Identification of human spinal cell types from spatial transcriptomics.

(A) Overview of the experimental workflow for spatial transcriptomics. (B) UMAP plot showing 17 cell types in the spinal cord. Dots, individual spots; colors, cell types. (C) Representative image showing the spatial distribution of these 17 cell types on Visium slides. (D) Dot plot showing the expression of representative marker genes across all cell types. The dot size represents the percentage of barcodes within a cluster, and the color scale indicates the average expression across all barcodes within a cluster for each gene shown. (E) UMAP plot showing the expression of representative marker genes. (F) UMAP plot showing 5 major cell types in the spinal cord. Glia include oligodendrocytes, astrocytes, microglia, and OPCs. Dots, individual spots; colors, cell types. (G) Representative image showing the spatial distribution of 5 major cell types in spinal slices. Dots, individual spots; colors, cell types. (H) The number of spots in each cluster. (I) The number of genes detected per cluster. Oligo, oligodendrocytes; Menin, meningeal cells; OPC, oligodendrocyte precursor cells; UMAP, uniform manifold approximation and projection.

Identification of spinal cell types using single-nucleus RNA-seq.

(A) Overview of the experimental workflow for single-nucleus RNA-seq. (B) UMAP plot showing 8 major cell types. Dots, individual cells; colors, cell types. (C) Dot plot showing the expression of representative marker genes across all 8 cell types. The dot size indicates the percentage of cells expressing the gene; the color scale indicates the average normalized expression level in each cluster. (D) UMAP plot showing the expression of representative marker genes. (E) Representative section showing the spatial distribution of 8 clusters in the spinal cord. (F) Representative immunofluorescence images of NeuN, MBP, GFAP, and Iba1 in a coronal cryosection of the lumber spinal cord. Oligo, oligodendrocytes; OPC, oligodendrocyte precursor cells; Menin, meningeal cells; UMAP, uniform manifold approximation and projection.

Identification of neuronal subtypes in the human spinal cord.

(A) UMAP plot showing 21 neuronal clusters. Dots, individual cells; colors, neuronal clusters. (B) Dot plot showing the expression of selected marker genes across all 21 neuronal clusters. (C) UMAP plot of spinal cord neurons based on excitatory, inhibitory, and cholinergic marker genes (left). Dot plot showing the expression of representative marker genes of excitatory, inhibitory, and cholinergic clusters (right). (D) UMAP plot showing the expression of representative marker genes of excitatory, inhibitory, and cholinergic clusters. (E) Representative cartoons of subregions in coronal slices of the spinal cord. (F) Representative section showing the spatial distribution of the excitatory clusters (left) and representative immunofluorescence image of the excitatory (VGLUT2) marker in a coronal cryosection of lumber spinal cord (right). (G) Representative section showing the spatial distribution of the inhibitory clusters (left) and representative immunofluorescence image of the inhibitory (GAD67) marker in a coronal cryosection of lumber spinal cord (right). (H) Representative section showing the spatial distribution of motor neurons (C20) and representative immunofluorescence image of its marker gene (CHAT) in a coronal cryosection of lumber spinal cord. (I) Gene set variation analysis (GSVA) showing the spatial distribution patterns of neuronal clusters in different subregions of coronal sections from the lumber spinal cord. (J) Representative section showing the spatial distribution of C8 and representative immunofluorescence image of its marker gene (PDYN) in a coronal cryosection of lumber spinal cord. (K) A summary of the characteristics of neuronal clusters, including their location, functional status, putative neuronal type, and representative marker genes. SD, superficial dorsal horn; DD, deep dorsal horn; V, ventral horn; E, excitatory; I, inhibitory; M, mixed; C, cholinergic; UMAP, uniform manifold approximation and projection.

Human-mouse cell type homology.

