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A Tppp3+Pdgfra+ tendon stem cell population contributes to regeneration and reveals a shared role for PDGF signalling in regeneration and fibrosis

Nature Cell Biology volume 21, pages 1490–1503 (2019)Cite this article

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Abstract

Tendon injuries cause prolonged disability and never recover completely. Current mechanistic understanding of tendon regeneration is limited. Here, we use single-cell transcriptomics to identify a tubulin polymerization-promoting protein family member 3-expressing (Tppp3+) cell population as potential tendon stem cells. Through inducible lineage tracing, we demonstrate that these cells can generate new tenocytes and self-renew upon injury. A fraction of Tppp3+ cells expresses platelet-derived growth factor receptor alpha (Pdfgra). Ectopic platelet-derived growth factor-AA (PDGF-AA) protein induces new tenocyte production while inactivation of Pdgfra in Tppp3+ cells blocks tendon regeneration. These results support Tppp3+Pdgfra+ cells as tendon stem cells. Unexpectedly, Tppp3−Pdgfra+ fibro-adipogenic progenitors coexist in the tendon stem cell niche and give rise to fibrotic cells, revealing a clandestine origin of fibrotic scars in healing tendons. Our results explain why fibrosis occurs in injured tendons and present clinical challenges to enhance tendon regeneration without a concurrent increase in fibrosis by PDGF application.

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Fig. 1: Tendon harbours a self-renewing stem cell population.
Fig. 2: Tppp3 stem cells amplify early and generate tenocytes by the second week.
Fig. 3: Molecular characterization of tendon subpopulations.
Fig. 4: Tppp3+Pdgfra+ cell gene signature and contribution during regeneration.
Fig. 5: PDGF-AA drives Tppp3 lineage towards tenogenesis.
Fig. 6: PDGFRα signalling is required in Tppp3+ cells for regeneration.
Fig. 7: Tppp3+ mutant cells fail to differentiate following injury.
Fig. 8: Sheath subpopulations do not have identical properties in vitro.

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Data availability

RNA–seq and scRNA–seq data that support the findings of this study have been deposited at NCBI under accession code SRA accession: PRJNA506218. Previously published sequencing data that were re-analysed here are available under accession code GSE89633. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

Code availability

Programming code for RNA–seq analysis has been deposited in GitHub (https://github.com/ciwemb/fan-2019-tendon).

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Acknowledgements

We thank the Fan laboratory members and C. Lepper for critical reading of the manuscript. We also thank S. Satchell for technical assistance, C. Lepper and Y. Bai for assistance with FACS and R. Schweitzer for invaluable advice and sharing the ScxGFP mice. This research was supported by the Carnegie Institution for Science. C.-M.F. is supported by the NIH (grants R01AR060042, R01AR071976 and R01AR072644) and the Carnegie Institution for Science.

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Authors and Affiliations

  1. Carnegie Institution for Science, Department of Embryology, Baltimore, MD, USA

    Tyler Harvey & Chen-Ming Fan

  2. Johns Hopkins University, Department of Biology, Baltimore, MD, USA

    Tyler Harvey & Sara Flamenco

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  1. Tyler Harvey
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T.H. and C.-M.F. conceived and designed the study and wrote the manuscript. T.H. carried out all experiments with assistance by S.F.

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Correspondence to Chen-Ming Fan.

