Figure 1.
Lineage potential of MPP5 cells and HSC/MPP ontogeny analysis. (A) Workflow of functional and molecular analysis of HSC and MPP cells. (B) Analysis of HSC/MPP transplantations. Workflow of transplantation approach. The relative percentage of donor contribution to the PB, myeloid, B-cell, and T-cell lineage is shown; n = 4-11. (C) Analysis of HSCST/MPP5/6 transplantations. Workflow of transplantation approach. The relative percentage of donor contribution to the PB, myeloid, B-cell, and T-cell lineage is shown; n = 5-11. (D) CFU assay of HSC, MPP1, MPP2, and MPP5 cells showing colony numbers for first, second, and third plating.; n = 6-9 (first, second); n = 2-5 (third). (E) In vitro ontogeny analysis. Frequency of single cells in the respective HSPC subset is shown 6 hours, 16 hours, 24 hours and 48 hours after plating as identified in flow cytometry analysis. Statistical analysis between the populations (respective color) or overall output (black) is shown compared with HSCs; n = 8 (48 hours: n = 3). (F) High-throughput simultaneous division and differentiation tracking per ancestor. Cells were stained with distinct carboxyfluorescein diacetate succinimidyl ester (CFSE)/CellTrace Violet (CTV) combinations and HSC/MPP populations were index sorted combining 4 cells of distinct cell trace colors per well. Twenty-four hours after culture, phenotypic identification was performed allowing analysis of differentiation trajectories from a founding ancestor. (G) In vivo ontogeny analysis. Endpoint analysis of recipient animals 1 or 2 weeks following transplantation in sublethally irradiated recipients. HSPC engraftment in the BM is shown as frequency of single cells. Statistical analysis between the populations (respective color) or overall engraftment (black) is shown compared with HSCs; n = 5-9. For all panels, mean plus standard deviation (SD) is shown. Two-way analysis of variance (ANOVA) (B,D-F). (B) Statistical analysis of MPP5 vs HSC is shown. *P < .05; **P < .01; ***P < .001. "n" indicates number of biological replicates. Two to 3 independent experiments (B-C), 2 to 4 independent experiments (D), 6/16/24 hours: 4 independent experiments; 48 hours: 1 independent experiment (E) 3 independent experiments (F-G). See also supplemental Figures 1 and 2. ns, not significant.

Lineage potential of MPP5 cells and HSC/MPP ontogeny analysis. (A) Workflow of functional and molecular analysis of HSC and MPP cells. (B) Analysis of HSC/MPP transplantations. Workflow of transplantation approach. The relative percentage of donor contribution to the PB, myeloid, B-cell, and T-cell lineage is shown; n = 4-11. (C) Analysis of HSCST/MPP5/6 transplantations. Workflow of transplantation approach. The relative percentage of donor contribution to the PB, myeloid, B-cell, and T-cell lineage is shown; n = 5-11. (D) CFU assay of HSC, MPP1, MPP2, and MPP5 cells showing colony numbers for first, second, and third plating.; n = 6-9 (first, second); n = 2-5 (third). (E) In vitro ontogeny analysis. Frequency of single cells in the respective HSPC subset is shown 6 hours, 16 hours, 24 hours and 48 hours after plating as identified in flow cytometry analysis. Statistical analysis between the populations (respective color) or overall output (black) is shown compared with HSCs; n = 8 (48 hours: n = 3). (F) High-throughput simultaneous division and differentiation tracking per ancestor. Cells were stained with distinct carboxyfluorescein diacetate succinimidyl ester (CFSE)/CellTrace Violet (CTV) combinations and HSC/MPP populations were index sorted combining 4 cells of distinct cell trace colors per well. Twenty-four hours after culture, phenotypic identification was performed allowing analysis of differentiation trajectories from a founding ancestor. (G) In vivo ontogeny analysis. Endpoint analysis of recipient animals 1 or 2 weeks following transplantation in sublethally irradiated recipients. HSPC engraftment in the BM is shown as frequency of single cells. Statistical analysis between the populations (respective color) or overall engraftment (black) is shown compared with HSCs; n = 5-9. For all panels, mean plus standard deviation (SD) is shown. Two-way analysis of variance (ANOVA) (B,D-F). (B) Statistical analysis of MPP5 vs HSC is shown. *P < .05; **P < .01; ***P < .001. "n" indicates number of biological replicates. Two to 3 independent experiments (B-C), 2 to 4 independent experiments (D), 6/16/24 hours: 4 independent experiments; 48 hours: 1 independent experiment (E) 3 independent experiments (F-G). See also supplemental Figures 1 and 2. ns, not significant.

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