Figure 2.
Figure 2. Phf6 deletion at the fetal stage enhances the repopulating capacity of adult HSCs. (A) PB cell counts in VC and VC;Phf6Δ/y mice. WBC, red blood cell (RBC), and platelet (PLT) counts and hemoglobin (HGB) levels in PB from VC (n = 6) and VC;Phf6Δ/y mice (n = 8) at 12 weeks of age. (B) The proportions of myeloid (My) (Mac-1+ and/or Gr-1+), B220+ B cells, and CD4+ or CD8+ T cells among CD45.2+ cells in PB from VC (n = 6) and VC;Phf6Δ/y (n = 8) mice at 12 weeks of age. (C) Absolute numbers of CD150+CD34−LSK LT-HSCs, CD34−LSK HSCs, myeloid progenitors (CMP, GMP, and MEP), and CLPs in a pool of femurs and tibias from VC (n = 5) and VC;Phf6Δ/y mice (n = 5) at 12 weeks of age. (D) Cell-cycle status of CD34−LSK HSCs identified by Ki67 and 7-aminoactinomycin D (7-AAD) staining at 12 weeks of age. Representative flow cytometric profiles (left panel). The proportion of G0 (Ki67−) cells in CD34−LSK HSCs is shown as the mean plus or minus SEM (n = 6 or 7) (right panel). (E) Competitive repopulating assays. A total of 5 × 106 BM cells from VC and VC;Phf6Δ/y mice (donor, CD45.2) mixed with the same number of BM cells (competitor, CD45.1) were transplanted into lethally irradiated CD45.1 congenic recipients. In serial transplantations, the same numbers of BM cells from all recipients were pooled and 2 × 106 BM cells were then injected into lethally irradiated CD45.1 congenic recipients. The chimerism of donor-derived CD45.2+ cells in the PB of primary (1°), secondary (2°), and tertiary (3°) recipients are shown as the mean plus or minus SEM (n = 5 or 6). (F) The chimerism of My, B-, and T-cell lineages in PB 16, 32, and 48 weeks after 1° transplantation are shown as the mean plus or minus SEM (n = 5 or 6). (G) The chimerism of LT-HSCs, CD34−LSK, GMPs, and CLPs 16, 32, and 48 weeks after 1° transplantation are shown as the mean plus or minus SEM (n = 5 or 6). (H) Growth of VC and VC;Phf6Δ/y CD150+CD34−LSK HSCs in vitro. Freshly sorted HSCs were transduced with a control retrovirus (pMYs) or retrovirus expressing Phf6 (Phf6). After GFP sorting, GFP+ cells were cultured under HSC culture conditions (SCF+TPO) for 7 days. Data are shown as the mean plus or minus SEM of triplicate cultures. Insets, Western blot data of cells in culture on day 7. *P < .05, **P < .01, ***P < .001 by the Student t test. BMT, BM transplantation.

Phf6 deletion at the fetal stage enhances the repopulating capacity of adult HSCs. (A) PB cell counts in VC and VC;Phf6Δ/y mice. WBC, red blood cell (RBC), and platelet (PLT) counts and hemoglobin (HGB) levels in PB from VC (n = 6) and VC;Phf6Δ/y mice (n = 8) at 12 weeks of age. (B) The proportions of myeloid (My) (Mac-1+ and/or Gr-1+), B220+ B cells, and CD4+ or CD8+ T cells among CD45.2+ cells in PB from VC (n = 6) and VC;Phf6Δ/y (n = 8) mice at 12 weeks of age. (C) Absolute numbers of CD150+CD34−LSK LT-HSCs, CD34−LSK HSCs, myeloid progenitors (CMP, GMP, and MEP), and CLPs in a pool of femurs and tibias from VC (n = 5) and VC;Phf6Δ/y mice (n = 5) at 12 weeks of age. (D) Cell-cycle status of CD34−LSK HSCs identified by Ki67 and 7-aminoactinomycin D (7-AAD) staining at 12 weeks of age. Representative flow cytometric profiles (left panel). The proportion of G0 (Ki67−) cells in CD34−LSK HSCs is shown as the mean plus or minus SEM (n = 6 or 7) (right panel). (E) Competitive repopulating assays. A total of 5 × 106 BM cells from VC and VC;Phf6Δ/y mice (donor, CD45.2) mixed with the same number of BM cells (competitor, CD45.1) were transplanted into lethally irradiated CD45.1 congenic recipients. In serial transplantations, the same numbers of BM cells from all recipients were pooled and 2 × 106 BM cells were then injected into lethally irradiated CD45.1 congenic recipients. The chimerism of donor-derived CD45.2+ cells in the PB of primary (1°), secondary (2°), and tertiary (3°) recipients are shown as the mean plus or minus SEM (n = 5 or 6). (F) The chimerism of My, B-, and T-cell lineages in PB 16, 32, and 48 weeks after 1° transplantation are shown as the mean plus or minus SEM (n = 5 or 6). (G) The chimerism of LT-HSCs, CD34−LSK, GMPs, and CLPs 16, 32, and 48 weeks after 1° transplantation are shown as the mean plus or minus SEM (n = 5 or 6). (H) Growth of VC and VC;Phf6Δ/y CD150+CD34−LSK HSCs in vitro. Freshly sorted HSCs were transduced with a control retrovirus (pMYs) or retrovirus expressing Phf6 (Phf6). After GFP sorting, GFP+ cells were cultured under HSC culture conditions (SCF+TPO) for 7 days. Data are shown as the mean plus or minus SEM of triplicate cultures. Insets, Western blot data of cells in culture on day 7. *P < .05, **P < .01, ***P < .001 by the Student t test. BMT, BM transplantation.

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