(A) UMAP plot showing the coclustering of mouse and human neurons. Dots, individual cells. Colors, species. (B) UMAP plot showing the distribution of putative homologous neuronal clusters of humans and mice. Dots, individual cells. Colors, clusters. (C) Heatmap of conserved cell-type-specific gene expression (columns) in human and mouse cell types (rows; m, mouse; h, human). Genes in each species are included in the heatmap if they are significantly enriched in a cluster compared to all other clusters (FDR < 0.01, top 50 genes by log2-fold change [FC] per cell type). (D-E) Dot plot showing the expression of classical marker genes in the human (D) and mouse (E) spinal cord. (F-G) Heatmap showing the expression of classical marker genes in the human (F) and mouse (G) spinal cord. (H) Representative image showing the distribution of NPY-positive spots in the human spinal cord (left) and representative immunofluorescence image of NPY in a coronal cryosection of the human lumber spinal cord. (I) Representative RNAscope in situ hybridization images of Npy and Rbfox3 in a coronal cryosection of mouse lumber spinal cord. (J) Representative image showing the distribution of TAC1-positive spots in the human spinal cord and representative immunofluorescence image of TAC1 in a coronal cryosection of the human lumber spinal cord. (K) Representative RNAscope in situ hybridization images of Tac1 and Rbfox3 in a coronal cryosection of mouse lumber spinal cord. UMAP, uniform manifold approximation and projection.

Sex differences in gene expression in human spinal neuronal types.

(A) UMAP plot showing barcodes in all spinal neuronal clusters from males and females. Dots, individual cells. Colors, sexes. (B) Volcano plot showing DEGs of all spinal neurons between males and females. (C) A summary table showing the number of DEGs for specific neuronal types between males and females. (D) Volcano plot showing DEGs in C20 (motor neurons) between males and females. (E) Violin plot showing the expression of DEGs in males and females within the motor neurons. (F) Representative immunofluorescence images of SCN10A with CHAT (a marker of motor neurons) in male and female spinal sections. (G-H) Summarized GO terms for upregulated DEGs (G) or downregulated DEGs (G) in male motor clusters compared to females. DEGs, differentially expressed genes. DEGs were considered if FC ≥ 1.33 and adjusted P < 0.05. GO, Gene Ontology; NS, no significance.

Identification of human DRG cell types from spatial transcriptomics.

(A) Overview of the experimental workflow for spatial transcriptomics in human DRG. (B) UMAP plot showing 16 cell types in the spinal cord. Dots, individual spots; colors, cell types. (C) Dot plot showing the expression of representative marker genes across all cell types. (D) UMAP plot showing the expression of representative marker genes. (E) UMAP plot showing 6 major DRG cell types and their percentages. Dots, individual spots; colors, cell types. (F) Representative section showing the spatial distribution of 6 major cell types in the DRG. Dots, individual spots; colors, cell types. RBCs, red blood cells; VECs, vascular endothelial cells; VSMCs, vascular smooth muscle cells; SGCs, satellite glial cells; UMAP, uniform manifold approximation and projection.

Identification of neuronal subtypes in human DRG.

(A) UMAP plot showing 13 neuronal clusters of human DRG neurons. Dots, individual spots; colors, neuronal clusters. (B) UMAP plot showing the expression of representative marker genes. (C) Putative DRG neuronal types and their representative marker genes. (D-E) Dot plot showing the expression of classical marker genes in human (D) and mouse (E) DRG neuronal clusters. (F) Representative RNAscope in situ hybridization images of PVALB and RBFOX3 in mouse (top) and human (bottom) DRG. (G) Representative immunofluorescence images of SST and NeuN in mouse (top) and human (bottom) DRG. NF, neurofilament neurons; PEP, peptidergic neurons; NP, nonpeptidergic neurons; UMAP, uniform manifold approximation and projection.

Ligand‒receptor interactions between the human DRG and spinal cord.

(A) Putative ligand‒receptor interactions of neuronal clusters between the human DRG and spinal cord. The thickness of connecting lines is proportional to the number of total ligand‒receptor interactions between the two connecting cell types. (B) DRG CALCA-spinal CALCRL interactions (left). The spatial location of the CALCRL-cluster (right). (C) DRG NGF-spinal NGFR interactions (left). The spatial location of the NGFR-cluster (right). (D) DRG SST-spinal SSTR1 interactions (left). The spatial location of the SSTR1-cluster (right). (E) DRG APOE-spinal LRP1 interactions. (F) DRG SLIT2-spinal ROBO2 interactions. (G) DRG TAC1-spinal TACR1 interactions. (H) DRG NRG1-spinal ERBB4 interactions. Dot size denotes relative expression of a gene in each cell type, and colors indicate cell type. Arrows between cell types denote the 10 highest ligand‒receptor scores. NF, neurofilament neurons; PEP, peptidergic neurons; NP, nonpeptidergic neurons.