Figure 2. Differential kinetics of HSC... | Rockefeller University Press

$Figure 2. Differential kinetics of HSC contribution to hematopoietic progenitors. Adult Pdzk1ip1-CreER R26tdTomato reporter mice were given a single dose of 0.5 mg tamoxifen, and Tom+ cells in the BM were analyzed in sacrificed individual mice. (A) The viSNE plot of gated Lin− (CD11b− Ter119− B220−) Tom+ cells from the experiment described in Fig. 1 (C and D). Left panels show major cell types indicated by color; right panels show cells from each time point highlighted in red. Each cell was plotted using two tSNE parameters (arbitrary units). LSK, pink; MkP, lavender; MEP, purple; MyP, light blue; Ery, green; Flt3+, yellow; CLP, orange; Ly6c+, blue. (B) Identification of Tom+ cells among the progenitor populations by flow cytometry. Shown are dot plots overlaying Tom+ cells (red) and total cells (gray) in the gated BM populations from representative animals at 2 wk (top) or 7 wk (bottom) after labeling. The definition of progenitor populations is indicated. (C) The fraction of Tom+ cells in the HSC/progenitor populations defined as in B at 1 or 4 wk after labeling. Shown are values from individual animals (n = 6–8); bars represent median. The significance of difference between MkPs and other progenitors as determined by Mann-Whitney test is indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. (D) The fraction of Tom+ cells in the HSC/progenitor populations defined as in B at 2 or 7 wk after labeling. Shown are values from individual animals (n = 3 per time point); bars represent the median. (E–G) Clonal growth of BM cells in liquid medium. Single cells were sorted into individual wells of 96-well plates, cultured in serum-free medium with cytokines SCF and TPO, and the growing colonies were scored at the indicated days of culture as follows: a single Mk (Mk*), multiple Mk’s (Mk), small undifferentiated cells (U), or undifferentiated cells and Mk’s (U+Mk). The fraction of each colony type is presented relative to the initial number of all colonies on days 3–4, which was taken as 100%. (E) The growth of single HSCs (LSK CD150+ CD48−), immature non-HSC progenitors (LSK cells other than HSCs), or MkPs (Lin− c-Kit+ Sca-1− CD150+ CD41+) sorted from wild-type mice. Data represent mean ± range of duplicate plates for HSCs (average 30 initial colonies per plate) and single plates for non-HSCs and MkPs (34 and 23 initial colonies, respectively). (F) The growth of single Lin− Tom+ cells sorted from individual Pdzk1ip1-CreER R26tdTomato reporter mice at the start, 1 or 2 wk after induction with tamoxifen. Data represent mean ± range of two animals per time point (average 182, 107, and 71 initial colonies per animal at the start, 1, or 2 wk, respectively). (G) Secondary growth of colonies derived from single Lin− Tom+ cells. U or U+Mk colonies from Tom+ cells at the start, 1, or 2 wk after labeling were harvested on day 7, replated in fresh liquid medium, and their ability to produce secondary U or U+Mk colonies was scored on day 10 after replating as a fraction of replated colonies of each type. A total of 16 colonies (equal number of U and U+Mk types) were picked per time point. Data represent mean ± range of two animals per time point.$

Figure 2.

Differential kinetics of HSC contribution to hematopoietic progenitors. Adult Pdzk1ip1-CreER R26^tdTomato reporter mice were given a single dose of 0.5 mg tamoxifen, and Tom⁺ cells in the BM were analyzed in sacrificed individual mice. (A) The viSNE plot of gated Lin⁻ (CD11b⁻ Ter119⁻ B220⁻) Tom⁺ cells from the experiment described in Fig. 1 (C and D). Left panels show major cell types indicated by color; right panels show cells from each time point highlighted in red. Each cell was plotted using two tSNE parameters (arbitrary units). LSK, pink; MkP, lavender; MEP, purple; MyP, light blue; Ery, green; Flt3⁺, yellow; CLP, orange; Ly6c⁺, blue. (B) Identification of Tom⁺ cells among the progenitor populations by flow cytometry. Shown are dot plots overlaying Tom⁺ cells (red) and total cells (gray) in the gated BM populations from representative animals at 2 wk (top) or 7 wk (bottom) after labeling. The definition of progenitor populations is indicated. (C) The fraction of Tom⁺ cells in the HSC/progenitor populations defined as in B at 1 or 4 wk after labeling. Shown are values from individual animals (n = 6–8); bars represent median. The significance of difference between MkPs and other progenitors as determined by Mann-Whitney test is indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. (D) The fraction of Tom⁺ cells in the HSC/progenitor populations defined as in B at 2 or 7 wk after labeling. Shown are values from individual animals (n = 3 per time point); bars represent the median. (E–G) Clonal growth of BM cells in liquid medium. Single cells were sorted into individual wells of 96-well plates, cultured in serum-free medium with cytokines SCF and TPO, and the growing colonies were scored at the indicated days of culture as follows: a single Mk (Mk*), multiple Mk’s (Mk), small undifferentiated cells (U), or undifferentiated cells and Mk’s (U+Mk). The fraction of each colony type is presented relative to the initial number of all colonies on days 3–4, which was taken as 100%. (E) The growth of single HSCs (LSK CD150⁺ CD48⁻), immature non-HSC progenitors (LSK cells other than HSCs), or MkPs (Lin⁻ c-Kit⁺ Sca-1⁻ CD150⁺ CD41⁺) sorted from wild-type mice. Data represent mean ± range of duplicate plates for HSCs (average 30 initial colonies per plate) and single plates for non-HSCs and MkPs (34 and 23 initial colonies, respectively). (F) The growth of single Lin⁻ Tom⁺ cells sorted from individual Pdzk1ip1-CreER R26^tdTomato reporter mice at the start, 1 or 2 wk after induction with tamoxifen. Data represent mean ± range of two animals per time point (average 182, 107, and 71 initial colonies per animal at the start, 1, or 2 wk, respectively). (G) Secondary growth of colonies derived from single Lin⁻ Tom⁺ cells. U or U+Mk colonies from Tom⁺ cells at the start, 1, or 2 wk after labeling were harvested on day 7, replated in fresh liquid medium, and their ability to produce secondary U or U+Mk colonies was scored on day 10 after replating as a fraction of replated colonies of each type. A total of 16 colonies (equal number of U and U+Mk types) were picked per time point. Data represent mean ± range of two animals per time point.

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