Figure 7. Evi1 heterozygosity causes... | Rockefeller University Press

Figure 7.

$Figure 7. Evi1 heterozygosity causes specific abrogation of self-renewal capacity in ST- and LT-HSCs. (A) Numbers of CFU-GM, CFU-GEMM, and BFU-E colonies derived from 100 sorted Evi1+/+ and Evi1+/− CD34+ LSK cells (n = 3). (B) Appearance and number of CFU-S colonies in the spleen 11 d after injection of 100 sorted Evi1+/+ or Evi1+/− CD34+ LSK cells into lethally irradiated recipients. (left) Representative appearance is shown. (right) Data are shown as a dot plot and each bar represents mean (n = 15–16 from 3 independent experiments). (C and D) Short-term in vivo repopulating assay, in which 500 sorted Evi1+/+ or Evi1+/− CD34+ LSK cells (Ly5.2) were transplanted into lethally irradiated recipients (Ly5.1) together with 2 × 105 competitor BM cells (Ly5.1). (C) Percentages of donor-derived myeloid and B cells (Ly5.2) in PB 2 wk after transplantation are shown (n = 3). (D) Short-term kinetics of the percentages of donor-derived cells (Ly5.2) in PB. Each dot indicates an individual recipient mouse (*, P < 0.05; n = 3). (E) Proliferation of 1,000 sorted Evi1+/+ and Evi1+/− Flk-2− CD34− LSK cells cultured in serum-free medium supplemented with 20 ng/ml SCF and 20 ng/ml TPO for 14 d (*, P < 0.01; ** P < 0.001; n = 3–4). (F) After 7 d of culture, the percentage of the remaining LSK fraction in cultured Evi1+/+ and Evi1+/− Flk-2− CD34− LSK cells was analyzed (*, P < 0.005; n = 3). (G) In vitro colony-forming assay was performed to assess the numbers of CFU-GM, CFU-GEMM, and BFU-E colonies after 1,000 Evi1+/+ and Evi1+/− Flk-2− CD34− LSK cells were cultured for 14 d (*, P < 0.01; ** P < 0.001; n = 3–4). (H) Single Evi1+/+ and Evi1+/− Flk-2− CD34− LSK cells were clone-sorted and cultured in serum-free medium. After 14 d of culture, cell numbers in each colony were analyzed. Their relative distribution is shown (*, P < 0.0001; n = 192 clones from 2 independent experiments). (I–J) Long-term in vivo repopulating assay, in which 200 sorted Evi1+/+ or Evi1+/− Flk-2− CD34− LSK cells (Ly5.2) were transplanted into lethally irradiated recipients (Ly5.1) together with 2 × 105 competitor BM cells (Ly5.1). (I) Percentages of donor-derived cells (Ly5.2) in PB after transplantation are shown. Each dot indicates an individual recipient mouse (*, P < 0.0001, n = 5–6). (J) Percentages of donor-derived cells (Ly5.2) in myeloid, B, and T cells of PB and LSK cells of BM 16 wk after transplantation (*, P < 0.001; ** P < 0.0001; n = 5–6 for PB and n = 3 for BM). (K) Noncompetitive repopulating assay, in which 200 sorted Evi1+/+ or Evi1+/− Flk-2− CD34− LSK cells (Ly5.2) were transplanted into lethally irradiated recipients (Ly5.1) without competitor. Percentages of donor-derived cells (Ly5.2) in PB of recipient mice that survived 12 wk after transplantation are shown (*, P < 0.05, n = 4–6). (L) Reciprocal transplantation assay was performed by transplantation of 2 × 105 WT BM cells (Ly5.1) into unirradiated Evi1+/+ or Evi1+/− mice (Ly5.2). Percentages of donor-derived cells (Ly5.1) in PB 16 wk after transplantation are shown (n = 6–8). Data represent mean ± SD.$

