Single Cell Transcriptomic Analysis Reveals Organ Specific Pericyte Markers and Identities

Abstract

Pericytes are mesenchymal-derived mural cells that wrap around capillaries and directly contact endothelial cells. Present throughout the body, including the cardiovascular system, pericytes are proposed to have multipotent cell-like properties and are involved in numerous biological processes, including regulation of vascular development, maturation, permeability, and homeostasis. Despite their physiological importance, the functional heterogeneity, differentiation process, and pathological roles of pericytes are not yet clearly understood, in part due to the inability to reliably distinguish them from other mural cell populations. Our study focused on identifying pericyte-specific markers by analyzing single-cell RNA sequencing data from tissue-specific mouse pericyte populations generated by the Tabula Muris Senis. We identified the mural cell cluster in murine lung, heart, kidney, and bladder that expressed either of two known pericyte markers, Cspg4 or Pdgfrb. We further defined pericytes as those cells that co-expressed both markers within this cluster. Single-cell differential expression gene analysis compared this subset with other clusters that identified potential pericyte marker candidates, including Kcnk3 (in the lung); Rgs4 (in the heart); Myh11 and Kcna5 (in the kidney); Pcp4l1 (in the bladder); and Higd1b (in lung and heart). In addition, we identified novel markers of tissue-specific pericytes and signaling pathways that may be involved in maintaining their identity. Moreover, the identified markers were further validated in Human Lung Cell Atlas and human heart single-cell RNAseq databases. Intriguingly, we found that markers of heart and lung pericytes in mice were conserved in human heart and lung pericytes. In this study, we, for the first time, identified specific pericyte markers among lung, heart, kidney, and bladder and reveal differentially expressed genes and functional relationships between mural cells.

Keywords: heart; lung; pericytes; single cell RNA sequencing; tissue-specific.

Figure 1

Pericyte clusters are identified from mouse lung, heart, kidney, and bladder. (A) The Tabula Muris Senis dataset of the lung, heart, kidney, and bladder were used to identify pericytes within the cell population of each tissue. Graph-based unsupervised clustering identified 22, 17, 27, and 15 clusters for the lung, heart, kidney, and bladder tissue, respectively. (B) Pericyte-enriched clusters for each tissue were identified based on the expression of previously accepted pericyte markers Cspg4 and Pdgfrb. Cells that co-expressed Cspg4 and Pdgfrb within the identified pericyte-enriched clusters were stringent pericytes (shown in red).

Figure 2

Specific pericyte markers in four organs are identified. Potential pericyte markers for each tissue. Potential pericyte markers were selected based on the differential expression (DE) analysis using the Wilcoxon rank sum test to compare the expression levels of transcripts in the tissue-specific stringent pericytes vs. all other cells within the corresponding tissue. Pericyte-enriched transcripts with an adjusted p-value below 0.05 were selected. The subset of these transcripts detected in more than 80% of the stringent pericytes and in <5% in all other cells were then chosen as potential pericyte markers for each tissue. pct3 represents the maximum percentage found across the clusters, excluding the pericyte-enriched cluster(s). Markers with pct3 below 10% are shown with an asterisk (*).

Figure 3

Pathway enrichment analysis of pericytes in four organs are shown. Pre-ranked gene set enrichment analysis (GSEA) with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database was conducted on the DE genes between the tissue-specific stringent pericytes and all other cells of its corresponding tissue. The DE genes were sorted based on their average log2(Fold Change) in a descending order to generate the pre-ranked gene list. Enriched pathways were identified for each tissue-specific stringent pericytes based on their adjusted q-value (q < 0.05). The identified enriched pathways of the tissue-specific stringent pericytes were compared across four tissue types.

Figure 4

The identified murine lung and pericyte markers are tested on human lung and heart scRNAseq data. The expression of human orthologs of the potential pericyte markers identified from the stringent pericytes of the mouse lung and heart tissue were tested against the (A) Human Lung Cell Atlas and the (B) collection of the cells of the adult human heart to validate whether they can be used to annotate pericytes in the human lung and heart tissue. Purple (fond color) indicated the annotated pericytes of the two human datasets. The Human Lung Cell Atlas annotated pericytes based on CSPG4, PDGFRB, and TRPC6, while the human heart pericytes were annotated based on RGS5, ABCC9, and KCNJ8. Light blue (fond color) genes indicated the human orthologs of the top 5 potential pericyte markers identified from the mouse lung. Pink (fond color) genes indicated the human orthologs of all the potential pericyte markers identified from the mouse heart.

Figure 5

Pericyte transcripts locate on capillaries in human heart tissue by spatial transcriptomic analysis. (A) The spatial transcriptomic data of the human heart tissue from the Spatial Gene Expression Dataset by 10 × Genomics Space Ranger (10 × Genomics Space Ranger 1.1.0) was used to investigate the pericyte spatial localization within the heart tissue and compare it with regions of the capillaries and the arteries. (B,C) Based on the expression of CSPG4 and PDGFRB, spots of clusters 1/6 were designated as pericyte spots (shown as red dots). Dark blue dots indicate spots that express HIGD1B. Cyan dots indicate spots that express NOTCH3. Sky blue dots indicate spots that co-express HIGD1B and NOTCH3 among the pericyte spots. Spots that expressed MYH11 were identified as smooth muscle coverage of arteries (shown as orange dots). Spots that expressed PECAM1 were identified as endothelial cells and capillary lumens (shown as dark pink dots).