (A) Experimental strategy used to examine the role of Mcl-1 in human SRC regenerative function and self-renewal in vivo. (B) Representative examples of flow cytometry analyses used to examine the effect of Mcl-1 knockdown on human SRC regenerative capacity in vivo. Percentages represent frequency of human engrafted cells with indicated cell surface phenotype. (C) Effect of Mcl-1 knockdown on human SRC regenerative capacity in vivo. Each dot represents the frequency of CD45+GFP+ human hematopoietic cells in the BM of 1 primary recipient mouse that received a transplant with Mcl-1 shRNA or vector control transduced SRCs. The frequencies represent the average frequency of GFP+ human hematopoietic cells in the BM of recipient mice. Bars represent the mean of 7 independent experiments, each with 3 mice per group. **P < .01. (D) Analysis of the effect of Mcl-1 knockdown on the viability of engrafted human hematopoietic cells. The average frequency of dead cells in the CD34+CD38− or CD34+CD38+ fraction of the transduced human hematopoietic graft (GFP+7AAD+) in the BM of primary recipient mice that received a transplant with Mcl-1 shRNA or vector control transduced SRCs. Error bars represent the mean ± SEM of 3 independent experiments, each with 3 mice per group. (E) Analysis of the effect of Mcl-1 knockdown on the CD34+CD38− stem cell and CD34+CD38+ progenitor compartments in vivo. The average frequency of CD34+CD38− and CD34+CD38+ cells within the GFP+ human hematopoietic graft in the BM of primary recipient mice reconstituted with Mcl-1 shRNA transduced SRCs is expressed relative to vector control. Error bars represent the mean ± SEM *P < .05. (F) Representative examples of flow cytometric analyses used to examine the effect of Mcl-1 knockdown on the human CD34+CD38− SRC–enriched fraction of the human hematopoietic graft in vivo. Percentages represent frequency of 34+38− cells. (G) Analysis of the effect of Mcl-1 knockdown on multilineage and primitive human hematopoietic engraftment. The average frequency of lymphoid (CD19+), myeloid (CD33+), and primitive (CD34+) cells within the GFP+ human hematopoietic graft (gated CD45+GFP+ cells) in the BM of primary recipient mice reconstituted with vector or Mcl-1 shRNA-transduced SRCs. Error bars represent the mean ± SEM of at least 3 independent experiments. (H) Effect of Mcl-1 knockdown on human SRC self-renewal capacity in vivo. Each dot represents the frequency of CD45+GFP+ human hematopoietic cells in the BM of 1 secondary recipient mouse that received a transplant with human-engrafted BM isolated from a primary recipient mouse that received a transplant with Mcl-1 shRNA or vector control transduced SRCs. The frequencies represent the average frequency of GFP+ human hematopoietic cells in the BM of secondary recipient mice. Equal numbers of GFP+CD45+CD34+ cells isolated from primary grafts representing 3 independent CB donors were used.$

A functional dependence on Mcl-1 for self-renewal capacity hierarchically distinguishes human HSCs from HPCs. (A) Experimental strategy used to examine the role of Mcl-1 in human SRC regenerative function and self-renewal in vivo. (B) Representative examples of flow cytometry analyses used to examine the effect of Mcl-1 knockdown on human SRC regenerative capacity in vivo. Percentages represent frequency of human engrafted cells with indicated cell surface phenotype. (C) Effect of Mcl-1 knockdown on human SRC regenerative capacity in vivo. Each dot represents the frequency of CD45⁺GFP⁺ human hematopoietic cells in the BM of 1 primary recipient mouse that received a transplant with Mcl-1 shRNA or vector control transduced SRCs. The frequencies represent the average frequency of GFP⁺ human hematopoietic cells in the BM of recipient mice. Bars represent the mean of 7 independent experiments, each with 3 mice per group. **P < .01. (D) Analysis of the effect of Mcl-1 knockdown on the viability of engrafted human hematopoietic cells. The average frequency of dead cells in the CD34⁺CD38⁻ or CD34⁺CD38⁺ fraction of the transduced human hematopoietic graft (GFP⁺7AAD⁺) in the BM of primary recipient mice that received a transplant with Mcl-1 shRNA or vector control transduced SRCs. Error bars represent the mean ± SEM of 3 independent experiments, each with 3 mice per group. (E) Analysis of the effect of Mcl-1 knockdown on the CD34⁺CD38⁻ stem cell and CD34⁺CD38⁺ progenitor compartments in vivo. The average frequency of CD34⁺CD38⁻ and CD34⁺CD38⁺ cells within the GFP⁺ human hematopoietic graft in the BM of primary recipient mice reconstituted with Mcl-1 shRNA transduced SRCs is expressed relative to vector control. Error bars represent the mean ± SEM *P < .05. (F) Representative examples of flow cytometric analyses used to examine the effect of Mcl-1 knockdown on the human CD34⁺CD38⁻ SRC–enriched fraction of the human hematopoietic graft in vivo. Percentages represent frequency of 34⁺38⁻ cells. (G) Analysis of the effect of Mcl-1 knockdown on multilineage and primitive human hematopoietic engraftment. The average frequency of lymphoid (CD19⁺), myeloid (CD33⁺), and primitive (CD34⁺) cells within the GFP⁺ human hematopoietic graft (gated CD45⁺GFP⁺ cells) in the BM of primary recipient mice reconstituted with vector or Mcl-1 shRNA-transduced SRCs. Error bars represent the mean ± SEM of at least 3 independent experiments. (H) Effect of Mcl-1 knockdown on human SRC self-renewal capacity in vivo. Each dot represents the frequency of CD45⁺GFP⁺ human hematopoietic cells in the BM of 1 secondary recipient mouse that received a transplant with human-engrafted BM isolated from a primary recipient mouse that received a transplant with Mcl-1 shRNA or vector control transduced SRCs. The frequencies represent the average frequency of GFP⁺ human hematopoietic cells in the BM of secondary recipient mice. Equal numbers of GFP⁺CD45⁺CD34⁺ cells isolated from primary grafts representing 3 independent CB donors were used.

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