Single-cell transcriptome profiling of an adult human cell atlas of 15 major organs

Abstract

Background: As core units of organ tissues, cells of various types play their harmonious rhythms to maintain the homeostasis of the human body. It is essential to identify the characteristics of cells in human organs and their regulatory networks for understanding the biological mechanisms related to health and disease. However, a systematic and comprehensive single-cell transcriptional profile across multiple organs of a normal human adult is missing.

Results: We perform single-cell transcriptomes of 84,363 cells derived from 15 tissue organs of one adult donor and generate an adult human cell atlas. The adult human cell atlas depicts 252 subtypes of cells, including major cell types such as T, B, myeloid, epithelial, and stromal cells, as well as novel COCH⁺ fibroblasts and FibSmo cells, each of which is distinguished by multiple marker genes and transcriptional profiles. These collectively contribute to the heterogeneity of major human organs. Moreover, T cell and B cell receptor repertoire comparisons and trajectory analyses reveal direct clonal sharing of T and B cells with various developmental states among different tissues. Furthermore, novel cell markers, transcription factors, and ligand-receptor pairs are identified with potential functional regulations in maintaining the homeostasis of human cells among tissues.

Conclusions: The adult human cell atlas reveals the inter- and intra-organ heterogeneity of cell characteristics and provides a useful resource in uncovering key events during the development of human diseases in the context of the heterogeneity of cells and organs.

Keywords: BCR; Human cell atlas; Single-cell RNA sequencing; TCR; Transcriptome.

Fig. 1

Overview of single-cell RNA sequencing of 15 organ tissues from a male adult donor. a An experiment schematic diagram highlighting the sites of the organs for tissue collection and sample processing. Live cells were collected using flow cytometry sorting (FACS) and subjected for cell barcoding. cDNA libraries for TCR, BCR, and 5′-mRNA expression were constructed independently, followed by high-throughput sequencing and downstream analyses. b t-SNE visualization of all cells (84,363) in organs. Each dot represents one cell, with colors coded according to the origin of organ. Labeled cell types are the predominant cell types in each cluster. c Dot plots showing the most highly expressed marker genes (x-axis) of major cell types (y-axis) in b. The depth of the color from white to blue and the size of the dot represent the average expression from low to high and the percent of cells expressing the gene

Fig. 2

The heterogeneity, development, and clonality of T cells in human organs. a, b t-SNE plots of 7006 CD4⁺ (a, 11 clusters) and 11,256 CD8⁺ (b, 21 clusters) T cells from 15 organ tissues. Each dot represents one cell. Each color-coded region represents one cell cluster, which is indicated on the right. c, d Violin plots showing the normalized expression of marker genes for each CD4⁺ (c) and CD8⁺ (d) T cell cluster as indicated at the bottom. For each panel, the y-axis shows the normalized expression level for a marker gene as indicated on the left. Marker genes were also grouped according to functional cell types. e Pseudo-time trajectory analysis of all CD4⁺ (left panel) and CD8⁺ T cells (right panel) with high variable genes. Each dot represents one cell and is colored according to their cluster above: a for CD4⁺ and b for CD8⁺. The inset t-SNE plot shows each cell with a pseudo-time score from dark blue to yellow, indicating early and terminal states, respectively. f, g Heat maps of the activation scores of each T cell cluster for expression regulated by transcription factors (TFs). T cell clusters are indicated on top, and the scores were estimated using SCENIC analysis. Only shows the top 15 TFs for CD4⁺ T cells (f) and the top 10 for CD8⁺ T cells (g), with the highest difference in expression regulation estimates between each cluster and all other cells, under a Wilcoxon rank-sum test. h Sharing intensity of TCR clones in CD4⁺ (top panel) and CD8⁺ (bottom panel) T cells between different organ samples. Each line represents a sharing of TCR between two organs at the ends, and the thickness of the line represents a migration-index score between paired organs calculated by STARTRAC. The sizes of the dots are shown according to the logarithm to the base 2 of the size of T cell clones in organs with different colors. i Migration- (left panel) and expansion-index (right panel) scores of CD4⁺ and CD8⁺ T cells of each tissue calculated and compared using STARTRAC with a paired Student’s t test

