Figure 6
Figure 6. Recombinant NXPH1 does not affect primitive hematopoietic cells in vivo. (A) Percent chimerism of donor cells in total BM, as well as several lineage-restricted populations in primary recipients after 7 months. In the competitive repopulation assay, pooled BM from 3 C57Bl/6 mice per treatment group was combined either 1:1 or 0.5:1 with BoyJ competitor cells 24 hours after treatment of mice with DPBS carrier or 5 μg/mouse recombinant NXPH1 and transplanted into 5 mice per treatment group (mean ± SD). (B) Percent chimerism in total BM, as well as several lineage-restricted populations in secondary recipients derived from either 1:1 or 0.5:1 primary competitive repopulation assay. After 7 months, 5 secondary recipients per treatment group were transfused with pooled BM from at least 4 members of the corresponding primary transplant group. Four months after the secondary transplant, BM was harvested and analyzed for percent chimerism was analyzed (mean ± SD). (C) Effect of in vitro exposure to recombinant NXPH1 on muBM total CFUs derived from mice treated with recombinant NXPH1 in vivo. Mice were intravenously injected with either DPBS carrier or 5 μg of recombinant NXPH1. Twenty-four hours after in vivo exposure, BM was harvested and treated with either mouse IgM or anti-DAG1 blocking Ab in vitro and then plated in SCF, GM-CSF, PWMSCM, and Epo ± 200 ng/mL recombinant NXPH1 (combined data of 3 animals each analyzed in triplicate; mean ± SD). (D) Fold change in vivo of phenotypically defined HSCs 24 and 48 hours after injection of 5 μg/mouse recombinant NXPH1 (combined data from 2 independent experiments performed in triplicate; mean ± SD). (E) Fold change in cycling status of populations heavily enriched for long (LSK CD34−) or short (LSK CD34+) term engrafting HSCs 24 hours after injection of 5 μg/mouse recombinant NXPH1(combined data from 2 independent experiments performed in triplicate; mean ± SD). (F) Fold change in phenotypically defined HSCs 24 hours after 5 μg/mouse intravenous recombinant NXPH1 exposure and 5 mg/mouse intraperitoneal rapamycin injection (combined data from 2 independent experiments performed in triplicate; mean ± SD). *P < .05, **P < .005, ***P < .0005.

Recombinant NXPH1 does not affect primitive hematopoietic cells in vivo. (A) Percent chimerism of donor cells in total BM, as well as several lineage-restricted populations in primary recipients after 7 months. In the competitive repopulation assay, pooled BM from 3 C57Bl/6 mice per treatment group was combined either 1:1 or 0.5:1 with BoyJ competitor cells 24 hours after treatment of mice with DPBS carrier or 5 μg/mouse recombinant NXPH1 and transplanted into 5 mice per treatment group (mean ± SD). (B) Percent chimerism in total BM, as well as several lineage-restricted populations in secondary recipients derived from either 1:1 or 0.5:1 primary competitive repopulation assay. After 7 months, 5 secondary recipients per treatment group were transfused with pooled BM from at least 4 members of the corresponding primary transplant group. Four months after the secondary transplant, BM was harvested and analyzed for percent chimerism was analyzed (mean ± SD). (C) Effect of in vitro exposure to recombinant NXPH1 on muBM total CFUs derived from mice treated with recombinant NXPH1 in vivo. Mice were intravenously injected with either DPBS carrier or 5 μg of recombinant NXPH1. Twenty-four hours after in vivo exposure, BM was harvested and treated with either mouse IgM or anti-DAG1 blocking Ab in vitro and then plated in SCF, GM-CSF, PWMSCM, and Epo ± 200 ng/mL recombinant NXPH1 (combined data of 3 animals each analyzed in triplicate; mean ± SD). (D) Fold change in vivo of phenotypically defined HSCs 24 and 48 hours after injection of 5 μg/mouse recombinant NXPH1 (combined data from 2 independent experiments performed in triplicate; mean ± SD). (E) Fold change in cycling status of populations heavily enriched for long (LSK CD34−) or short (LSK CD34+) term engrafting HSCs 24 hours after injection of 5 μg/mouse recombinant NXPH1(combined data from 2 independent experiments performed in triplicate; mean ± SD). (F) Fold change in phenotypically defined HSCs 24 hours after 5 μg/mouse intravenous recombinant NXPH1 exposure and 5 mg/mouse intraperitoneal rapamycin injection (combined data from 2 independent experiments performed in triplicate; mean ± SD). *P < .05, **P < .005, ***P < .0005.

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