Figure 2.

Hallmarks of accelerated HSC aging in the Pf4⁺FF1⁺ mice compared with control mice. Representative flow cytometry plots showing gating strategy (A) used to measure marrow Lin⁻cKit⁺Sca1⁺CD150⁺CD48⁻CD41⁺ myeloid-biased HSC frequency (B) in 6 months, 1 year, and 2-year-old Pf4-cre control and Pf4⁺FF1⁺ mice (6 months and 1 year old: n = 3 in each group; 2 years old: n = 6 in each group). (C) Methylcellulose colony formation: single cells from old Pf4-cre or old Pf4⁺FF1⁺ HSC population were sorted into individual wells of a 96-well plate containing complete methylcellulose medium. After 12-14 days, colonies were scored. The percentage of each type of colony out of the total cells plated is indicated. Data shown are representative from 1 of 2 independent experiments of 60 cells each, for a total of 120 cells each group. (D) Peripheral blood CD45.2 donor chimerism in recipients of competitive transplantation experiments during which donor marrow cells from 6 months old Pf4-cre or Pf4⁺FF1⁺ mice (CD45.2) were transplanted together with 8wk old CD45.1 wild-type competitor marrow cells into lethally irradiated CD45.1 recipients. (n=8 mice in each group from 2 independent experiments) (E) Peripheral blood CD45.2 donor chimerism in recipients of competitive transplantation experiments during which donor marrow cells from 2 years old Pf4-cre or Pf4⁺FF1⁺ (CD45.2) mice were transplanted together with 8wk old CD45.1 wild-type competitor marrow cells into lethally irradiated CD45.1 recipients. (n=8-9 mice in each group from 2 independent experiments) (F) Frequency of donor-derived myeloid and lymphoid cells in the blood of recipients of 2 years old Pf4-cre or Pf4⁺FF1⁺ marrow donors at 16wks post transplantation. Myeloid cells were defined as CD11b- and/or Gr1-positive events. Lymphoid cells were defined as CD3- or B220-positive events that were negative for CD11b or Gr1. The proportion of myeloid and lymphoid cells from Pf4-cre or Pf4⁺FF1⁺ donor (CD45.2) or wild-type competitor donor (CD45.1) was determined using CD45.2 versus CD45.1 for each cell type. (n = 8-9 mice in each group from 2 independent experiments) (G) HSCs from old Pf4⁺FF1⁺ mice are impaired in differentiation compared with HSCs from old Pf4-cre control mice, as shown by their donor-derived marrow HSC chimerism (left) and peripheral blood chimerism (right) (n = 5-6 mice in each group). (H) Scheme of serial marrow transplantation experiments. (I) Peripheral blood CD45.2 donor chimerism in secondary and tertiary transplant recipients after transplantation (n = 5 mice in each group).