Evi1 heterozygosity causes specific abrogation of self-renewal capacity in ST- and LT-HSCs. (A) Numbers of CFU-GM, CFU-GEMM, and BFU-E colonies derived from 100 sorted Evi1^+/+ and Evi1^+/− CD34⁺ LSK cells (n = 3). (B) Appearance and number of CFU-S colonies in the spleen 11 d after injection of 100 sorted Evi1^+/+ or Evi1^+/− CD34⁺ LSK cells into lethally irradiated recipients. (left) Representative appearance is shown. (right) Data are shown as a dot plot and each bar represents mean (n = 15–16 from 3 independent experiments). (C and D) Short-term in vivo repopulating assay, in which 500 sorted Evi1^+/+ or Evi1^+/− CD34⁺ LSK cells (Ly5.2) were transplanted into lethally irradiated recipients (Ly5.1) together with 2 × 10⁵ competitor BM cells (Ly5.1). (C) Percentages of donor-derived myeloid and B cells (Ly5.2) in PB 2 wk after transplantation are shown (n = 3). (D) Short-term kinetics of the percentages of donor-derived cells (Ly5.2) in PB. Each dot indicates an individual recipient mouse (*, P < 0.05; n = 3). (E) Proliferation of 1,000 sorted Evi1^+/+ and Evi1^+/− Flk-2⁻ CD34⁻ LSK cells cultured in serum-free medium supplemented with 20 ng/ml SCF and 20 ng/ml TPO for 14 d (*, P < 0.01; ** P < 0.001; n = 3–4). (F) After 7 d of culture, the percentage of the remaining LSK fraction in cultured Evi1^+/+ and Evi1^+/− Flk-2⁻ CD34⁻ LSK cells was analyzed (*, P < 0.005; n = 3). (G) In vitro colony-forming assay was performed to assess the numbers of CFU-GM, CFU-GEMM, and BFU-E colonies after 1,000 Evi1^+/+ and Evi1^+/− Flk-2⁻ CD34⁻ LSK cells were cultured for 14 d (*, P < 0.01; ** P < 0.001; n = 3–4). (H) Single Evi1^+/+ and Evi1^+/− Flk-2⁻ CD34⁻ LSK cells were clone-sorted and cultured in serum-free medium. After 14 d of culture, cell numbers in each colony were analyzed. Their relative distribution is shown (*, P < 0.0001; n = 192 clones from 2 independent experiments). (I–J) Long-term in vivo repopulating assay, in which 200 sorted Evi1^+/+ or Evi1^+/− Flk-2⁻ CD34⁻ LSK cells (Ly5.2) were transplanted into lethally irradiated recipients (Ly5.1) together with 2 × 10⁵ competitor BM cells (Ly5.1). (I) Percentages of donor-derived cells (Ly5.2) in PB after transplantation are shown. Each dot indicates an individual recipient mouse (*, P < 0.0001, n = 5–6). (J) Percentages of donor-derived cells (Ly5.2) in myeloid, B, and T cells of PB and LSK cells of BM 16 wk after transplantation (*, P < 0.001; ** P < 0.0001; n = 5–6 for PB and n = 3 for BM). (K) Noncompetitive repopulating assay, in which 200 sorted Evi1^+/+ or Evi1^+/− Flk-2⁻ CD34⁻ LSK cells (Ly5.2) were transplanted into lethally irradiated recipients (Ly5.1) without competitor. Percentages of donor-derived cells (Ly5.2) in PB of recipient mice that survived 12 wk after transplantation are shown (*, P < 0.05, n = 4–6). (L) Reciprocal transplantation assay was performed by transplantation of 2 × 10⁵ WT BM cells (Ly5.1) into unirradiated Evi1^+/+ or Evi1^+/− mice (Ly5.2). Percentages of donor-derived cells (Ly5.1) in PB 16 wk after transplantation are shown (n = 6–8). Data represent mean ± SD.