Fig. 3

The heterogeneity and clonality of B cells in human organs. a t-SNE plots showing 14 clusters (10,100 cells) of B and plasma cells. Each dot represents a cell, colored according to the origin of tissue (top panel) and cell subtype (bottom panel). b Distribution of B and plasma cells in each organ. Pie charts on top illustrate the proportions of B and plasma cells in each organ. The stacked bars represent the percentage of each cluster in the indicated organ. c Violin plots of the normalized expression of marker genes for B (MS4A1), plasma cells (SDC1), naïve B cell (TCL1A), and memory B cells (CD27). For each panel, the y-axis shows the normalized expression level for a marker gene as indicated on the title, and the x-axis indicates cell clusters. d Gene Ontology enrichment analysis results of B and plasma cell clusters. Cell clusters as indicated at the bottom are colored according to their −log₁₀P values in columns. Only the top 20 significant GO terms (P value < 0.05) are shown in rows. e Heat map of the activation scores of each B and plasma cell cluster for expression regulated by transcription factors (TFs). Cell clusters are indicated on top, and the scores were estimated using SCENIC analysis. It shows the top 10 TFs with the highest difference in expression regulation estimates between each cluster and all other cells, tested with a Wilcoxon rank-sum test. f Sharing intensity of BCR clones between different organs. Each line represents a sharing of BCR between two organs at the ends, and the thickness of the line represents a migration-index score between paired organs calculated using STARTRAC. The size of the dot is shown as the logarithm to the base 2 of the size of B and plasma cell clones in each organ. g Expansion- (top panel) and transition-index (bottom panel) scores of each B and plasma cell cluster calculated using STARTRAC

Fig. 4

Heterogeneity and developmental stages of myeloid cells. a t-SNE plots of 5587 myeloid cells. Each dot represents one cell, colored according to their tissue origins (top panel) or cell clusters (bottom panel) as indicated on the right. b t-SNE plots of the normalized expression of marker genes for monocytes (S100A8/9/12 and VCAN) and macrophages (pan-marker: C1QC, C1QB, and VSIG4), cDC1 (CLEC9A), cDC2 (FCER1A), and Langerhans (CD207) as well as subpopulation-specific genes (CD14 and FCGR3A). Each dot represents one cell, with a color from gray to blue representing the expression level from low to high. c Dendrogram of 18 clusters based on their normalized mean expression values (correlation distance metric, complete linkage). Only genes with ln(fold change) above 0.25, p.adjust < 0.05, and pct.1 ≥ 0.2 in each cluster were included in the calculations. d Heat map showing the expression profiles of each myeloid cell cluster as indicated on top. The expression of 640 genes in each cell cluster with FC ≥ 2 and p.adjust < 0.05 are shown as lines, colored from blue to red according to the expression from low to high. e Pseudo-time trajectory analysis of monocytes/macrophages with high variable genes. Each dot represents one cell and is colored according to their clustering in a. The inset t-SNE plot shows each cell with a pseudo-time score from dark blue to yellow, indicating early and terminal states, respectively. f Heat map of the activation scores of each monocyte and macrophage subtype for gene expression regulated by transcription factors (TFs). Cell clusters are indicated on top, and the scores were estimated using SCENIC analysis. Only the top 10 TFs are shown with the highest difference in expression regulation estimates between each cluster and all other cells, tested with a Wilcoxon rank-sum test. g Plots showing the normalized expression of representative TFs in f along the pseudo-time trajectory maps corresponding to e. Each dot in one plot shows the expression of the indicated gene in the plot, colored from gray to red, indicating low and high expression, respectively. SPIP1, HCFC1, and ELF2 for classical monocytes; enhanced expression of STAT1 and TCF7L2 for non-classical monocytes and SDC3_Mac (3); MAF, PRDM1, and EVT5 for most non-classical monocytes

