scRNA-seq of tendon cells from 2-week-old and 6-week-old mice and the identification of surface markers of TSPC.

(A) Integrated uniform manifold approximation and projection (UMAP) scRNA-seq clustering of cells harvested from 2-week-old and 6-week-old mouse Achilles tendons.

(B) Dot plot of average gene expression levels of the indicated genes in each scRNA-seq cluster. The size of the dot reflects the percentage of cells in the cluster that express each gene. TC, tenocyte; SP1, tendon stem/progenitor cell_1; CA, cartilage; RB, ribosomal RNA; SP2, tendon stem/progenitor cell_2; LC1, lymphocyte_1; EC, endothelial cell; RBC, red blood cell; SM, smooth muscle cell; PC, proliferating cell; LC2, lymohocytes_2; MTJ, myotendinous junction cell; SW, Schwann cell; VEC, vascular endothelial cell; MC, macrophage.

(C) Proportions of cells in clusters identified from scRNA-seq. Clusters are colored according to cluster type.

(D) Volcano plot of gene expression in the SP2 cluster and the identification of candidate TSPC marker genes (red under line).

(E) Feature plot of Cd55 and Cd248 expression.

(F) Correlation of gene expression of TSPC candidate genes in 2-week data.

(G) Violin plots presenting the gene expression changes for a selection of differentially expressed genes.

snATAC-seq of tendon cells from a 2-week-old mouse and the validation of Cd55 and Cd248 as candidate markers of TSPC.

(A) UMAP snATAC-seq clustering of cells derived from the Achilles tendon of a 2-week-old mouse. Annotation was based on each gene activity (ground-truth annotation). SP1, tendon stem/progenitor cell_1; TC1, tenocyte_1; MTJ, myotendinous junction cell; SM, smooth muscle cell; MC, macrophage; CA, cartilage; TC2, tenocyte_2; SK, skeletal muscle cell; SW, Schwann cell; LC, lymphocyte; NC, neutrophil; EC, endothelial cell; RBC, red blood cell; UC, unspecified cell; SP2, tendon stem/progenitor cell_2; SP3, tendon stem/progenitor cell_3; VEC, vascular endothelial cell.

(B) Dot plot of average gene activity of the indicated genes in each snATAC-seq cluster. The size of the dot reflects the percentage of cells in the cluster that express each gene.

(C) UMAP visualization and predicted annotation of 2-week snATAC-seq after integration and label transfer of 2-week snRNA-seq data.

(D) Identification of matching cell clusters between the 2-week snRNA- and 2-week snATAC-seq data from visualized as heatmap. The heatmap shows the proportions of cells from each snATAC-seq cluster across all sample conditions assigned to each snRNA-seq cluster as part of the label-transfer process.

(E) Violin plot of tenocytes and TSPC-related gene expression in each cluster.

Trajectory analysis and peak visualization of snRNA-seq and snATAC-seq data for the tendon and tendon stem/progenitor cell-related cluster.

(A) UMAP representation of snRNA-seq differentiation trajectory of tenocytes and TSPC lineage and pseudotime-dependent gene expression changes of Tppp3, Cd55, Cd248, Mkx, and Scx, as inferred using Monocle3.

(B) UMAP representation of snATAC-seq differentiation trajectory of tenocytes and the TSPC lineage and pseudotime-dependent gene expression changes, as inferred using Cicero.

(C) Coverage plots of Mkx, Scx, Cd55, and Cd248. Selected peaks that differ across each cluster are highlighted.

Transcription factor landscapes of 2-week mouse Achilles tendons.

SCENIC analysis of transcription factor activity based on 2-week snRNA-seq data for tenocytes and the TSPC lineage (Left). Validation was performed based on the gene activity and motif activity of 2-week snATAC-seq and gene expression of 2-week snRNA-seq (right).

Estimated TF activity in each gene

In vitro analysis of CD55+/CD248+ TSPCs.

(A) Immunohistochemical image of 2-week mouse Achilles tendons. CD55 and CD248, green; DAPI, blue.

(B) Schema of the in vitro assessment of the capacity of CD55+/CD248+ TSPCs as the differentiation toward tenocytes.