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Extended data

Extended Data Fig. 1

a, Cells expressing >1 UMI of Acta2 from (Fig. 1a). b, Cartoon summary (right) from cell atlas (Fig. 1a) and immunofluorescence (Fig. 1b); midsubstance (maroon), sheath (gray), and key (left). c, Fluorescence images of Tppp3CG/+;R26RtdT tendon; upper, no TMX control with anti-GFP antibody (Ab.); middle, +TMX and Ab.; lower, +TMX and no Ab. control–eGFP expressed by the Tppp3CG driver is only detectable with Ab. staining; 3. Tppp3CG driver labels 38.3±2.6 (Mean±SEM)% of sheath cells. d, Tppp3CG driver labeling efficiency is 79.5±3.4 (Mean±SEM)%; n=3 animals. e, Fluorescent images of Tppp3CG/+;R26RtdT tendons: digit flexor (leftmost), tail (left), Achilles (right) or Patellar (rightmost); TMX pulsed at embryonic day (E)15.5 and chased to E17.5; Col I, collagen-I antibody stained; dashed line, midsubstance-sheath boundary; 3 animals/tendon. f, Fluorescent images of Tppp3CG/+;R26RtdT Patellar tendon; TMX pulsed at postnatal day (P)5 and chased to P8; dashed line, midsubstance-sheath boundary; 3 animals. g, Sheath cell fractions (key; right) over time; n=3 animals/time point; all ns. h, Midsubstance cell fractions (key same as g) over time; n=3 animals/time point; all ns. i, Midsubstance cell fractions (key; right) over time; n=3 animals/time point; all non-significant by Chi-square test. j, Wholemount multiphoton images of uninjured and biopsy punched (immediately after (T0)) Patellar tendon; 3 animals; collagen fibers visualized by second harmonic generation (SHG). k, Regenerated tendon immuno-stained for Tenascin-C (TNC); asterisks, proliferated Tppp3-lineage cells in TNC matrix of midsubstance; ^, self-renewed Tppp3-lineage in sheath with lower TNC signal; 3 animals. (l,m) Zoomed in FMOD images from Fig. 1g; 3 independent repeats; asterisks, proliferated Tppp3-lineage in midsubstance (l); ^, self-renewed Tppp3-lineage in sheath (m). Scale bar = 30 (c), 15 (flexor) 50 (tail, Achilles, Patellar) (e), 40 (f), 200 (j), 50 (k), 10 (l,m) μm. Error bars = SEM (d,g,h,i). Two-tailed Student’s t-test (g,h).

Extended Data Fig. 2

a, Tppp3ECE/+;R26RtdT;ScxGFP tendon showed TMX-dependent cell marking (tdT+) in sheath; 3 independent repeats. Given the proportion of labeled sheath cells (22.7 ± 3.7 (Mean±SEM) %) relative to Tppp3CG, Tppp3ECE driver labeling efficiency is ~60%; dashed line, midsubstance-sheath boundary. ScxGFP signal was detected without antibody–not every midsubstance cell is ScxGFP+. b, Fractions of sheath tdT+ScxGFP+ cells at given dpi; n=3 animals. c, Midsubstance images at specified regeneration windows related to Fig. 2b, 3 independent repeats. d, Bar graph (top) for average number of midsubstance tdT+ cells at specified time point. Line graph (bottom) for % of proliferated (EdU+) midsubstance Tppp3-lineage marked and ScxGFP+ cells (keys, upper right); n=3 animals/time point; comparisons for a, tdT+ScxGFP− vs tdT+ScxGFP+, b, tdT+ScxGFP− vs tdT−ScxGFP+, c, tdT+ScxGFP+ vs tdT−ScxGFP+ at 28 d; all other time comparisons not-significant. e, Regeneration assay for 30 d with daily EdU throughout; n=3 animals/condition; asterisks, tdT+ScxGFP+EdU+ cells in midsubstance; <, tdT+EdU+ cells in sheath. Below, quantified sheath and midsubstance cell fractions (key; right) in uninjured (U) and injured (I) conditions; Chi-squared test ## P < 0.01. t-test for specified cell fraction in Sheath and Midsubstance comparisons, respectively, found in Source Data. f, Fluorescent images of FACS isolated cells from 30 d regenerated Tppp3ECE/+;R26RtdT;ScxGFP tendons related to Fig. 2c, 3 independent repeats. g, Averaged, normalized log10 counts from DESeq comparison between tdT+ScxGFP+ and tdT−ScxGFPr+ cells: arranged in boxes by transcription factors (blue), collagens (green), and proteoglycans/glycoproteins (magenta); *, for FDR-adjusted q-value < 0.05, by DESeq package. h, Table for selected gene categories: log2(fold-change) directionality of enrichment in tdT+ScxGFP+r (yellow) versus uninjured tdT+ (red) cells; n=2 replicates; FDR-adjusted q-value by DESeq package are shown. Unpaired two-tail Student’s t-test (b,d,e); error bars = SEM (b,d,e); scale bars= 30 (a,c), 50 (e,f) μm.

