Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Subjects

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

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.

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).

References

  1. Elliott, D. H. Structure and function of mammalian tendon. Biol. Rev. Camb. Philos. Soc. 40, 392–421 (1965).

    Article  CAS  PubMed  Google Scholar 

  2. Voleti, P. B., Buckley, M. R. & Soslowsky, L. J. Tendon healing: repair and regeneration. Annu. Rev. Biomed. Eng. 14, 47–71 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Jozsa, L. & Kannus, P. Human Tendons: Anatomy, Physiology and Pathology. (Human Kinetics, 1997).

  4. Harvey, T. & Fan, C.-M. Origins of tendon stem cells in situ. Front. Biol. 13, 263–276 (2018).

    Article  CAS  Google Scholar 

  5. Howell, K. et al. Novel model of tendon regeneration reveals distinct cell mechanisms underlying regenerative and fibrotic tendon healing. Sci. Rep. 7, 45238 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Loiselle, A. E. et al. Remodeling of murine intrasynovial tendon adhesions following injury: MMP and neotendon gene expression. J. Orthop. Res. 27, 833–840 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Kim, H. M. et al. Technical and biological modifications for enhanced flexor tendon repair. J. Hand Surg. Am. 35, 1031–1037 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Juneja, S. C., Schwarz, E. M., O’Keefe, R. J. & Awad, H. A. Cellular and molecular factors in flexor tendon repair and adhesions: a histological and gene expression analysis. Connect. Tissue Res. 54, 218–226 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Manning, C. N. et al. The early inflammatory response after flexor tendon healing: a gene expression and histological analysis. J. Orthop. Res. 32, 645–652 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Loiselle, A. E., Kelly, M. & Hammert, W. C. Biological augmentation of flexor tendon repair: a challenging cellular landscape. J. Hand Surg. Am. 41, 144–149 (2016).

    Article  PubMed  Google Scholar 

  11. Lin, T. W., Cardenas, L., Glaser, D. L. & Soslowsky, L. J. Tendon healing in interleukin-4 and interleukin-6 knockout mice. J. Biomech. 39, 61–69 (2006).

    Article  PubMed  Google Scholar 

  12. Bi, Y. et al. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat. Med. 13, 1219–1227 (2007).

    Article  CAS  PubMed  Google Scholar 

  13. Dyment, N. A. & Galloway, J. L. Regenerative biology of tendon: mechanisms of renewal and repair. Curr. Mol. Bio. Rep. 1, 124–131 (2015).

    Article  Google Scholar 

  14. Fiel, R., Wagner, J., Metzger, D. & Chambon, P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophy. Res. Commun. 237, 752–757 (1997).

    Article  Google Scholar 

  15. Madisen, L. et al. A robust and high-throughput cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. Dyment, N. A. et al. Lineage tracing of resident tendon progenitors during growth and natural healing. PLoS One 9, e96113 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Ansorge, H. L., Adams, S., Birk, D. E. & Soslowsky, L. J. Mechanical, compositional and structural properties of the post-natal mouse achilles tendon. Ann. Biomed. Eng. 39, 1904–1913 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Beason, D. P., Kuntz, A. F., Hsu, J. E., Miller, K. S. & Soslowsky, L. J. Development and evaluation of multiple tendon injury models in mouse. J. Biomech. 45, 1550–1553 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Staverosky, J. A., Pryce, B. A., Watson, S. S. & Schweitzer, R. Tubulin polymerization-promoting protein family member 3, Tppp3, is a specific marker of the differentiating tendon sheath and synovial joints. Dev. Dyn. 238, 685–692 (2013).

    Article  CAS  Google Scholar 

  20. Wang, Y. et al. Osteocalcin expressing cells from tendon sheaths in mice contribute to tendon repair by activating hedgehog signaling. eLife 6, e30474 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Chen, J., Renia, L. & Ginhoux, F. Constructing cell lineages from single-cell transcriptomes. Mol. Aspects Med. 59, 95–113 (2017).

    Article  PubMed  CAS  Google Scholar 

  22. Uezumi, A., Fukada, S., Yamamoto, N., Takeda, S. & Tsuchida, L. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle. Nat. Cell Biol. 12, 143–152 (2010).

    Article  CAS  PubMed  Google Scholar 

  23. Joe, A. W. et al. Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis. Nat. Cell Biol. 12, 153–163 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ameye, L. et al. Abnormal collagen fibrils in tendons of biglycan/fibromodulin-deficient mice lead to gait impairment, ectopic ossification and osteoarthritis. FASEB J. 16, 673–680 (2002).

    Article  CAS  PubMed  Google Scholar 

  25. Jepsen, K. J. et al. A syndrome of joint laxity and impaired tendon integrity in lumican- and fibromodulin-deficient mice. J. Biol. Chem. 277, 35532–35540 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Docheva, D., Hunziker, E. B., Fassler, R. & Brandau, O. Tenomodulin is necessary for tenocyte proliferation and tendon maturation. Mol. Cell. Biol. 25, 699–705 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hauser, N., Paulsson, M., Kale, A. A. & DiCesare, P. E. Tendon extracellular matrix contains pentameric thrombospondin-4 (TSP-4). FEBS Lett. 368, 307–310 (2003).

    Article  Google Scholar 

  28. Kohrs, R. T. et al. Tendon fascicle gliding in wild type, heterozygous and lubricin knockout mice. J. Orthop. Res. 29, 384–389 (2011).

