Figures and data
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Potential tendon differentiation ability of human myogenic progenitor cells
a. UMAP plot of all cells colored by cell types.
b. fDot blot of representative genes in each cell type.
c. Pseudotime trajectory inference of muscle progenitor cells, tenocytes and muscle cells. Different celltypes were annotated.
d. Heatmap showing the different expression levels of specific markers during the trajectory.
e. RNA FISH of PAX7 and SCX in human skeletal muscle section. Scale bars, 10μm.
f. Statistical analysis of the percentage of PAX7+ or SCX+ cells by RNA FISH in human skeletal muscle section. Error bars indicated standard deviation (n=5).
g. Immunofluorescence staining of PAX7, MYOD1 and MYF5 in primary human myogenic progenitor cells. Scale bars, 100µm.
h. Statistical analysis of the percentage of PAX7+, MYOD1+ and MYF5+ cells in the isolated myogenic progenitor cells. Error bars indicated standard deviation (n=5).
i. Immunofluorescence staining of MyHC in myotubes differentiated from human myogenic progenitor cells. Primary human myogenic progenitor cells were isolated and differentiated to myotubes for 5 days followed by MyHC immunofluorescence staining. Scale bars, 100 µm.
j. Statistical analysis of the percentage of the percentage of nuclei in MyHC+ myotubes after differentiation of the myogenic progenitor cells. Error bars indicated standard deviation (n=5).
k. Relative expression level of PAX7, MYF5, and MYOD1 in human myogenic progenitor cells and differentiated myotubes. RT-qPCR assays were performed with human myogenic progenitor cells. Error bars indicated standard deviation (n=3). *** indicated p<0.001.
l. Relative expression level of MYOG, MYH1, and MYH3 in human myogenic progenitor cells and differentiated myotubes. RT-qPCR assays were performed with human muscle myogenic progenitor cells after myogenic differentiation. Error bars indicated standard deviation (n=3). *** indicated p<0.001.
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Human myogenic progenitor cells display tendon differentiation ability in vitro
a. Immunofluorescence staining of tendon marker TNC and SCX in human myogenic progenitor cells induced for myogenic and tenogenic differentiation, respectively. Scale bars, 100 µm.
b. Quantification of TNC and SCX fluorescent intensity in human myogenic progenitor cells undergone myogenic and tenogenic differentiation, respectively. Error bars indicated standard deviation (n=5).
c. Relative expression levels of genes enriched in tendon cells. RT-qPCR assays were performed with human myogenic progenitor cells upon myogenic and tenogenic differentiation, respectively. Error bars indicated standard deviation (n=3). *** indicated p<0.001.
d. Scheme of clonal proliferation and differentiation assay.
e. Representative immunofluorescence staining images of MyHC as the marker for successful myogenic differentiation. Scale bars, 100 µm.
f. Representative immunofluorescence staining images of SCX as the marker for successful tenogenic differentiation. Scale bars, 100 µm.
g. Statistical analysis of the myogenic and tenogenic differentiation efficiency of human myogenic progenitor cells. Error bars indicated standard deviation (n=5).
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Human myogenic progenitor cells have tendon differentiation potential
a. Heat map of gene expression profiles of human myogenic progenitor cells, human muscle myogenic progenitor cells after myogenic differentiation, human myogenic progenitor cells after tenogenic differentiation, and primary tenocytes.
b.cVolcano plot of genes enriched in myogenic differentiation of human myogenic progenitor cells.
c. Volcano plot of genes enriched in tenogenic differentiation of human myogenic progenitor cells.
d. Bubble chart of GO analysis of cellular process up-regulated in myogenic differentiation of human myogenic progenitor cells.
e. Bubble chart of GO analysis of cellular process up-regulated in tenogenic differentiation of human myogenic progenitor cells.
