Figure 2.
The dynamics of gene expression over the first division of LT-HSC ex vivo at single-cell resolution. (A) Uniform manifold approximation and projection (UMAP) of 429 single EXPER_CB LT-HSCs over a time course of 0, 6, 24, and 72 hours (n = 2 independent experiments). The UMAP was generated using the Seurat 4 pipeline after cell cycle regression. (B) A 2-dimensional pseudotime rank plot of EXPER_CB LT-HSCs over the time course generated after cell cycle regression. (C) Transcriptional allocation of cell cycle status at different time points during EXPER_CB LT-HSC culture; n = 429 single cells. (D) The number of differentially expressed genes (false discovery rate < 0.05) at each time point in comparison with the 0 hours time point. Upregulated genes are shown in blue, and downregulated genes are shown in brown. A full list of genes is available in supplemental Table 4. (E) The broad patterns of gene expression that were identified over the time course (8966 genes classified after filtering by the DEG report algorithm). Numbers indicate the percentage of genes that showed the specific patterns of gene expression displayed to the right of the bar. (F-J) GSVA score of c2 curated pathways showing the following specific expression patterns: (F) continuous up; (G) continuous down; (H) transient up; (I) transient down; and (J) up later than 6 hours. The GSVA score that was calculated per single cell with a line at the median and upper and lower whiskers indicating the 25th and 75th percentile of expression. (K) The GSVA scores of indicated published gene signatures, representative of specific HSCs and progenitor cell (HSPC) subsets. The median and interquartile range are shown. ∗P < .001. (L) The scEntropy value at each time point is shown (calculated for both batches combined; Wilcoxon rank sum test shown; 0 vs 6 hours; P = .835). The median and interquartile range are shown. ∗P < .001. (M) Shows the number of maximally variable genes (MVGs) at each time point (supplemental Methods; 2792 genes total). (N) Shows selected biological pathways that are significantly enriched from MVGs (–log10[adjusted P value] < .05). A full list of the pathways available in supplemental Table 7. CMP, common myeloid progenitor; GMP, granulocyte monocyte progenitor; MEP, myeloid erythroid progenitor.

The dynamics of gene expression over the first division of LT-HSC ex vivo at single-cell resolution. (A) Uniform manifold approximation and projection (UMAP) of 429 single EXPER_CB LT-HSCs over a time course of 0, 6, 24, and 72 hours (n = 2 independent experiments). The UMAP was generated using the Seurat 4 pipeline after cell cycle regression. (B) A 2-dimensional pseudotime rank plot of EXPER_CB LT-HSCs over the time course generated after cell cycle regression. (C) Transcriptional allocation of cell cycle status at different time points during EXPER_CB LT-HSC culture; n = 429 single cells. (D) The number of differentially expressed genes (false discovery rate < 0.05) at each time point in comparison with the 0 hours time point. Upregulated genes are shown in blue, and downregulated genes are shown in brown. A full list of genes is available in supplemental Table 4. (E) The broad patterns of gene expression that were identified over the time course (8966 genes classified after filtering by the DEG report algorithm). Numbers indicate the percentage of genes that showed the specific patterns of gene expression displayed to the right of the bar. (F-J) GSVA score of c2 curated pathways showing the following specific expression patterns: (F) continuous up; (G) continuous down; (H) transient up; (I) transient down; and (J) up later than 6 hours. The GSVA score that was calculated per single cell with a line at the median and upper and lower whiskers indicating the 25th and 75th percentile of expression. (K) The GSVA scores of indicated published gene signatures, representative of specific HSCs and progenitor cell (HSPC) subsets. The median and interquartile range are shown. ∗P < .001. (L) The scEntropy value at each time point is shown (calculated for both batches combined; Wilcoxon rank sum test shown; 0 vs 6 hours; P = .835). The median and interquartile range are shown. ∗P < .001. (M) Shows the number of maximally variable genes (MVGs) at each time point (supplemental Methods; 2792 genes total). (N) Shows selected biological pathways that are significantly enriched from MVGs (–log10[adjusted P value] < .05). A full list of the pathways available in supplemental Table 7. CMP, common myeloid progenitor; GMP, granulocyte monocyte progenitor; MEP, myeloid erythroid progenitor.

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