(C) Colony-forming efficiency of CD55+/CD248+ and CD55-/CD248- (negative) TSPCs. Colonies were stained with crystal violet (n = 6). CD55+/CD248+ TSPC exhibited higher clonogenic capacity. Data are presented as means ± SEM. **p < 0.01.

(D) Morphological changes of CD55+/CD248+ and negative TSPCs after tenogenic induction.

(E) Quantitative PCR of tendon-related gene expression in CD55+/CD248+ and negative TSPCs after tenogenic induction (n = 3). Data are presented as means ± SD. **p < 0.01, *p < 0.05.

(F) SEM and TEM imaging of artificial tendons derived from CD55+/CD248+ and negative TSPCs. Data are presented as means ± SD. **p < 0.01, *p < 0.05.

(G) Proportions of fiber alignment for each artificial tendon (n = 4). Data are presented as means ± SEM. ***p < 0.005, **p < 0.01, *p < 0.05.

(H) Diameter of collagen fiber in each artificial tendon based on TEM imaging (n = 4). Data are presented as means ± SD. ***p < 0.005.

(I) Tensile strength (MPa) of each artificial tendon (n = 5). Data are presented as means ± SEM. ***p < 0.005.

Comparison of 2w- and 6w-scRNA seq results.

(A) Violin plot of gene expression enriched in the limb bud (Development (2014) 141, 3683-3696) in each cluster.

(B) Gene Ontology (GO) terms associated with genes with upregulated expression in the SP2 cluster.

(C) Number of inferred interactions and interaction strength of genes expressed within 2w- and 6w-mouse scRNA-seq data.

Analysis of 2-week snRNA-seq data.

(A) UMAP snRNA-seq clustering of cells harvested from 2-week mouse Achilles tendons.

(B) Dot plot of average gene expression levels of the indicated genes in 2-week snRNA-seq clusters. RB, ribosomal RNA; TC1, tenocyte_1; SP1, stem/progenitor cell_1; SP2, stem/progenitor cell_2; SM, smooth muscle cell; MTJ, myotendinous junction cell; CA, cartilage; SP3, stem/progenitor cell_3; LC, lymphocyte; MC, macrophage; RBC, red blood cell; SW, Schwann cell; EC, endothelial cell; TC2, tenocyte_2; PC, proliferating cell; NC, neutrophil; UC, unspecified cell.

(C) Violin plot of tenocytes and TSPC-related gene expression in each cluster.

(D) Feature plot of TSPC-related gene expression.

Analysis of 6-week snATAC-seq.

(A) UMAP snATAC-seq clustering of cells harvested from 6-week mouse Achilles tendons. Annotation was based on gene activity (ground-truth annotation, left) and the predicted annotation inferred from 2-week snATAC-seq (right).

(B) Identification of matching cell clusters between the 2-week and 6-week snATAC-seq data, visualized as a heatmap.

(C) Violin plot of tenocytes and TSPC-related gene activity in each cluster.

Analysis of 6-week snRNA-seq.

(A) UMAP snRNA-seq clustering of cells harvested from 6-week mouse Achilles tendons. Annotation was based on each gene expression (ground-truth annotation, left) and predicted annotation inferred from 2-week snRNA-seq (right).

(B) Identification of matching cell clusters between the 2-week and 6-week snRNA-seq data visualized as a heatmap.

(C) Violin plot of tenocytes and TSPC-related gene expression in each cluster.

Comparison of 2-week and 6-week snATAC-seq.

(A) Circle plot of annotated differentially accessible regions for each data.

(B) Integrated UMAP snATAC-seq clustering of cells harvested from 2-week and 6-week mouse Achilles tendons.

(C) Feature plot of TSPC-related gene activity in each dataset.

Gene expression changes in CD55+/CD248+ and negative TSPCs.

Quantitative PCR of gene expression in CD55+/CD248+ and negative TSPCs (n = 4). Data are presented as means ± SEM. *p < 0.05, ***p < 0.005.

Chondrogenic and osteogenic induction of CD55+/CD248+ and negative TSPCs.

Quantitative PCR of tendon-related genes in CD55+/CD248+ and negative TSPCs after chondrogenic and osteogenic induction (n = 4). Data are presented as means ± SEM. **p < 0.01, *p < 0.05.