Transcriptome analysis of up- and downregulated genes and pathways in normal and human tendinopathic tendons.

(A) Volcano plot of differentially expressed genes (DEGs) comparing tendinopathic to normal human tendons. Genes colored in red have a log2 (fold change) > 1, a p-value < 0.05, and are therefore considered to be significantly increased. Genes colored in blue have a log2 (fold change) < −1, a p-value < 0.05, and are therefore considered to be significantly decreased. The log2 and p-value thresholds are represented by the dashed lines. Annotated genes are part of the IL-6 cytokine superfamily, the IL-6 signaling cascade, or involved in matrix turnover. (B) Unsupervised hierarchical clustering of expression values from members of the IL-6 cytokine superfamily, their receptors, and parts of the IL-6 signaling cascade (N=16 normal, N=17 tendinopathic). Genes are clustered by color with positive (red) or negative (blue) row-scaled z-scores. (C) Dotplot showing significantly enriched gene sets (p-value < 0.05) as determined by GSEA based on the MSigDB human hallmark gene sets. The color of the circles represents their p-value, the size the number of enriched genes (count), the position on the x-axis the number of enriched genes in ratio to the total number of genes annotated to the gene set (gene ratio), and the +/-signs the direction of the enrichment. (D) GSEA plot for the IL-6/JAK/STAT3 signaling hallmark contained in the MSigDB human hallmark gene sets. The green line traces the running enrichment score on the y-axis while going down the rank of genes listed on the x-axis. (E) Enrichment map plot clustering the top 30 biological processes significantly enriched by overlapping DEG sets. The color of the circles represents their adjusted p-value, the size represents the number of enriched genes (count), and the grey lines connect GO annotations that share the same gene subsets. The manually mapped cluster borders are indicated by dashed lines and the detailed annotations are listed in the Supporting Figure 3 (F) Dotplot showing significantly enriched gene sets (p-value < 0.05) as determined by GSEA based on the MSigDB human cell type signature gene sets. The color of the circles represents their p-value, the size the number of enriched genes (count), the position on the x-axis the number of enriched genes in ratio to the total number of genes annotated to the gene set (gene ratio), and the +/- signs the direction of the enrichment.

Effect sizes and p-values for selected transcripts.

The data describes the differences between tendinopathic and healthy control human tendons.

Concept behind the in vitro hybrid explant // hydrogel assembloid system.

(A) Abstract representation of the in vivo load-bearing tendon core subunits (light blue / white) surrounded by the extrinsic compartment (white) containing i.e. extrinsic fibroblast progenitors (light brown). (B) Sources of the in vitro model system components with the IL-6 knock-out core (KO core) in violet, the IL-6 wildtype core (WT core) in light blue, the IL-6 wildtype fibroblast progenitors in light brown, and the ScxGFP+ fibroblast progenitors in green. Core explants were clamped and the fibroblast progenitors embedded in a (liquid) collagen solution before crosslinking the mixture into a hydrogel around the clamped core explants in various combinations. (C) Photographic and light microscopic images of the in vitro assembloid model system. Lid of a 15 ml Falcon® tube (Ø: 17 mm) used for scale.

Transcript changes in hydrogel-embedded fibroblast progenitors seeded around an IL-6 knock-out (KO) core explant compared those seeded around a wildtype (WT) core.

(A) Illustration of the assembloid combinations compared here (KO core // progenitors vs. WT core // progenitors), the assessed timepoint (d7), and the analyzed compartment (extrinsic fibroblast progenitors only). (B) RNA-seq volcano plot of differentially expressed genes (DEGs). Genes colored in red have a log2 (fold change) > 0.5, a p-value < 0.05, and are considered to be significantly increased in the extrinsic compartment of KO core // progenitor assembloids. Genes colored in blue have a log2 (fold change) < - 0.5, a p-value < 0.05, and are considered to be significantly increased in the extrinsic compartment of WT core // progenitor assembloids. The log2 and p-value thresholds are represented by the dashed lines. (C) Unsupervised hierarchical clustering of the top 50 differentially expressed genes. Genes are clustered by color with positive (red) or negative (blue) row-scaled z-scores. Columns represent individual samples. N=6. (D) Dotplots depicting a selection of GO annotations significantly enriched (adjusted p-value < 0.05) by the DEGs. The selection was biased by GO biological process annotations enriched in the human dataset (Figure 1E). The color of the circles represents their adjusted p-value, the size the number of enriched genes (count), and the x-axis the number of enriched genes in ratio to the total number of genes annotated to the gene set (gene ratio).

Cell proliferation around and ScxGFP+ fibroblast progenitor recruitment to core explants.

