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. 2023 Dec 12;5(2):114–131. doi: 10.1158/2643-3230.BCD-23-0195

Figure 2.

Figure 2. Combined single-cell RNA sequencing and genotyping reveal distinct transcriptomic profiles of IDH1-mutant pHSCs in AML. A, Schematic overview of the single-cell RNA sequencing workflow with specific amplification of mRNA and gDNA for parallel genotyping of IDH1 mutations using ddPCR. Ficoll separated mononuclear cells from two patients with AML bone marrow aspirates at the time of diagnosis were FACS isolated into 96-well plates containing lysis buffer. The mixed population of HSCs and pHSCs was defined as CD19−CD20−CD34+CD38lowCD99−TIM3− and separated based on subsequent genotyping, the LSC population was defined as CD19−CD20−CD34+CD38lowCD99+TIM3+. B, Identification of pHSCs and HSC based on single-cell IDH1 genotyping by ddPCR, a total of 356 single cells were genotyped. C, UMAP of transcriptomic single-cell data from sorted LSC and rHSC form five Leiden clusters. D, UMAP of transcriptomic single-cell data within the CD19−CD20−CD34+CD38low compartment, annotated as HSC (CD99−TIM3−IDH1wt), pHSC (CD99−TIM3−IDH1R132H), and LSC (CD99+TIM3+) based on immunophenotype and genotype. E, Cell state frequencies in the five Leiden clusters. F, Top differentially expressed genes in the three cell types. G, Three of the most overexpressed genes in pHSCs compared with HSCs. H, Three of the most downregulated genes related to MHC class II antigen presentation on pHSCs compared with HSCs. Mut, mutant; UMAP, Uniform Manifold Approximation and Projection for Dimension Reduction.

Combined single-cell RNA sequencing and genotyping reveal distinct transcriptomic profiles of IDH1-mutant pHSCs in AML. A, Schematic overview of the single-cell RNA sequencing workflow with specific amplification of mRNA and gDNA for parallel genotyping of IDH1 mutations using ddPCR. Ficoll separated mononuclear cells from two patients with AML bone marrow aspirates at the time of diagnosis were FACS isolated into 96-well plates containing lysis buffer. The mixed population of HSCs and pHSCs was defined as CD19−CD20−CD34+CD38lowCD99−TIM3− and separated based on subsequent genotyping, the LSC population was defined as CD19−CD20−CD34+CD38lowCD99+TIM3+. B, Identification of pHSCs and HSC based on single-cell IDH1 genotyping by ddPCR, a total of 356 single cells were genotyped. C, UMAP of transcriptomic single-cell data from sorted LSC and rHSC form five Leiden clusters. D, UMAP of transcriptomic single-cell data within the CD19−CD20−CD34+CD38low compartment, annotated as HSC (CD99−TIM3−IDH1wt), pHSC (CD99−TIM3−IDH1R132H), and LSC (CD99+TIM3+) based on immunophenotype and genotype. E, Cell state frequencies in the five Leiden clusters. F, Top differentially expressed genes in the three cell types. G, Three of the most overexpressed genes in pHSCs compared with HSCs. H, Three of the most downregulated genes related to MHC class II antigen presentation on pHSCs compared with HSCs. Mut, mutant; UMAP, Uniform Manifold Approximation and Projection for Dimension Reduction.