Figure 3.
Comparative transcriptomic analysis of lung and BM HSCs reveals shared and unique gene expression profiles. (A) Uniform manifold approximation and projection (UMAP) projection of BM and lung Lin− CD34+ progenitor hierarchy from 8 human donors highlighting the HSC/MPP cluster (purple). The pie graph indicates the proportion of cells from the BM (blue) and lung (red) within the MP subset. (B) Grouping of gene expression patterns into modules using Monocle3. Aggregate expression values of genes in the module highly specific for HSCs (supplemental Figure 7) are shown individually for the BM and lung. (C) Pseudotime calculation for each cell within the BM and lung using Monocle3 to infer progression through different cellular differentiation to provide insights into the developmental trajectory. (D) Scatterplot of median gene expression of cells in the HSC/MPP cluster from the lung (red) and BM (blue) to visualize consistent (gray) and differentially (highlighted) expressed genes. (E) Venn diagram and top 10 differentially expressed genes. The number in each circle represents the amount of differentially expressed genes between lung (red) and BM (blue), and the overlapping number indicates mutual differentially expressed genes based on the Wilcoxon rank-sum test in Seurat’s “FindMarkers” function. (F) Box and violin plots showing the distribution of selected genes upregulated in pulmonary HSCs. Wilcoxon adjusted P < .001. (G) Selection of marker genes shared between lung and BM as box and violin plots, respectively. (H) Box and violin plots showing the distribution of markers genes upregulated in BM HSCs, Wilcoxon adjusted P < .001. (I) T.statistic of single-sample gene set enrichment analysis (ssGSEA) scores for selected gene sets (Hallmark, Reactome, Biocarta, KEGG) enriched in pulmonary HSCs categorized by recurring functions. (J) Enrichment ridge plots comparing the distribution of enrichment scores in HSCs from lung (red) and BM (blue) of selected Reactome pathways. Rug plots indicate the scores of individual cells along the ridge plot. P values are given in the figure, FDR R-HSA-9027277 = 2.38 × 10−4; FDR R-HSA-9006335 = 0.09; FDR R-HSA-8936459 = 0.03; R-HSA-76002 = 2.03 × 10−10. (K) Enrichment ridge plots showing the distribution of enrichment scores in lung (red) and BM (blue) with individual cell placement on the rug plot to compare selected Gene Ontology Biological Process gene set enrichments. P values are given in the figure, FDR GO:00025 = 1.77 × 10−6; FDR GO:0001816 = 2.42 × 10−7; FDR GO:0006955 = 6.70 × 10−7; GO:0050729 = 2.02 × 10−8. earlyEry, early erythroid progenitor; ECM, extracellular matrix; EMP, erythroid megakaryocytic progenitor; Eo/Ba/Ma, eosinophil/basophil/mast cell progenitor; FDR, false discovery rate; GFR, growth factor receptor; lateEry, late erythroid progenitor; MultiLin, multilineage; My, myeloid cell; nd, not determined; ns, not significant; prog/stroma mix, progenitor stroma cell mix.

Comparative transcriptomic analysis of lung and BM HSCs reveals shared and unique gene expression profiles. (A) Uniform manifold approximation and projection (UMAP) projection of BM and lung Lin− CD34+ progenitor hierarchy from 8 human donors highlighting the HSC/MPP cluster (purple). The pie graph indicates the proportion of cells from the BM (blue) and lung (red) within the MP subset. (B) Grouping of gene expression patterns into modules using Monocle3. Aggregate expression values of genes in the module highly specific for HSCs (supplemental Figure 7) are shown individually for the BM and lung. (C) Pseudotime calculation for each cell within the BM and lung using Monocle3 to infer progression through different cellular differentiation to provide insights into the developmental trajectory. (D) Scatterplot of median gene expression of cells in the HSC/MPP cluster from the lung (red) and BM (blue) to visualize consistent (gray) and differentially (highlighted) expressed genes. (E) Venn diagram and top 10 differentially expressed genes. The number in each circle represents the amount of differentially expressed genes between lung (red) and BM (blue), and the overlapping number indicates mutual differentially expressed genes based on the Wilcoxon rank-sum test in Seurat’s “FindMarkers” function. (F) Box and violin plots showing the distribution of selected genes upregulated in pulmonary HSCs. Wilcoxon adjusted P < .001. (G) Selection of marker genes shared between lung and BM as box and violin plots, respectively. (H) Box and violin plots showing the distribution of markers genes upregulated in BM HSCs, Wilcoxon adjusted P < .001. (I) T.statistic of single-sample gene set enrichment analysis (ssGSEA) scores for selected gene sets (Hallmark, Reactome, Biocarta, KEGG) enriched in pulmonary HSCs categorized by recurring functions. (J) Enrichment ridge plots comparing the distribution of enrichment scores in HSCs from lung (red) and BM (blue) of selected Reactome pathways. Rug plots indicate the scores of individual cells along the ridge plot. P values are given in the figure, FDR R-HSA-9027277 = 2.38 × 10−4; FDR R-HSA-9006335 = 0.09; FDR R-HSA-8936459 = 0.03; R-HSA-76002 = 2.03 × 10−10. (K) Enrichment ridge plots showing the distribution of enrichment scores in lung (red) and BM (blue) with individual cell placement on the rug plot to compare selected Gene Ontology Biological Process gene set enrichments. P values are given in the figure, FDR GO:00025 = 1.77 × 10−6; FDR GO:0001816 = 2.42 × 10−7; FDR GO:0006955 = 6.70 × 10−7; GO:0050729 = 2.02 × 10−8. earlyEry, early erythroid progenitor; ECM, extracellular matrix; EMP, erythroid megakaryocytic progenitor; Eo/Ba/Ma, eosinophil/basophil/mast cell progenitor; FDR, false discovery rate; GFR, growth factor receptor; lateEry, late erythroid progenitor; MultiLin, multilineage; My, myeloid cell; nd, not determined; ns, not significant; prog/stroma mix, progenitor stroma cell mix.

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