(A) Illustrative depictions and representative fluorescence microscopy images of wildtype (WT) core explants surrounded by fibroblast progenitor populations from ScxGFP+ mice cultured with or without Tocilizumab (10 µg/ml), and IL-6 knock-out (KO) explants cultured with or without recombinant IL-6 (25 ng/ml) for 7 days. All cells are colored in blue (NucBlue), ScxGFP+ fibroblast progenitors in green (GFP), and dead cells in red (EthD). (B, C, D) Boxplots depicting the total number of cells, the number of ScxGFP+ cells, and the ratio between core-resident and extrinsic ScxGFP+ cells normalized to the WT median. Each datapoint was calculated from 3 representative fluorescence microscopy images taken from the same sample. The upper and lower hinges correspond to the first and third quartile (25th and 75th percentile), the middle one to the median, the whiskers extend from the hinges no further than 1.5 times the interquartile range, and the points beyond the whiskers are treated as outliers. (E) Lineplot depicting the cumulative percentage of ScxGFP+ cells depending on their distance from the center line of the core explant. The points and the line represent the mean cumulative percentages and the error bands the standard error of the mean (sem). The dashed line indicates locations inside the core area. N=12. Results of the statistical analysis are indicated as follows: *p < 0.05, **p < 0.01. The applied statistical test was the Mann-Whitney-Wilcoxon Test.

Total cell numbers, ScxGFP+ cell numbers, and the ratios between core-resident and extrinsic ScxGFP+ cells in assembloids.

The values were normalized to the WT median and are given as median(IQR).

Cell proliferation around and Scx+ fibroblast progenitor recruitment to damaged mouse Achilles tendons.

(A) Illustrative depiction of the experimental setup and the time schedule. (B) Representative fluorescence microscopy images of mouse hindleg sections from wildtype (WT) Achilles tendons that underwent tenotomy (left), the contralateral untreated control (middle), as well as sections from IL-6 knock-out (KO) Achilles tendons that underwent tenotomy (right). (C) Total number of cells stained with NucBlue. (D) Number of Scx+ cells. (E) Ratio between core-resident and extrinsic Scx+ cells depicted on a logarithmic y-axis. N=7. The upper and lower hinges correspond to the first and third quartile (25th and 75th percentile), the middle one to the median, the whiskers extend from the hinges no further than 1.5 times the interquartile range, and the points beyond the whiskers are treated as outliers. (F) Lineplot depicting the cumulative percentage of Scx+ cells depending on their distance from the Achilles tendon center. The points and the line represent the mean cumulative percentages and their error bands the standard error of the mean (sem). The dashed line indicates locations inside the Achilles tendon stump. Results of the statistical analysis are indicated as follows: *p < 0.05. The applied statistical tests was the Mann-Whitney-Wilcoxon Test.

Total cell number, Scx+ percentage, and the ratios between Achilles tendon (AT)- and neotendon (Nt)-resident Scx+ cells in WT and KO mice after Achilles tenotomy.

The values were determined from sections isolated seven days after the Achilles tenotomy and are given as median(IQR). Statistically significant differences of the KO compared to the WT are indicated as follows: *p < 0.05, n.s.p > 0.05.

Transcript analysis of differentially regulated genes and pathways in wildtype (WT) core explants surrounded by a hydrogel seeded with fibroblast progenitors compared to a WT core surrounded by a cell-free hydrogel.

(A) Illustration depicting the assembloid combinations compared here (WT core // progenitors vs. WT core // cell-free), the assessed timepoint (d7), and the analyzed compartment (core only). (B) RNA-seq volcano plot of differentially expressed genes (DEGs). Genes colored in red have a log2 (fold change) > 0.5, an adjusted p-value < 0.05, and are considered to be significantly increased in the core of WT core // progenitor assembloids. Genes colored in blue have a log2 (fold change) < - 0.5, an adjusted p-value < 0.05, and are considered to be significantly increased in in the core of WT core // cell-free assembloids. The log2 and p-value thresholds are represented by the dashed lines. (C) Unsupervised hierarchical clustering of the top 50 differentially expressed genes. Genes are clustered by color with positive (red) or negative (blue) row-scaled z-scores. Columns represent individual samples. N=4. (D) Dotplots depicting a selection of GO annotations significantly enriched (adjusted p-value < 0.05) by the DEGs. The selection was biased by GO biological processes and GSEA hallmark annotations enriched in the human dataset (Figure 1C & E). The color of the circles represents their adjusted p-value, the size the number of enriched genes (count), and the x-axis the number of enriched genes in ratio to the total number of genes annotated to the gene set (gene ratio).

Effect sizes and p-values for selected transcripts.

The data describes differences in transcripts between the core explants from WT core // progenitors and those from WT core // cell-free assembloids.

Transcript analysis of differentially regulated genes and pathways in IL-6 knock-out (KO) core explants surrounded by a hydrogel seeded with fibroblast progenitors compared to a wildtype (WT) core surrounded by fibroblast progenitors.

(A) Illustration depicting the assembloid combinations compared here (KO core // progenitors vs. WT core // progenitors), the assessed timepoint (d7), and the analyzed compartment (core only). (B) RNA-seq volcano plot of differentially expressed genes (DEGs). Genes colored in red have a log2 (fold change) > 0.5, a p-value < 0.05, and are considered to be significantly increased in the core of KO core // progenitor assembloids. Genes colored in blue have a log2 (fold change) < - 0.5, a p-value < 0.05, and are considered to be significantly increased in the core of WT core // progenitor assembloids. The log2 and p-value thresholds are represented by the dashed lines. (C) Unsupervised hierarchical clustering of the top 50 differentially expressed genes. Genes are clustered by color with positive (red) or negative (blue) row-scaled z-scores. Columns represent individual samples. N=4. (D) Dotplots depicting a selection of GO annotations significantly enriched (adjusted p-value < 0.05) by the DEGs in both the WT core // cell-free vs. WT core // progenitor assembloid comparison (red to black gradient) and the KO core // progenitor vs. WT core // progenitor assembloid comparison (light blue to black gradient). The selection was biased by enriched GO biological process and GSEA hallmark annotations in the human dataset (Figure 1C & E). The color gradient of the circles represents their adjusted p-value, the size the number of enriched genes (count), and the x-axis the number of enriched genes in ratio to the total number of genes annotated to the gene set (gene ratio). (E) Venn-Diagramm depicting the number and the overlap (violet) of significantly enriched GO annotations for biological processes between the WT core // cell-free vs. WT core // progenitor assembloid comparison (red) and the KO core // progenitor vs. WT core // progenitor assembloid comparison (blue). (F) Linear elastic moduli of devitalized (Dev.), IL-6 knock-out (KO), and wildtype (WT) core explants surrounded by hydrogel-embedded fibroblast progenitor populations at day 21 normalized to day 0. N=8. The data are displayed as barplots with mean ± standard error of the mean (sem). The applied statistical test was the Mann-Whitney-Wilcoxon Test and yielded no significant differences.

Human patient metadata.

GEO accession number, patient sex, source tissue, patient age, donor number, and disease state of the isolated tissue ordered by GEO accession number. Samples colored in red were considered as outliers and as such excluded from further analysis.

PCA plots of the human tendon microarray data.

(A) Principal components 1 and 2 for the full dataset with tendinopathic (red) and normal (blue) tendons. (B) Principal components 1 and 2 for the same dataset after excluding outliers based on tendon subtype and clustering in A.

Detailed transcriptome analysis of up- and downregulated genes and pathways in normal and human tendinopathic tendons.

(A) Detailed annotation of the enrichment map plot clustering the top 30 biological processes significantly enriched by overlapping DEG sets. The color of the circles represents their adjusted p-values, the size represents the number of enriched genes (count), and the grey lines connect GO annotations that share the same gene subsets. (B) Dotplot showing the top 20 GO gene sets for biological processes significantly enriched by transcripts increased in human tendinopathic tendons. (C) Dotplot showing all the GO gene sets for biological processes enriched by transcripts decreased in human tendinopathic tendons. (D) Dotplot showing all the GO gene sets for molecular functions enriched by transcripts increased in human tendinopathic tendons. (E) Dotplot showing all the GO gene sets for molecular functions enriched by transcripts decreased in human tendinopathic tendons. In all the dotplots, the color of the circles represents their adjusted p-value, the size the number of enriched genes (count), and the position on the x-axis the number of enriched genes in ratio to the total number of genes annotated to the gene set (gene ratio).

Detailed transcriptome analysis of genes up- and down-regulated in hydrogel-embedded fibroblast progenitors seeded around an IL-6 knock-out (KO) core explant compared to those seeded around a wildtype (WT) core.

(A) Illustration of the assembloid combinations compared here (KO core // progenitors vs. WT core // progenitors), the assessed timepoint (d7), and the analyzed compartment (extrinsic fibroblast progenitors only). (B) Dotplot showing significantly enriched gene sets (p-value < 0.05) as determined by GSEA based on the MSigDB mouse hallmark gene sets. The +/- signs indicate the direction of the enrichment. (C) Dotplot showing the top 20 GO gene sets for biological processes significantly enriched by transcripts increased in fibroblast progenitors seeded around a KO core. (D) Dotplot showing the top 20 GO gene sets for biological processes significantly enriched by transcripts decreased in fibroblast progenitors seeded around a KO core. In all the dotplots, the color of the circles represents their p-value, the size the number of enriched genes (count), and the position on the x-axis the number of enriched genes in ratio to the total number of genes annotated to the gene set (gene ratio).