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Murine MuSCs display poor tenogenic differentiation ability
a. Immunofluorescence staining of murine MuSCs directed towards myogenic and tenogenic differentiation, respectively. The undifferentiated murine MuSCs were stained with Pax7 and DAPI. Murine MuSCs after myogenic differentiation were stained with MyHC and DAPI. Murine MuSCs after tenogenic differentiation were stained with Scx and DAPI. Scale bars, 100 µm.
b. Statistical analysis of the percentage of nuclei in MyHC+ myotubes. Error bars indicated standard deviation (n=5).
c. Relative expression levels of myogenic differentiation specific genes MyoG, Myh1, and Myh3. Mouse MuSCs were induced to differentiate towards muscle and tendon, respectively. Mouse MuSCs before and after differentiation together with primary tenocytes were harvested and subjected for RT-qPCR analysis. Error bars indicated standard deviation (n=3). * indicated p<0.05, ** indicated p<0.01, n.s. indicated n>0.05.
d. Relative expression levels of tenogenic differentiation marker genes Scx, Tnc, Col I, Mkx and Thbs4. Mouse MuSCs were induced to differentiate towards muscle and tendon, respectively. Mouse MuSCs before and after differentiation together with primary tenocytes were harvested and subjected for RT-qPCR analysis. Error bars indicated standard deviation (n=3). n.s. indicated n>0.05.
e. Scheme of Pax7+ MuSC progeny lineage tracing.
f. Immunofluorescence staining of tendon tissue 4 months after injury. tdTomato+ cells indicated the progeny of Pax7+ MuSCs. TNC+ cells indicated tendon cells. MyHC+ cells indicated muscle fibers. DAPI indicated nuclei staining. Merged indicated the merged images of tdTomato, TNC, MyHC, and DAPI. The upper panel indicated the low magnification images. Scale bars, 100 µm. The lower panel indicated the high magnification images of the region label by white square in the upper panel. Scale bars, 10 µm.
g. The statistical analysis of the percentage of tdTomato+ TNC+ cell 4 months after tendon injury. Error bars indicated standard deviation (n=5).
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Transplantation of human myogenic progenitor cells facilitates tendon regeneration
a. Scheme of muscle stem/progenitor cell transplantation.
b. Immunofluorescence staining of the regenerated tendon-like tissue after human myogenic progenitor cell transplantation. Tendon injury was induced in recipient NOD/SCID mice and 50,000 human myogenic progenitor cells were transplanted to the injured tendon at the injured site. The regenerated tendon-like tissue, the connected muscle, and the surrounding soft tissues were harvested to make continuous cryosections. One of the continuous cryosections were subjected for immunofluorescence staining of tendon marker human specific TNC and human specific Lamin A/C. DAPI indicated nuclei staining. Merge indicated the merged images of human TNC, human Lamin A/C, and DAPI. The white lines indicated the location of regenerated tendon-like tissues from human myogenic progenitor cells based on human TNC staining. The yellow dashed lines indicated the superimposed location of muscle based on MyHC staining in panel c. Scale bars, 100 µm.
c. Immunofluorescence staining of MyHC and human Lamin A/C. The regenerated tendon-like tissue, the connected muscle, and the surrounding soft tissues were harvested and subjected for continuous cryosection. One of the continuous cryosections were stained for MyHC which was specifically expressed in skeletal muscle, and human Lamin A/C to label human cells. DAPI indicated nuclei staining. Merge indicated the merged images of MyHC, human Lamin A/C, and DAPI. The white lines indicated the location of regenerated tendon-like tissue from human myogenic progenitor cells based on human TNC staining in panel b. The yellow dashed lines indicated the location of muscle based on MyHC staining. Scale bars, 100µm.
d. Statistical analysis of the percentage of human cells expressing skeletal muscle marker MyHC or tendon marker TNC after being transplanted to the injured tendon. Error bars indicated standard deviation (n=5).
e. Immunofluorescence staining of Col I, Col III and human Lamin A/C. Two months after human cell transplantation, continuous cryosections containing the regenerated tendon-like tissue and native tendon tissue was stained with Col I, Col III and human Lamin A/C. DAPI indicated the staining of nuclei. T, native tendon tissue; W, wound tendon tissue. Scale bars, 50 µm.
f. Immunofluorescence staining of tendon tissue after transplantation of tdTomato+ murine MuSCs. tdTomato indicated the progenies of murine MuSCs. TNC indicated immunofluorescence staining of tendon marker TNC. MyHC indicated immunofluorescence staining of myofiber marker MyHC. DAPI indicated nuclei staining. Merge indicated merged images of tdTomato, TNC, MyHC, and DAPI. The upper panel indicated low magnification images. Scale bars, 100 µm. The lower panel indicated the amplified images of the region indicated by the white square. Scale bars, 10 µm.
g. Statistical analysis of TNC+ tdTomato+ cells in tendon tissue after transplantation of murine MuSCs. Error bars indicated standard deviation (n=5).
