American Society of Hematology

Figure 2

$Figure 2. Repopulation activity of Fbxw7α-transduced HSCs after ex vivo culture. (A) In vitro colony formation assays of EGFP or Fbxw7α virus-transduced EGFP+ cells. Colonies were counted and classified as granulocyte, erythroid, monocyte, and megakaryocyte (GEMM), granulocyte and monocyte (GM), or erythroid (E) on day 9 of culture (mean ± SD, n = 3). (B) Number of highly proliferative colony-forming EGFP or Fbxw7α virus-transduced EGFP+ cells on day 14 of culture (mean ± SD, n = 3). *P < .005. (C) CFU-S12 assay. EGFP or Fbxw7α virus-transduced EGFP+ cells were transplanted into 8.5-Gy irradiated recipient mice. Spleens were harvested after 12 days, fixed with Bounin solution, and CFU-S12 colonies were counted (mean ± SD, n = 5). (D) Study design for transplantation experiment. EGFP virus or Fbxw7α virus was transduced into LSK cells (CD45.1+). After 48 hours, EGFP+ cells were sorted and cultured for 1 week (18 000 LSK cells/well). Cultured cells were transplanted into lethally irradiated recipient mice (CD45.2+) with 4 × 105 CD45.2+ competitors. PB chimerism was analyzed monthly, and BM chimerism was analyzed at 4 months after transplantation. Donor-derived LSK cells were used for serial transplantation after 4 months. (E) Relative numbers of total, Lin−, LSK, and CD150+ LSK EGFP or Fbxw7α virus-transduced EGFP+ cells after 1 week of culture. Cell number was analyzed by flow cytometry (mean ± SD, n = 6 for EGFP, n = 2 for Fbxw7). (F) Quantitative PCR analysis of various cell cycle–related genes in EGFP or Fbxw7α virus-transduced LSK cells after 1 week of culture (n = 4). (G) PB chimerism in primary BMT recipients of EGFP or Fbxw7α virus-transduced cells after 1 week of culture at the indicated times after BMT (mean ± SEM, n = 4 or 5). *P < .05. (H) Differentiation status (CD3+ or CD4/8+ T cells, B220+ B cells, or Mac-1/Gr-1+ myeloid cells) of PB in primary BMT recipients of cultured EGFP or Fbxw7α virus-transduced cells at 4 months after BMT. (I) Donor-derived (Ly5.1+) chimerism in BM mononuclear cells, Lin−, LSK, or CD34− LSK fractions in primary BMT recipients of cultured EGFP or Fbxw7α virus-transduced cells at 4 months after BMT (mean ± SD, n = 4 or 5). *P < .05.$

Repopulation activity of Fbxw7α-transduced HSCs after ex vivo culture. (A) In vitro colony formation assays of EGFP or Fbxw7α virus-transduced EGFP⁺ cells. Colonies were counted and classified as granulocyte, erythroid, monocyte, and megakaryocyte (GEMM), granulocyte and monocyte (GM), or erythroid (E) on day 9 of culture (mean ± SD, n = 3). (B) Number of highly proliferative colony-forming EGFP or Fbxw7α virus-transduced EGFP⁺ cells on day 14 of culture (mean ± SD, n = 3). *P < .005. (C) CFU-S₁₂ assay. EGFP or Fbxw7α virus-transduced EGFP⁺ cells were transplanted into 8.5-Gy irradiated recipient mice. Spleens were harvested after 12 days, fixed with Bounin solution, and CFU-S₁₂ colonies were counted (mean ± SD, n = 5). (D) Study design for transplantation experiment. EGFP virus or Fbxw7α virus was transduced into LSK cells (CD45.1⁺). After 48 hours, EGFP⁺ cells were sorted and cultured for 1 week (18 000 LSK cells/well). Cultured cells were transplanted into lethally irradiated recipient mice (CD45.2⁺) with 4 × 10⁵ CD45.2⁺ competitors. PB chimerism was analyzed monthly, and BM chimerism was analyzed at 4 months after transplantation. Donor-derived LSK cells were used for serial transplantation after 4 months. (E) Relative numbers of total, Lin⁻, LSK, and CD150⁺ LSK EGFP or Fbxw7α virus-transduced EGFP⁺ cells after 1 week of culture. Cell number was analyzed by flow cytometry (mean ± SD, n = 6 for EGFP, n = 2 for Fbxw7). (F) Quantitative PCR analysis of various cell cycle–related genes in EGFP or Fbxw7α virus-transduced LSK cells after 1 week of culture (n = 4). (G) PB chimerism in primary BMT recipients of EGFP or Fbxw7α virus-transduced cells after 1 week of culture at the indicated times after BMT (mean ± SEM, n = 4 or 5). *P < .05. (H) Differentiation status (CD3⁺ or CD4/8⁺ T cells, B220⁺ B cells, or Mac-1/Gr-1⁺ myeloid cells) of PB in primary BMT recipients of cultured EGFP or Fbxw7α virus-transduced cells at 4 months after BMT. (I) Donor-derived (Ly5.1⁺) chimerism in BM mononuclear cells, Lin⁻, LSK, or CD34⁻ LSK fractions in primary BMT recipients of cultured EGFP or Fbxw7α virus-transduced cells at 4 months after BMT (mean ± SD, n = 4 or 5). *P < .05.

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