Fig 1: SEC-seq measuring the transcriptome and VEGF-A secretion of normoxic or hypoxic MSCs.a, Schematic of the detection of secreted VEGF-A protein and corresponding global gene expression for individual MSCs using the SEC-seq method. b, UMAP dimensionality reduction based on transcriptomes from SEC-seq experiments on normoxic and hypoxic MSCs in nanovials. Cells are labeled according to the culture condition. c, UMAP displaying VEGF-A secretion level, shown as log transformation of the UMI collapsed anti-VEGF-A oligo-barcode reads per cell. d, UMAP displaying VEGFA transcript levels, shown as normalized transcripts per cell. e, Distribution of VEGF-A secretion for cell-loaded nanovials in normoxic and hypoxic conditions, detected by FACS using a fluorescent anti-VEGF-A antibody (top) or by the SEC-seq experiment in (b) using the oligo-barcoded anti-VEGF-A antibody (middle). The last plot shows distribution of VEGFA transcript levels from the SEC-seq experiment cells in (b) (bottom). f, UMAP displaying cluster assignment. g, Violin plots by cluster showing VEGFA transcripts and VEGF-A secretion levels for all cells in the normoxic clusters (N1-N5, red shades), hypoxic clusters (H1–4, blue shades), and mixed clusters (M1–3, green shades) from (f). For mixed clusters, the levels are shown separately for normoxic and hypoxic cells. The dashed line represents the mean across all cells for each plot. Data below this threshold are lightened to highlight differences. h, Scatter plots showing the relationship between VEGFA transcript and VEGF-A secretion levels for individual normoxic (left) and hypoxic (right) cells from the experiment in (b). Best fit regression lines and Pearson correlation coefficients are shown. i, Plot showing the ranking of all detected genes based on the correlation of their transcript levels to the VEGF-A secretion level per cell for normoxic (top) and hypoxic (bottom) MSCs. The rank of the VEGFA gene is highlighted, and the top three genes per sample are also noted.
Fig 2: Characterization of the high-VEGF-A secreting MSC subpopulation.a, Scatter plot of the transcript to VEGF-A secretion correlation for all genes from SEC-seq experiments for normoxic and hypoxic MSCs from Figure 3. The 10 most highly correlating genes based on both experiments are labeled. b, Table giving the ranking (based on average correlation), gene name, correlation to secretion in normoxic and hypoxic cells, and the average of those two values for the ten top genes from (a). c, UMAPs showing VEGF-A secretion levels and expression of 5 select correlated genes from (b) in normoxic and hypoxic MSCs from Figure 3. The VEGF-A secretion UMAP is given from Fig. 3c for comparison. d, As in (c), for a separate SEC-seq experiment performed on MSCs in the normoxic culture condition. e, Cell clusters projected onto the UMAP of the replicate SEC-seq experiment f, Heatmap of the top 10 differentially expressed genes from each cluster (indicated on top) of the SEC-seq experiment in (c,d) (rows=genes, columns=individual cells). Displayed at the top are the log transformed VEGF-A secretion and VEGFA transcript levels. Right: top 3 genes differentially expressed gene for each cluster. g, Heatmap of the top GO terms found for all of the differentially expressed genes from the clusters in (e). The (–logP) value indicates if the term was enriched for a given cluster. h, Venn diagram showing the overlap of differentially expressed genes from the highly secreting cluster in 3 SEC-seq experiments (top left: normoxic MSCs from (3b), top right: hypoxic MSCs from (3b), bottom: normoxic MSCs from (4e)). Overlapping genes form the Vascular Regenerative Signal (VRS). i, Gene ontology analysis for VRS genes from (h). Similar terms were collapsed. j, Average of the normalized transcripts level of VRS genes per cell, displayed for the SEC-seq experiment in (e) and (3b). k, As in (j), for MSCs loaded in oligo-barcoded nanovials (see Fig. 2j-l). l, As in (j), for a standard scRNA-seq experiment on unsorted, suspended MSCs. m, Comparison of gene type classification for VRS genes and genes differentially expressed in all clusters in (e) except for those from cluster c5. n, Enrichment of possible TF regulators of the VRS genes based on the TRRUST database. o, Consensus rank of VEGF-A secretion to gene correlation based the SEC-seq experiments used in (h), with red dots displaying all VRS genes. p, Schematic depicting the heterogeneity of VEGF-A secretion in MSCs under normoxic and hypoxic conditions, highlighting the importance of the VRS genes for marking high VEGF-A secretion.
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