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Transplantation of human myogenic progenitor cells improves the locomotor function after tendon injury
a. TEM images of collagen fibrils in the injured tendon with PBS injection, injured tendon with human myogenic progenitor cell transplantation, and uninjured tendon. Scale bars, 500nm.
b. Statistical analysis of the collagen fibril diameter and percentage of collagen fibril area for the injured tendon with PBS injection, injured tendon with human myogenic progenitor cell transplantation, and uninjured tendon. Error bars indicated standard deviation (n=5). * indicated p<0.05, *** indicated p<0.001.
c. Statistical analysis of the max load and stiffness of the injured tendon with PBS injection, injured tendon with human myogenic progenitor cell transplantation, and uninjured tendon. Error bars indicated standard deviation (n=5). * indicated p<0.05, ** indicated p<0.01, *** indicated p<0.001.
d. Twitch and tetanus plantarflexion force of the involved limb with PBS injection after tendon injury, human myogenic progenitor cell transplantation after tendon injury, and uninjured tendon. Error bars indicated standard deviation (n=5). * indicated p<0.05, ** indicated p<0.01, *** indicated p<0.001.
e. The treadmill exercise for tendon injured mice with or without human myogenic progenitor cell transplantation. The endurance time and max fatigue speed were compared. Error bars indicated standard deviation (n=4). * indicated p<0.05, ** indicated p<0.01, ***indicated p<0.001 and n.s. indicated p>0.05.
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TGFβ signaling pathway contributes to tenogenic differentiation of human myogenic progenitor cells
a. Bubble chart of KEGG enrichment analysis of upregulated genes in human myogenic progenitor cells when compared with mouse muscle stem cells.
b. Heatmap of detailed upregulated genes in human myogenic progenitor cells which were enriched in TGFβ signaling pathway.
c. Protein levels of SMAD2/SMAD3 and phosphorylated SMAD2/SMAD3. Human myogenic progenitor cells were cultured in growth medium, tenogenic induction medium with or without TGFβ signaling pathway inhibitor SB-431542 for 2 hours. Total protein was extracted from cells with different treatments and subjected for total SMAD2/SMAD3 and phosphorylated SMAD2/SMAD3 immunoblotting. GAPDH was served as loading control.
d. Immunofluorescence staining of tendon marker TNC and SCX in human myogenic progenitor cells induced for tenogenic differentiation with or without TGFβ signaling pathway inhibitor SB-431542 for 12 days, respectively. Scale bars, 100 µm.
e. Protein levels of TNC and SCX. Human myogenic progenitor cells were induced towards tenogenic differentiation with or without TGFβ signaling pathway inhibitor SB-431542 for 12 days, respectively. Total protein was extracted from cells before and after differentiation and subjected for TNC and SCX immunoblotting. GAPDH was served as loading control.
f. Relative expression levels of tendon related genes. RT-qPCR assays were performed with human myogenic progenitor cells upon tenogenic differentiation with or without TGFβ signaling pathway inhibitor SB-431542 for 12 days, respectively. Error bars indicated standard deviation (n=3). *** indicated p<0.001.
g. Immunofluorescence staining of myogenic differentiation marker MyHC in human myogenic progenitor cells induced for tenogenic differentiation with or without TGFβ signaling pathway inhibitor SB-431542 for 12 days, respectively. Scale bars, 50 µm.
h. Relative expression levels of muscle related genes. RT-qPCR assays were performed with human myogenic progenitor cells upon tenogenic differentiation with or without TGFβ signaling pathway inhibitor SB-431542 for 12 days, respectively. Error bars indicated standard deviation (n=3). *** indicated p<0.001.