    Article  PubMed  Google Scholar 

  29. Evans, C. J. et al. G-TRACE: rapid Gal4-basd cell lineage analysis in Drosophila. Nat. Methods 6, 603–605 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Pryce, B. A., Brent, A. E., Murchison, N. D., Tabin, C. J. & Schweitzer, R. Generation of transgenic tendon reporters, ScxGFP and ScxAP, using regulatory elements of the scleraxis gene. Dev. Dyn. 236, 1677–1682 (2007).

    Article  CAS  PubMed  Google Scholar 

  31. Dyment, N. A. et al. The paratenon contributes to scleraxis-expressing cells during patellar tendon healing. PLoS One 8, e59944 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. BestK. T. & LoiselleA. E. Scleraxis lineage cells contribute to organized bridging tissue during tendon healing and identify a subpopulation of resident tendon cells. FASEB J. 33, 8578–8587 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kishimoto, Y. et al. Wnt/b-catenin signaling suppresses expression of Scx, Mkx and Tnmd in tendon-derived cells. PLoS One 12, e0182051 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. D’Souza, D. & Patel, K. Involvement of long- and short-range signaling during early tendon development. Anat. Embryol. 200, 367–375 (1999).

    Article  Google Scholar 

  35. Dyment, N. A. et al. Gdf5 progenitors give rise to fibrocartilage cells that mineralize via hedgehog signaling to form the zonal enthesis. Dev. Biol. 405, 96–107 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Schwartz, A. G., Galatz, L. M. & Thomopoulos, S. Enthesis regeneration: a role for Gli+ progenitor cells. Development 144, 1159–1164 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Heldin, C.-H. & Lennartsson, J. Structural and functional properties of platelet-derived growth factor and stem cell factor receptors. Cold Spring Harb. Perspect. Biol. 5, a009100 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Qiu, X. et al. Reversed graph embedding resolves complex single-cell trajectories. Nat. Methods 14, 979–982 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. HamiltonT. G., KlinghofferR. A., CorrinP. D. & SorianoP. Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms. Mol. Cell. Biol. 23, 4013–4025 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Dominici, M. et al. Minimal criteria for defining multi-potent mesenchymal stromal cells: the international society for cellular therapy position statement. Cytotherapy 8, 315–317 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Sung, J. H. et al. Isolation and characterization of mouse mesenchymal stem cells. Transplant. Proc. 40, 2649–2654 (2008).

    Article  CAS  PubMed  Google Scholar 

  42. Pittenger, M. F. et al. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147 (1999).

    Article  CAS  PubMed  Google Scholar 

  43. Franchi, M., Trire, A., Quaranta, M., Orsini, E. & Ottani, V. Collagen structure of tendon relates to function. Sci. World J. 7, 404–420 (2007).

    Article  CAS  Google Scholar 

  44. Richardson, S. H. et al. Tendon development requires regulation of cell condensation and cell shape via cadherin-11-mediated cell–cell junctions. Mol. Cell. Biol. 27, 6218–6228 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Starborg, T. et al. Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization. Nat. Protoc. 8, 1433–1448 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Buschmann, J. & Bürgisser, G. Biomechanics of Tendons and Ligaments: Tissue Reconstruction and Regeneration. (Elsevier, 2017).

  47. Baksh, N., Hannon, C. P., Murawski, C. D., Smyth, N. A. & Kennedy, J. G. Platelet-rich plasma in tendon models: a systematic review of basic science literature. Arthroscopy 29, 596–607 (2013).

    Article  PubMed  Google Scholar 

  48. Evrova, O. & Buschmann, J. In vitro and in vivo effect of PDGF-BB delivery strategies on tendon healing: a review. Eur. Cell Mater. 34, 15–39 (2017).

    Article  CAS  PubMed  Google Scholar 

  49. Rodriguez, C. I. et al. High-efficiency deleter mice show that FLPe is an alternative to Cre-loxP. Nat. Genet. 25, 139–140 (2000).

    Article  CAS  PubMed  Google Scholar 

  50. Tallquist, M. D. & Soriano, P. Cell autonomous requirement of PDGFRa in populations of cranial and cardiac neural crest cells. Development 130, 507–518 (2003).

    Article  CAS  PubMed  Google Scholar 

  51. Liu, P., Jenkins, N. A. & Copeland, N. G. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res. 13, 476–484 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Wu, S., Ying, G., Wu, Q. & Capecchi, M. R. A protocol for constructing gene targeting vectors: generating knockout mice for the cadherin family and beyond. Nat. Protoc. 3, 1056–1076 (2008).

    Article  CAS  PubMed  Google Scholar 

  53. Matsuda, T. & Cepko, C. L. Controlled expression of transgenes introduced by in vivo electroporation. Proc. Natl Acad. Sci. USA 104, 1027–1032 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Gronthos, S., Mankani, M., Brahim, J., Robey, P. G. & Shi, S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc. Natl Acad. Sci. USA 97, 13625–13630 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Chien et al. Optimizing a 3D model system for molecular manipulation of tenogenesis. Connect. Tissue Res. 4, 295–308 (2018).

    Article  CAS  Google Scholar 

  56. Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Satija, R., Farrell, J. A., Gennert, D., Schier, A. F. & Regev, A. Spatial reconstruction of single-cell gene expression. Nat. Biotechnol. 33, 495–502 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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.

Author information

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

Authors
  1. Tyler Harvey
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sara Flamenco
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chen-Ming Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Chen-Ming Fan.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Source data

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-019-0417-z

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing