A tppp3+pdgfra+ tendon stem cell population contributes to regeneration and reveals a shared role for pdgf signalling in regeneration and fibrosis

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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your

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subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS A CD26+ TENDON STEM PROGENITOR CELL POPULATION CONTRIBUTES TO TENDON REPAIR AND HETEROTOPIC

OSSIFICATION Article Open access 16 January 2025 ENDOGENOUS TENOCYTE ACTIVATION UNDERLIES THE REGENERATIVE CAPACITY OF THE ADULT ZEBRAFISH TENDON Article Open access 19 September 2023

PI3K-AKT SIGNALLING REGULATES _SCX_-LINEAGE TENOCYTES AND _TPPP3_-LINEAGE PARATENON SHEATH CELLS IN NEONATAL TENDON REGENERATION Article Open access 20 April 2025 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 * Elliott, D. H. Structure and function of mammalian

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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 * Carnegie Institution for Science, Department of Embryology, Baltimore, MD, USA Tyler

Harvey & Chen-Ming Fan * Johns Hopkins University, Department of Biology, Baltimore, MD, USA Tyler Harvey & Sara Flamenco Authors * Tyler Harvey View author publications You can also

search for this author inPubMed Google Scholar * Sara Flamenco View author publications You can also search for this author inPubMed Google Scholar * Chen-Ming Fan View author publications

You can also search for this author inPubMed 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 _Tppp3_CG/+;_R26R_tdT tendon; upper, no TMX control with anti-GFP antibody (Ab.); middle, +TMX and Ab.; lower, +TMX and no Ab. control–eGFP expressed by the _Tppp3_CG

driver is only detectable with Ab. staining; 3. _Tppp3_CG driver labels 38.3±2.6 (Mean±SEM)% of sheath cells. D, _Tppp3_CG driver labeling efficiency is 79.5±3.4 (Mean±SEM)%; n=3 animals. E,

Fluorescent images of _Tppp3_CG/+;_R26R_tdT 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 _Tppp3_CG/+;_R26R_tdT 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, _Tppp3_ECE/+;_R26R_tdT;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 _Tppp3_CG, _Tppp3_ECE 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 _Tppp3_ECE/+;_R26R_tdT;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 _Tppp3_ECE/+;_R26R_tdT;_Pdgfra_H2B-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 _Pdgfra_H2B-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 _Tppp3_CG/+;_R26R_tdT;_Pdgfra_H2B-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 _Pdgfra_H2B-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; _Tppp3_CG

efficiency is 77.0±1.6 (Mean±SEM)% and _Tppp3_ECE 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 _Tppp3_ECE/+;_R26R_tdT;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 _Tppp3_CG/+;_Pdgfra_fl/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, _Tppp3_CG/+;_R26R_tdT, and R-cKO,

_Tppp3_CG/+;_R26R_tdT;_Pdgfra_fl/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 STATISTICAL SOURCE DATA FIG. 1 STATISTICAL SOURCE DATA FIG. 2 STATISTICAL SOURCE DATA FIG. 3 STATISTICAL

SOURCE DATA FIG. 4 STATISTICAL SOURCE DATA FIG. 5 STATISTICAL SOURCE DATA FIG. 6 STATISTICAL SOURCE DATA FIG. 7 STATISTICAL SOURCE DATA FIG. 8 STATISTICAL SOURCE DATA EXTENDED DATA FIG. 1

STATISTICAL SOURCE DATA EXTENDED DATA FIG. 2 STATISTICAL SOURCE DATA EXTENDED DATA FIG. 3 STATISTICAL SOURCE DATA EXTENDED DATA FIG. 4 STATISTICAL SOURCE DATA EXTENDED DATA FIG. 5 RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE 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: 09

November 2018 * Accepted: 27 September 2019 * Published: 25 November 2019 * Issue Date: December 2019 * DOI: https://doi.org/10.1038/s41556-019-0417-z SHARE THIS ARTICLE Anyone you share the

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