Figure 3
Effects of Stat5-deficiency on hematopoietic stem cells and progenitors expressing Jak2V617F. (A) Flow cytometric analysis revealed a marked reduction in CD71+Ter119+ erythroid precursors in the BM and spleens of induced MxCre;Jak2V617F/+;Stat5fl/fl mice compared with induced MxCre; Jak2V617F/+ mice; the CD71+Ter119+ population in the BM and spleens of MxCre;Jak2V617F/+;Stat5fl/fl mice was comparable with that of control mice. Representative dot plots from 7 independent experiments are shown. (B) Flow cytometric analysis of the LSK compartment (Lin−Sca1+c-kit+) and subsets of myeloid progenitors (MP) including CMP (Lin−Sca1−c-kit+CD34+FcγRII/IIIlo), GMP (Lin−Sca1−c-kit+CD34+FcγRII/IIIhigh), and MEP (Lin−Sca1−c-kit+CD34−FcγRII/III−) in the BM from control (n = 4), MxCre;Jak2V617F/+ mice (n = 4) and MxCre;Jak2V617F/+; Stat5fl/fl (n = 5) mice. (C) HSC-enriched LSK compartments from the BM of control, MxCre;Jak2V617F/+ and MxCre;Jak2V617F/+; Stat5fl/fl mice were further analyzed for LT-HSC (LSKCD34−Flk2−), ST-HSC (LSKCD34+Flk2−), and MPP (LSKCD34+Flk2+). Representative contour plots from 3 independent experiments are shown. (D) Percentages of LSK, LT-HSC. ST-HSC, MPP, CMP, GMP, and MEP are shown in histogram as mean ± SEM Data are presented as percentage of total cells. Asterisks show significant differences by 1-way ANOVA (**P < .005). Note that a significant decrease in LSK, LT-HSC, ST-HSC, and MEP compartments in the BM from MxCre;Jak2V617F/+;Stat5fl/fl mice compared with MxCre;Jak2V617F/+ mice and those populations were comparable with that observed in controls. (E) Analysis of hematopoietic progenitor colonies. BM (2 × 104) and spleen (1 × 105) cells from control (n = 3), MxCre;Jak2V617F/+ mice (n = 5), and MxCre;Jak2V617F/+;Stat5fl/fl mice (n = 5) were seeded in complete methylcellulose medium (Methocult M3434). BFU-E, CFU-GM, and CFU-GEMM colonies were counted on day 7. (F) Epo-independent CFU-E colonies. BM (1 × 105) or spleen (1 × 105) cells were plated in methylcellulose medium without any cytokine (Methocult M3234). CFU-E colonies were counted after 2 days. For BM CFU-E colonies, n = 4 for control and n = 6 for both MxCre;Jak2V617F/+ and MxCre;Jak2V617F/+;Stat5fl/fl mice; for spleen CFU-E colonies, n = 6 for control and n = 8 for both MxCre;Jak2V617F/+ and MxCre;Jak2V617F/+; Stat5fl/fl mice. Data are presented as mean ± SEM. Asterisks show significant differences by 1-way ANOVA (*P < .05; **P < .005).

Effects of Stat5-deficiency on hematopoietic stem cells and progenitors expressing Jak2V617F. (A) Flow cytometric analysis revealed a marked reduction in CD71+Ter119+ erythroid precursors in the BM and spleens of induced MxCre;Jak2V617F/+;Stat5fl/fl mice compared with induced MxCre; Jak2V617F/+ mice; the CD71+Ter119+ population in the BM and spleens of MxCre;Jak2V617F/+;Stat5fl/fl mice was comparable with that of control mice. Representative dot plots from 7 independent experiments are shown. (B) Flow cytometric analysis of the LSK compartment (Lin−Sca1+c-kit+) and subsets of myeloid progenitors (MP) including CMP (Lin−Sca1−c-kit+CD34+FcγRII/IIIlo), GMP (Lin−Sca1−c-kit+CD34+FcγRII/IIIhigh), and MEP (Lin−Sca1−c-kit+CD34−FcγRII/III−) in the BM from control (n = 4), MxCre;Jak2V617F/+ mice (n = 4) and MxCre;Jak2V617F/+; Stat5fl/fl (n = 5) mice. (C) HSC-enriched LSK compartments from the BM of control, MxCre;Jak2V617F/+ and MxCre;Jak2V617F/+; Stat5fl/fl mice were further analyzed for LT-HSC (LSKCD34−Flk2−), ST-HSC (LSKCD34+Flk2−), and MPP (LSKCD34+Flk2+). Representative contour plots from 3 independent experiments are shown. (D) Percentages of LSK, LT-HSC. ST-HSC, MPP, CMP, GMP, and MEP are shown in histogram as mean ± SEM Data are presented as percentage of total cells. Asterisks show significant differences by 1-way ANOVA (**P < .005). Note that a significant decrease in LSK, LT-HSC, ST-HSC, and MEP compartments in the BM from MxCre;Jak2V617F/+;Stat5fl/fl mice compared with MxCre;Jak2V617F/+ mice and those populations were comparable with that observed in controls. (E) Analysis of hematopoietic progenitor colonies. BM (2 × 104) and spleen (1 × 105) cells from control (n = 3), MxCre;Jak2V617F/+ mice (n = 5), and MxCre;Jak2V617F/+;Stat5fl/fl mice (n = 5) were seeded in complete methylcellulose medium (Methocult M3434). BFU-E, CFU-GM, and CFU-GEMM colonies were counted on day 7. (F) Epo-independent CFU-E colonies. BM (1 × 105) or spleen (1 × 105) cells were plated in methylcellulose medium without any cytokine (Methocult M3234). CFU-E colonies were counted after 2 days. For BM CFU-E colonies, n = 4 for control and n = 6 for both MxCre;Jak2V617F/+ and MxCre;Jak2V617F/+;Stat5fl/fl mice; for spleen CFU-E colonies, n = 6 for control and n = 8 for both MxCre;Jak2V617F/+ and MxCre;Jak2V617F/+; Stat5fl/fl mice. Data are presented as mean ± SEM. Asterisks show significant differences by 1-way ANOVA (*P < .05; **P < .005).

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