Extended Data Fig. 3

a, Immunofluorescence of novel sheath markers (LAMININ for Lama4; SCA-1 for Ly6a/e; PLIN for Plin2) identified in Fig. 3b; dashed line, midsubstance-sheath boundary; 3 independent repeats; scale bar = 50 μm. b, Table for genes in selected matrix categories from Fig. 3a; same organization as for Fig. 3b, n=2 replicates; FDR adjusted q-values by Cufflinks package are shown; samples represent Tppp3+ (tdT+ScxGFP−; red) and tenocyte (tdT−ScxGFP+; green). c, Table for differentially expressed signaling pathway genes from Fig. 3a, n=2 replicates; FDR adjusted q-values by Cufflinks package are shown; samples represent Tppp3+ (tdT+ScxGFP−; red) and tenocyte (tdT−ScxGFP+; green). d, Canonical Pathways list generated by Ingenuity Pathway Analysis® (IPA) on DE analysis gene list filtered by q-value < 0.05; positive z-score (orange) indicates predicted activation; PDGF signaling (red boxed) and its downstream effector branches ERK/MAPK and PI3K/AKT are enriched (indicated by asterisks), n=2 replicates; FDR adjusted q-value calculated by whole transcriptome normalization by IPA package. e, Relative expression of genes (as indicated) plotted in pseudotime, colored by state, and with expression level trend (line), related to Fig. 3d. In b,c,d samples were pooled from 14 tendons.

Extended Data Fig. 4

a, Novel Sheath markers: Log10 gene expression level specified per individual cell plotted in pseudotime trajectory of cluster 2 and 4 cells. Red circles (enrichment in state 1), correlating with Tppp3 and Pdgfra (Fig. 3d), suggesting a unique role for state 1-Tppp3+Pdgfra+ cells (see Fig. 3d). b, Fluorescent images of Tppp3ECE/+;R26RtdT;PdgfraH2B-eGFP tendon 5 d after TMX-induced marking; 3 independent repeats; dashed line, midsubstance-sheath boundary; yellow arrowheads, tdT+H2B-eGFP+ cells; red arrowheads, tdT+H2B-eGFP-; green arrowheads, tdT-H2B-eGFP+; blue arrowheads, negative; direct fluorescent imaging to visualize GFP signal from PdgfraH2B-eGFP. c, Relative cell fractions (keys; bottom) within Tppp3+(tdT+) population (bar graph) and within the sheath (pie chart) from data in (b); n=3 animals. d, (Left) Fluorescent images of Tppp3CG/+;R26RtdT;PdgfraH2B-eGFP Achilles or tail tendon 5 d after TMX-induced marking; n=6-9 animals; dashed line, midsubstance-sheath boundary; arrowheads same code as b; direct fluorescent imaging to visualize GFP signal from PdgfraH2B-eGFP. (Right) Bar graph of fraction of sheath cell populations across respective tendon types; A, Achilles (n=6); P, Patellar (n=9); T, tail (n=8); circles represent individual n. e, Stem cell (Tppp3+PDGFRα+) recombination efficiency per Tppp3 driver; Tppp3CG efficiency is 77.0±1.6 (Mean±SEM)% and Tppp3ECE efficiency is 56.1±2.9 (Mean±SEM)%; determined in situ by tdT+ co-localization with anti-TPPP3 antibody+PDGFRaH2BeGFP+ cells; n=4 samples. f, Bar graph of proliferation indices from entrant populations corresponding to Fig. 4f, all comparisons non-significant; circles indicate n; n=3 animals. g, Fluorescent images of midsubstance with specified treatment on Tppp3ECE/+;R26RtdT;ScxGFP tendon; 3 independent repeats. Error bars = SEM (c,d,ef); scale bar = 30 (d,g), 50 (b) μm; two-tailed Student’s t-test (d,f).