Fig. 5

The heterogeneity of epithelial cells inter- and intra-organ tissues. a t-SNE plots of 17,436 epithelial cells. Each dot represents one cell, colored according to their origins of tissues (top panel) or cell clusters (bottom panel). b Dot plot visualizing the normalized expression of marker genes for each epithelial cluster. Cell cluster at y-axis was coded in numbers on the left, corresponding to that in a. Marker genes are shown at the x-axis. The size of the dot represents the percentage of cells with a cell type, and the color represents the average expression level. c Dendrogram of 34 clusters based on their normalized mean expression values (correlation distance metric, complete linkage). Only genes with fold change above 1.5, p.adjust < 0.05, and pct.1 ≥ 0.2 in each cluster were included in the analysis. d Volcano plot shows the DEGs between the 14 digestive and 20 non-digestive related clusters. Labeled genes are markers for each cluster in b. e, f Gene Ontology enrichment analysis results of each epithelial cell cluster in the digestive organs (e) and non-digestive organs (f). Cell clusters in columns are coded as numbers at the bottom, correspond to that in a, and are colored according to their −log₁₀P values, with white to red for low to high enrichment of a GO term in a row indicated on the right. Only the top 20 significant GO terms (P value < 0.05) are shown. g Heat map of the activation scores of epithelial cell subtypes for gene expression regulated by transcription factors (TFs). Cell clusters are indicated on top, and the scores were estimated using SCENIC analysis. Only the top 10 TFs are shown with the highest difference in expression regulation estimates between each cluster and all other cells, tested with a Wilcoxon rank-sum test. The cluster numbers are in reference to those in a. Cell clusters are grouped according to the origin of organ and their digestive or non-digestive function as indicated on top

Fig. 6

Intercellular communication networks among tissues. a-c The top 10 significant ligand-receptor interactions between cells among different organs for epithelial and myeloid cell subtypes (a), myeloid and CD8⁺ T cell subtypes (b), and CD8⁺ T and stromal cell subtypes (c; fibroblast, smooth muscle cell, and FibSmo cell). An interaction is indicated as color-filled circle at the cross of interacting cell types in a tissue (x-axis) and a ligand-receptor pair (y-axis), with circle size representing the significance of −log₁₀P values in a permutation test and colors representing the means of the average expression level of the interacting pair. The naming system is as follows: taking an example of “EGFR_TGFB1” in “cholang_FXYD2.Mac.Commonbileduct,” the ligand-receptor pair is EGFR (red) and TGFB1 (black), and the circle is colored based on the expression levels of EGFR in cholang_FXYD2 cluster and TFGB1 in Mac cluster in the tissue Commonbileduct. Mon, monocyte; Mac, macrophage; DC, dendritic cell; TRM, tissue-resident memory T cell; TEFF, effector T cell; TGD, γδ T cell; MAIT, mucosal-associated invariant T cell; TEM, effector memory T cell; TIEL, intraepithelial T lymphocyte; TN, naïve T cell; Fib, fibroblast; Smo, smooth muscle cell; FibSmo, novel cell type named FibSmo cell; Commonbileduct, common bile duct; Lymphnode, lymph node; Smallintestine, small intestine. For epithelial cells, the full names of each cluster refer to Table S47. d Connection graph showing the intensity of interactions between one organ to another in colored circles. Interactions were evaluated between major cell types including CD4⁺ T cell, CD8⁺ T cell, γδ T cell, B cell, plasma cell, myeloid cell, NK cell, epithelial cell, fibroblast, smooth muscle cell, FibSmo cell, and endothelial cell. Numbers in red show the total counts of ligand-receptor pairs between the indicated organ and all others, which include only the unique significant interacting pairs between them (average expression > 0 and P value < 0.05). e Connection graph showing the intensity of interactions within a major cell type or between two major cell types in colored circles. Numbers in red show the total counts of ligand-receptor pairs within or between cell types, which only included the unique significant interacting pairs between them (average expression > 0 and P value < 0.05). CD4, CD4⁺ T cell; CD8, CD8⁺ T cell; γδ, γδ T cell; B, B cell; Plasma, plasma cell; Myeloid, myeloid cell; NK, NK cell; Epi, epithelial cell; Fib, fibroblast; Smo, smooth muscle cell; FibSmo, FibSmo cell; Endo, endothelial cell