Enforced expression of Kdm2b/Jhdm1b is sufficient for BM transformation. (A) Enforced expression of Kdm2b/Jhdm1b is capable of immortalizing BM cells in vitro. Shown are colony numbers of c-kit⁺ HPCs transduced with lentiviral vectors expressing GFP, Kdm2b/Jhdm1b, or Hoxa9-Meis1 at each round of methylcellulose replating. (B) Growth curve of c-kit⁺ hematopoietic progenitors transduced with lentiviral vectors expressing GFP, Kdm2b/Jhdm1b, or Hoxa9-Meis1 in suspension culture. (C) FACS analysis of the genetically modified Ly5.2⁺ donor cells, LacZ and Jhdm1b overexpression (Jhdm1b OE) in the peripheral blood of recipients. The results show that the percentage of Kdm2b/Jhdm1b-overexpressing Ly5.2⁺ cells increases gradually after transplantation. (D) Methylcellulose replating assay shows demonstrates that Kdm2b/Jhdm1b-overexpressing BM cells isolated from recipient mice can form increased numbers of colonies. In contrast, mock-transplanted BM cells fail to grow continuously in the methylcellulose replating assay. Colony numbers for each round of replating are shown. (E) Flow cytometric analysis shows that Kdm2b/Jhdm1b-overexpressed colonies express a high level of c-kit and low levels of myeloid lineage markers Mac-1/Gr-1. (F) Splenomegaly is observed in mice that received a transplant with Kdm2b/Jhdm1b-overexpressing BM cells. Shown is a representative picture of spleens harvested from mice 6 weeks after transplantation of BM cells transduced with lentiviral vectors expressing LacZ or Kdm2b/Jhdm1b. Bar size represents 1.0 cm. (G) May-Grünwald/Giemsa staining showing typical leukoblasts in the BM of mice that received a transplant with Kdm2b/Jhdm1b-overexpressing BM cells. Bar size represents 10 μm. (H) Survival curve shows mice that received a transplant with mock cells survived ≥ 210 days after transplantation, whereas most mice that received a transplant with Kdm2b/Jhdm1b-overexpressing cells died within 120-180 days after transplantation.