Extended Data Fig. 5

a, Immunofluorescence for indicated sheath markers on Tppp3CG/+;Pdgfrafl/fl tendon with same experimental scheme as (Fig. 6c) except for without punch and EdU; 3 independent repeats; dashed line, midsubstance-sheath boundary. b, Experimental scheme and fluorescent images of R-control, Tppp3CG/+;R26RtdT, and R-cKO, Tppp3CG/+;R26RtdT;Pdgfrafl/fl; chased 30 d after TMX; 3-4 animals/condition; EdU daily throughout; dashed line, midsubstance-sheath boundary; asterisk, tdT+EdU+ cell. c, Fluorescent images of R-control and R-cKO of sheath compartment in regenerate area; harvested at 14 dpi and EdU daily throughout; n=3 (R-control), n=4 (R-cKO) animals/condition; dashed line, midsubstance-sheath boundary; arrowheads, eGFP-tdT+ cells. Stacked column for the distribution (in %) of various cell populations (keys at side); mean (%) for specified population (Negative, eGFP+, tdT+, eGFP+tdT+) as follows: (R-control; 48.3, 8.0, 2.2, 41.5), (R-cKO; 53.0, 15.1, 10.6, 21.2). Bar graph for average number of sheath cells per injury area; mean = (R-control, 1013; R-cKO, 667). d, Controls for SCX immunofluorescence; anti-SCX antibody reacts strongly to midsubstance cells (arrowheads); n=3 independent repeats. e, Fluorescent micrographs of ScxGFP tissue stained with anti-SCX antibody, 93.6±1.9 (mean±SEM) % of ScxGFP+ cells are anti-SCX+; 3 independent repeats; dashed line, midsubstance-sheath boundary; arrowheads, anti-SCX+ScxGFP+ cells. f, Stacked columns for the distribution (in %) of various cell populations (keys at side); n=3 animals; mean (%) per population (tdT-, tdT+) as indicated: R-control (16.7, 16.8), R-cKO (32.7, 13.8). Unpaired two-tail Student’s t-test (c,f); error bars = SEM (c,f); scale bar = 20 (d,e), 30 (b), 50 (a,c) μm.

Extended Data Fig. 6

a, Fluorescent micrographs of purified tdT+H2B-eGFP+ cultured for 24 h in TSPC conditions stained with anti-PDGFRα or anti-TPPP3. b, Fluorescent micrographs of anti-TPPP3 antibody validation on cultured sub-populations. 3 independent repeats (a, b). Scale bars = 100 (b), 200 (a) μm.

Extended Data Fig. 7 Cells were selected in FSC/SSC dot plot to remove debris; single cells were gated using the FSC-H/FSC-W dot plot.

GFP+ (FITC-A), tdT+ (PE-A) cells were gated and compared with a control sample without tamoxifen induction or carrying ScxGFP. Tomato+-only cells were gated from tomato+GFP+ cells by population segregation.

Supplementary information

Reporting Summary

Supplementary Table 1

Differentially expressed genes per cell cluster from scRNA–seq data (Fig. 1a, n = 2,451 cells) derived from Loupe Cell Browser using Cell Ranger clustering and statistical ranking. Average, normalized UMI counts per each gene ID (with corresponding colour code in Fig. 1a) are listed as well as cluster log2fold enrichment value, cluster assignment number and cell type identity.

Supplementary Table 2

Mouse alleles used in this study with abbreviated names as they appear in the manuscript as well as the full names. Allele supplier is indicated as well as stock number and citations (where applicable).

Supplementary Table 3

Primary antibodies used in this study with dilution factors and special conditions for antigen retrieval.

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Harvey, T., Flamenco, S. & Fan, CM. A Tppp3+Pdgfra+ tendon stem cell population contributes to regeneration and reveals a shared role for PDGF signalling in regeneration and fibrosis. Nat Cell Biol 21, 1490–1503 (2019). https://doi.org/10.1038/s41556-019-0417-z

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