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Human myogenic progenitor cells display tenogenic differentiation potential
a. Phase contrast images of human myogenic progenitor cells before and after differentiation induction. Scale bars, 100 µm.
b. Immunofluorescence staining of myogenic differentiation marker genes MYOG and MyHC. Human myogenic progenitor cells were induced for tenogenic differentiation. The differentiated cells were stained with MYOG and MyHC antibody. Red indicated MYOG or MyHC; DAPI indicated nuclei staining; merge indicated the merged images of red and blue staining. Scale bars, 100µm.
c. Statistical analysis of the percentage of human cells expression MYOG or MyHC under tenogenic differentiation condition. Error bars indicated standard deviation (n=5).
d. Expression level of genes marking myogenic differentiation. Human myogenic progenitor cells were induced for myogenic and tenogenic differentiation, respectively. The undifferentiated and differentiated cells were subjected for RT-qPCR analysis of MYH1, MYH3, DESMIN, and MYL1. Error bars indicated standard deviation (n=5). *** indicated p<0.001.
e. Protein levels of TNC and MyHC. Human myogenic progenitor cells were induced towards myogenic and tenogenic differentiation, respectively. Total protein was extracted from cells before and after differentiation and subjected for TNC and MyHC immunoblotting. HSP70 and GAPDH were served as loading control.
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Murine MuSCs display poor tenogenic differentiation ability
a. Immunofluorescence images of Pax7, MyoD1 and Vcam1 for mouse muscle CD29+/CD56+ cells and Vcam1+ cells isolated by FACs. Scale bars, 50μm.
b. Statistical analysis of percentage of Pax7, MyoD1 and Vcam1 positive cells in mouse muscle CD29+/CD56+ cells and Vcam1+ cells isolated by FACs. *** indicated p<0.001.
c. Relative expression levels of tendon related genes. RT-qPCR assays were performed with murine muscle CD29+/CD56+ cells before and after tenogenic differentiation, and tenocytes. Error bars indicated standard deviation (n=3). *** indicated p<0.001.
d. Schematic illustration of the mouse line for murine MuSCs lineage tracing.
e. The surgery in mice mimicking human peroneus longus tendon removal surgery. The medial gastrocnemius tendon was completely removed. Scale bars, 1mm.
f. Immunofluorescence images of injured muscle adjacent to the removed tendon. The MuSCs were labeled with tdTomato before injury. The tdTomato positive myofibers after muscle injury were analyzed to evaluate the tracing system. Scale bars, 100μm.
g. Statistical analysis of the tdTomato positive myofibers adjacent to the removed tendon. Error bars indicated standard deviation (n=5).
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Transplantation of human myogenic progenitor cells facilitates tendon regeneration and transplantation of human/murine myogenic progenitor cells improve muscle regeneration
a. Immunofluorescence staining of SCX and human specific Lamin A/C in injured tendon. Two months after human cells transplantation, the injured tendon was stained with SCX and human specific Lamin A/C. DAPI indicated nuclei staining. Merge indicated the merged images of SCX, human specific Lamin A/C, and DAPI. Scale bars, 20 µm.
b. Immunofluorescence staining of TNMD and human specific Lamin A/C in injured tendon. Two months after human cell transplantation, the injured tendon was stained with TNMD and human specific Lamin A/C. DAPI indicated nuclei staining. Merge indicated the merged images of TNMD, human Lamin A/C, and DAPI. Scale bars, 20 µm.
c. Statistical analysis of the percentage of human cells expressing tendon related markers SCX or TNMD after being transplanted to the injured tendon. Error bars indicated standard deviation (n=5).
d. Scheme of human myogenic progenitor cell transplantation after muscle injury. TA muscles were first irradiated to kill the local MuSCs in NOD/SCID mice. Then transplantation of human myogenic progenitor cells to the irradiated pre-injured recipient mice was performed and TA muscles were harvested after 28 days.
e. Immunofluorescence staining of MyHC and human specific Lamin A/C after human myogenic progenitor cells transplantation. Scale bars, 100μm.
f. Statistical analysis of the engraftment efficiency of human myogenic progenitor cell transplantation in muscle. Error bars indicated standard deviation (n=5).
g. Scheme of murine MuSC transplantation after muscle injury. TA muscles were first irradiated to kill the local MuSCs in NOD/SCID mice. Then transplantation of tdTomato positive MuSCs to the irradiated pre-injured recipient mice was performed and TA muscles were harvested after 28 days.
h. Immunofluorescence staining of Laminin and murine MuSCs constitutively expressing tdTomato after murine tdTomato positive MuSCs transplantation. Scale bars, 100μm.
i. Statistical analysis of the engraftment efficiency of murine MuSCs transplantation in muscle. Error bars indicated standard deviation (n=5).