Figure 2.
Molecular analysis of HSPCs and LSK cells on the population and single-cell level. (A) Workflow generation population RNA-seq data. (B) Principal component analysis. (C) Relative distances are shown based on numbers of DEGs in pairwise comparisons. (D) Gene-expression clusters. DEGs were grouped into 6 clusters based on higher expression (enriched) in the respective cell population compared with mean expression level across all cell populations. Number of genes in the respective gene cluster is shown. (E) Hallmark-term analysis of gene sets enriched in MPP5 compared with HSCs, HSCs compared with MPP1 and MPP1 compared with MPP5. Normalized enrichment score (NES) for significant pathways (adjusted P [padj] < .05) is shown. (F) Workflow generation scRNAseq data. (G) Uniform manifold approximation and projection (UMAP) projection. Clusters are color-coded based on enrichment scores, and clusters were associated to the respective gene signatures identified in the population RNA-seq analysis (based on panel H). (H) Expression of HSPC gene signatures in the respective clusters. Red circles indicate maximal enrichment ± 0.02 considering the signature scoring. Analysis of maximal enrichment was used to assign colors and MPP identity to the respective clusters. (I) UMAP projection. Coloring is derived from the cell-cycle status. (J) UMAP projection. Trajectory analysis is depicted. (K) Pseudotime analysis. Zoom in in gray box shows analysis for selected clusters 0, 1, 2, 4, and 6. (L) Workflow in vitro barcoding and single-cell (sc) profiling,27 Clone label transference within HSPC clusters between 2 and 4 days of in vitro culture was analyzed based on the respective gene signatures identified in the population RNA-seq data. (M) Workflow in vitro barcoding and in vitro/in vivo single-cell profiling.27 Fate probability matrix is shown. HSPC identity of clones (2 days) was identified based on population RNA-seq gene signatures and lineages were assigned based on marker expression (2-3 weeks after transplantation). See also supplemental Figures 3 and 4. DC, dendritic cell; FACS, fluorescence-activated cell sorting; Mk/Ery, megakaryocyte/erythrocyte; Mono, monocyte; Neu, neutrophil; QC, quality control.

Molecular analysis of HSPCs and LSK cells on the population and single-cell level. (A) Workflow generation population RNA-seq data. (B) Principal component analysis. (C) Relative distances are shown based on numbers of DEGs in pairwise comparisons. (D) Gene-expression clusters. DEGs were grouped into 6 clusters based on higher expression (enriched) in the respective cell population compared with mean expression level across all cell populations. Number of genes in the respective gene cluster is shown. (E) Hallmark-term analysis of gene sets enriched in MPP5 compared with HSCs, HSCs compared with MPP1 and MPP1 compared with MPP5. Normalized enrichment score (NES) for significant pathways (adjusted P [padj] < .05) is shown. (F) Workflow generation scRNAseq data. (G) Uniform manifold approximation and projection (UMAP) projection. Clusters are color-coded based on enrichment scores, and clusters were associated to the respective gene signatures identified in the population RNA-seq analysis (based on panel H). (H) Expression of HSPC gene signatures in the respective clusters. Red circles indicate maximal enrichment ± 0.02 considering the signature scoring. Analysis of maximal enrichment was used to assign colors and MPP identity to the respective clusters. (I) UMAP projection. Coloring is derived from the cell-cycle status. (J) UMAP projection. Trajectory analysis is depicted. (K) Pseudotime analysis. Zoom in in gray box shows analysis for selected clusters 0, 1, 2, 4, and 6. (L) Workflow in vitro barcoding and single-cell (sc) profiling,27  Clone label transference within HSPC clusters between 2 and 4 days of in vitro culture was analyzed based on the respective gene signatures identified in the population RNA-seq data. (M) Workflow in vitro barcoding and in vitro/in vivo single-cell profiling.27  Fate probability matrix is shown. HSPC identity of clones (2 days) was identified based on population RNA-seq gene signatures and lineages were assigned based on marker expression (2-3 weeks after transplantation). See also supplemental Figures 3 and 4. DC, dendritic cell; FACS, fluorescence-activated cell sorting; Mk/Ery, megakaryocyte/erythrocyte; Mono, monocyte; Neu, neutrophil; QC, quality control.

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