CD146+ and TPPP3+ fibroblast progenitor recruitment to damaged mouse Achilles tendons.

(A) Representative fluorescence microscopy images of mouse hindleg sections from wildtype (WT) and IL-6 knock-out (KO) Achilles tendons that underwent unilateral tenotomy. (B) Boxplot reporting the number of CD146+ cells normalized to the WT median. (C) Boxplot reporting the number of TPPP3+ cells normalized to the WT median. In these boxplots, the upper and lower hinges correspond to the first and third quartile (25th and 75th percentile), the middle one to the median, the whiskers extend from the hinges no further than 1.5 times the interquartile range, and the points beyond the whiskers are treated as outliers. (D, E) Lineplots depicting the cumulative percentages of CD146+ and TPPP3+ cells depending on their distance from the Achilles tendon center. The points and the line represent the mean cumulative percentages and the error bands their standard error of the mean (sem). The dashed line indicates locations inside the Achilles tendon stump. N=4. The applied statistical test was the non-parametric Wilcoxon Rank Sum Test and no significant differences were detected.

Detailed transcriptome analysis of genes up- and down-regulated in wildtype (WT) core explants surrounded by a hydrogel seeded with fibroblast progenitors compared to a WT core surrounded by a cell-free hydrogel.

(A) Illustration of the assembloid combinations compared here (WT core // progenitors vs. WT core // cell-free), the assessed timepoint (d7), and the analyzed compartment (core only). (B) Dotplot depicting the top 6 significantly enriched gene sets as determined by GSEA based on the MSigDB mouse cell type signature gene sets. The color of the circles represents their p-value, the size the number of enriched genes (count), the position on the x-axis the number of enriched genes in ratio to the total number of genes annotated to the gene set (gene ratio), and the +/- signs the direction of the enrichment. (C) Detailed annotation of the enrichment map plot clustering the top 30 biological processes significantly enriched by DEG sets. The color of the circles represents their adjusted p-values, the size represents the number of enriched genes (count), and the grey lines connect GO annotations that share the same gene subsets.

Overlap between differentially expressed transcripts in in vitro assembloids and differentially expressed transcripts between Achilles tendon fibroblast progenitors from the extrinsic compartment and the tendon core in vivo.

(A) Illustrative depiction of the comparisons whose overlap was investigated here: The core explants of WT core // progenitor vs. the WT core // cell-free progenitor assembloids (in vitro, red) and the extrinsic (paratenon-derived) vs. the core (tendon proper-derived) fibroblast progenitors (in vivo, blue). (B) Venn-Diagramm depicting the number and the overlap (violet) of DEGs as well as the top 7 GO gene sets for biological processes significantly enriched by the overlapping DEGs. (C) Venn-Diagramm depicting the number and the overlap (violet) of significantly enriched GO annotations for biological processes. (D) Dotplot depicting the top 30 biological processes significantly enriched by DEGs in tendon fibroblast progenitors derived from the extrinsic compartment (paratenon-derived) compared to those derived from the core compartment (tendon proper-derived) colored in a blue to black gradient. The plot is augmented by the data of matched biological processes also significantly enriched in the core of WT core // progenitor compared to that of the WT core // cell-free progenitor assembloids colored in a red to black gradient. The color gradient of the circles represents their adjusted p-values, the size the number of enriched genes (count), and the x-axis the number of enriched genes in ratio to the total number of genes annotated to the gene set (gene ratio).

Detailed transcriptome analysis of genes up- and down-regulated in knock-out (KO) core explants surrounded by a hydrogel seeded with fibroblast progenitors compared to a wildtype (WT) core surrounded by fibroblast progenitors.

(A) Illustration depicting the assembloid combinations compared here (KO core // progenitors vs. WT core // progenitors), the assessed timepoint (d7), and the analyzed compartment (core only). (B) Dotplot depicting the top 6 significantly enriched gene sets as determined by GSEA based on the MSigDB mouse cell type signature gene sets. The +/- signs indicate the direction of the enrichment. (C) Dotplot showing the top 20 GO gene sets for biological processes significantly enriched by transcripts increased in the core of KO core // progenitor assembloids. (D) Dotplot showing the top 20 GO gene sets for biological processes significantly enriched by transcripts decreased in the core of KO core // progenitor assembloids. (E) Dotplot showing the top 20 GO gene sets for molecular functions significantly enriched by transcripts increased in the core of KO core // progenitor assembloids. (F) Dotplot showing the top 20 GO gene sets for molecular functions significantly enriched by transcripts decreased in the core of KO core // progenitor assembloids. In all the dotplots, the color of the circles represents their p-value, the size the number of enriched genes (count), and the position on the x-axis the number of enriched genes in ratio to the total number of genes annotated to the gene set (gene ratio).