H3.3^K27Mexpression in human HSPCs leads to expansion of phenotypically immature cells through inhibition of H3K27me2/3. (A) Lentiviral expression of H3.3^KM mutants induces specific reductions in H3 methylation in cultured human HSPCs. After 48 hours in culture, human umbilical cord blood (UCB)–derived CD34⁺ cells were infected with lentiviruses expressing the indicated H3.3 variants and IRES GFP under control of the EF1a promoter. After 10 days of total culture, GFP⁺ cells were FACS sorted, lysed in Laemmli buffer, and subjected to western analysis at 10⁵ cells per lane. Decreased H3K27me2, H3K27me3, or H3K9me3 was detected upon expression of H3^K27M or H3^K9M, respectively. (B) Phenotypic characterization of H3.3^KM mutant–expressing human HSPCs. UCB-derived CD34⁺ cells were infected and cultured for 10 days as in panel A, stained with the indicated antibodies, and FACS analyzed. Differences in the CD34⁺/CD45RA⁻ population (top panels) and CD34⁺/CD45RA⁻/CD90⁺/CD133^-− or CD34⁺/CD45RA⁻/CD90⁺/CD133⁺ populations (bottom) within infected (GFP⁺) cells were assessed. Percentages of each gate are indicated. (C) Summary of phenotypic changes in HSPCs expressing H3.3^K27M. Population frequencies were calculated based on analysis in panel B. Quadruplicate infections/cultures of CD34⁺ cells expressing H3.3^K27M revealed a prominent expansion of CD34⁺CD45RA⁻CD90⁺CD133⁻ cells in H3.3^K27M-infected HSPCs. (D) Addition of EZH1/2 inhibitors (GSK126 and UNC1999; 1 µM) leads to no increased expansion of the CD34⁺/CD45RA⁻/CD90⁺/CD133⁻ population in H3.3^KM-expressing cells compared with controls (DMSO; 10⁻⁴ and UNC2400; 1 µM). Cells were analyzed at day 10 postinfection and EZH1/2 inhibitor treatment. All analyses were restricted to the transduced (GFP⁺) subsets. (E-F) Assessment of myeloid progenitor activity in response to H3.3^K27M expression in HSPCs. The experimental strategy is depicted in panel E. GFP⁺ cells were FACS isolated and plated in methylcellulose media supporting erythromyeloid differentiation. Colonies were morphologically assessed and scored after 14 days. Granulo/erythoid/monocytic/megakaryocytic colony-forming units (CFU-GEMM) and granulocytic/granulomonocytic colony-forming units (CFU-G/GM) were scored. Combined results from 2 independent experiments are presented in panel F. ***P < .001, ****P < .0001; 2-way analysis of variance statistics with Tukey adjustment. (G) Experimental strategy for transplantation of HSPCs expressing H3.3^KM mutants. Cells were infected with lentiviruses as in panel A and cultured for 10 days in the presence of UM171. Progenies of 10⁴ CD34⁺ UCB cells were transplanted into NSG recipients (8 mice per condition). (H) Summary of human hematopoietic stem-cell engraftment from the experiment depicted in panel G. Bone marrow chimerism of NSG mice receiving transplants was determined by FACS analysis based on GFP and human CD45 surface expression at the specified posttransplantation time points. Individual mouse identifiers are indicated at the bottom of the graph. (I) Lineage contribution of H3.3^KM-expressing HSPCs in transplant-recipient NSG mice. Presented data refer to the distribution of myeloid (CD33), B-lymphoid (CD19), and immature (CD34) surface phenotypes within the GFP⁺ compartments. CFC, colony-forming cell; n.s., not significant.