Fig. 4: TMIGD2 enriches for functional LSCs.

a Schematic for flow cytometry sorting and subsequent CFC assay and in vivo xenotransplantation. The highest (TMIGD2⁺) and lowest (TMIGD2^-) TMIGD2-expressing blasts (top and bottom 15%, respectively) from the CD34⁺CD38^-, CD34⁺CD38⁺ and CD34⁻ AML specimens were FACS-purified and used for CFC assays, xenotransplantation and RNA-seq analysis. b Representative photo of CFC plates seeded with FACS-purified TMIGD2⁺ and corresponding TMIGD2^- AML patient cells with respect to colony formation after 12 days culture. Left, Pt#31; right, Pt#21. c First round (left) and second round (right) CFC assays using FACS-purified TMIGD2⁺ or TMIGD2^- cells of CD34⁺CD38⁻ (Pt#27 and Pt#31), CD34⁺CD38⁺ (Pt#40), CD34^- (Pt#21) and Lin^- (Pt#24 and Pt#28) subpopulations from individual AML patients. d Limiting dilution assay. Table (left) showing different numbers of FACS-purified cells and recipient NSG mice used for the xenotransplantation. Graph (right) showing the frequency of LSC in TMIGD2⁺ and TMIGD2⁻ subpopulations. The limiting dilution analysis was performed using ELDA (Extreme Limiting Dilution Analysis) software. e The volcano plot showing differentially expressed genes from RNA-seq analysis on CD34⁺TMIGD2⁺ and CD34⁺TMIGD2^- cells sorted from six primary AML patient samples. f Scattergrams of the top pathways that were significantly enriched in CD34⁺TMIGD2⁺ cells based on GSEA. g GSEA plots showing enrichment of gene sets for E2F targets, Myc targets and G2M checkpoints in CD34⁺TMIGD2⁺ versus CD34⁺TMIGD2^- groups from six primary AML patient samples. h GSEA plots showing the stemness signatures were enriched in CD34⁺TMIGD2⁺ cells. i CFC assay was performed with FACS-purified TMIGD2⁺ or TMIGD2^- cells from normal CD34⁺ HSPCs. j Percentages of hCD45⁺, hCD33⁺ and hCD19⁺ cells in the BM of NSG-recipient mice at 3 months post transplantation of FACS-purified TMIGD2⁺ or TMIGD2^- cells from CD34⁺ HSPCs. Mean ± SEM values are shown for Fig. 4c, i, j. ns by two-tailed Student’s t test. Normalized enrichment score (NES) and false discovery rate (FDR) are shown for Fig. 4g, h. Color dots in c and i represent technical replicates. Color dots in j represent individual mice. Results are representative of three independent experiments. Source data are provided in the Source Data file.