Assessment of functional HPC repopulating ability and in vitro functional assessment of HPC numbers and cytokine-proliferative responses from WT and STAT3^−/− mice. Results for primary mouse engraftment are shown as means ± SEM for 2 completely separate experiments. In experiment number 1, pooled male and female C57Bl/6 BM cells (CD45.2⁺) from 3 WT littermates or 3 Stat3^−/− mice were injected at a 1:1 ratio with B6.BoyJ (CD45.1⁺) competitor cells each into 5 lethally irradiated B6.BoyJ recipient mice. (Ai) Only one time point is shown for the secondary transplantations because all lethally irradiated secondary transplantation recipients (WT and STAT3^−/−) died before the fourth month after transplantation. (Aii) In experiment number 2, C57Bl/6 cells from 2 individual WT littermates or Stat3^−/− female mice were each injected at a 1:1 ratio with B6.BoyJ competitor cells into 5 lethally irradiated B6.BoyJ recipient mice. Results for secondary engraftment are shown as means ± SEM. For the secondary transplantations, 10⁶ cells from each primary group were injected into 5 lethally irradiated secondary female recipients. *P < .05; **P < .01; ***P < .001. (Bi) Influence of STAT3^−/− on absolute numbers of HPCs. Results shown are the average ± SEM for 11 WT and 10 STAT3^−/− mice individually assessed from a total of 3 different experiments. Cells were stimulated in vitro with erythropoietin, pokeweed mitogen mouse spleen cell conditioned medium, SCF, and hemin to detect the more immature subsets of progenitors. a indicates P < .001 compared with control. (Bii) Influence of STAT3^−/− on cycling status of HPC for the same cells as in panel Bi. The percentage of HPCs in the S-phase was determined with the high specific activity tritiated thymidine kill technique. (Ci) Response in vitro of BM CFU-GM from WT and STAT3^−/− mice to a colony stimulating factor (CSF) and a costimulating cytokine (SCF or FL). Results are presented as means ± SEM ratio shown for 7 WT and 6 STAT3^−/− mice from a total of 2 separate experiments. *P < .001 for numbers of colonies stimulated by only a CSF plus either SCF or FL compared with the additive number of colonies stimulated by a CSF plus that only stimulated by SCF or FL. (Cii) Comparative replating capacity in vitro of HPC-derived colonies from WT and STAT3^−/− BM cells. Results are based on 107 replated WT primary and 114 STAT3^−/− primary colonies from a total of 3 different experiments. For CFU-M, primary and secondary colonies were grown in methylcellulose culture with M-CSF and SCF. For CFU-GM, BFU-E, and CFM-GEMM, primary and secondary colonies were grown with erythropoietin, SCF, pokeweed mitogen mouse spleen cell conditioned medium, and hemin. For CFU-M, colonies were scored after 7 days incubation. (D) Phenotype analysis of hematopoietic progenitors from WT and STAT3^−/− mouse BM. Flow cytometric surface marker analysis was done as previously described.^27,28