Single-cell transcriptomics identifies the molecular consequences of low O2 concentration on HSC cultures. (A) Schematic of the scRNA-seq analysis of hematopoietic stem and progenitor cells (HSPCs) cultured at different O2 concentrations. (B) UMAP projections of all samples with color-coded cluster memberships. (C) Manual annotation of the clusters in panel B based on marker gene expression. (D) Uniform Manifold Approximation and Projection (UMAP) of all cells (gray) and cells from the indicated conditions (blue). In each case, an equal cell number was randomly selected for each sample. (E) Bar plot indicating relative cell abundance in the landscape areas for each sample. Areas were chosen as follows: HSC, cluster 1; Intermediate prog, clusters 0 and 2 to 4; Ery/Bas/MC/Meg prog, clusters 6, 7, and 17; Neu/Mono/DC, clusters 5, 11, and 14; and other, remaining clusters. (F) UMAP projection color-coded with diffusion pseudotime values, overlaid with arrows indicating putative paths of differentiation using random walks estimation with CellRank Pseudotime Kernel. (G) UMAP projection color-coded by the cell fate probability of cells differentiating into the tip of cluster 17 (megakaryocytes). (H) Cell density along the trajectory shown in panel G for each sample and pseudotime values between 0 and 0.05 are shown. Vertical lines indicate the regions of interest where cells at 5% and 1% O2 disappear. (I) Heatmap of genes differentially expressed between the beginning and end of the region of interest shown in panel H. (J) Enrichr gene enrichment analysis of upregulated genes within cluster 1 at 5% O2, compared with 20% (left) and 1% O2 (right). ∗ in panel E indicates a statistically significant change in cell abundance (FDR < 0.05) compared with the 20% O2 condition. Bas, basophil; Ery, erythroid; ILC, innate lymphoid cell; Ly/DC, lymphoid/dendritic cell; meg, megakaryocyte; MC, mast cell; monotyp, typical monocyte; neu, neutrophil; prog, progenitor.
