Research ArticleHEALTH AND MEDICINE

Synthetic matrix enhances transplanted satellite cell engraftment in dystrophic and aged skeletal muscle with comorbid trauma

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Science Advances  15 Aug 2018:
Vol. 4, no. 8, eaar4008
DOI: 10.1126/sciadv.aar4008
  • Fig. 1 RGD-presenting PEG-4MAL hydrogels promote MuSC survival, proliferation, and differentiation.

    (A) Schematic of cell-laden hydrogel. Synthetic hydrogel platform can be biofunctionalized with cell-adhesive ligands and protease-degradable cross-linkers. (B) Representative fluorescence-activated cell sorting plot of MuSCs. SSC, side scatter; PI, propidium iodide; ITGB1, β1 integrin. (C) Representative z-projections of myogenic colonies formed in PEG-4MAL hydrogels presenting synthetic cell adhesive peptides (day 6). Scale bars, 100 μm. (D) Quantification of myogenic colony circularity. n = 9 and 10 colonies. *P < 0.05, ***P < 0.001, and ****P < 0.0001 versus RGD via Kruskal-Wallis with Dunn’s test. (E) Quantification of myogenic colony cell packing density. n = 9 and 10 colonies. ****P < 0.0001 versus all groups via one-way analysis of variance (ANOVA) with Tukey’s test. (F) Quantification of myogenic colony size. n = 9 and 10 colonies. ****P < 0.0001 versus all groups via one-way ANOVA with Tukey’s test. (G) Quantification of myogenic colony proliferation. n = 9 and 10 colonies. *P < 0.05 via two-way ANOVA with Sidak’s test. (H) Representative z-projections of DNA and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining (day 1). Scale bars, 50 μm. (I) Quantification of TUNEL+ cells. n = 5 hydrogels. #P < 0.05 via unpaired two-tailed t test. (J) Representative z-projections of green fluorescent protein–positive (GFP+) and TdTomato+ MuSCs. Cells were cultured in growth media for 6 days and then in differentiation media for 4 days. GFP/TdTomato-fused cells appear yellow. Scale bars, 100 μm.

  • Fig. 2 PEG-4MAL macromer density modulates MuSC proliferation.

    (A) Schematic describing the changes in hydrogel mesh size and mechanical properties as a function of PEG-4MAL macromer density. (B) Storage modulus of hydrogels for different macromer densities. n = 3 hydrogels. *P < 0.05 (solid line) and **P < 0.01 (dashed line) via one-way ANOVA with Tukey’s test. (C) Estimation of hydrogel mesh size based on the measured storage modulus. n = 3 hydrogels. *P < 0.05 (solid line) and **P < 0.01 (dashed line) via one-way ANOVA with Tukey’s test. (D) Representative z-projections of myogenic colonies formed in PEG-4MAL hydrogels as a function of macromer density (day 4). Scale bars, 100 μm (top row) and 250 μm (bottom row). Percentage values indicate PEG-4MAL macromer density. (E and F) Quantification of myogenic colony (n = 18 colonies) and density (n = 3 and 4 hydrogels). *P < 0.05, **P < 0.01, and ***P < 0.001 via one-way ANOVA with Tukey’s test. (G) Representative z-stack projections of EdU-labeled myogenic colonies (day 4). Scale bars, 100 μm. (H) Quantification of EdU+ nuclei. n = 12 to 15 colonies. *P < 0.05 and **P < 0.01 via one-way ANOVA with Tukey’s test. (I) Representative z-projections of MuSCs cultured in 4% 20-kDa and 2.5% 10-kDa PEG-4MAL hydrogels (day 4). Scale bars, 100 μm. (J) Quantification of myogenic colonies. n = 10 colonies. Bar graphs are presented as mean ± SD.

  • Fig. 3 Protease degradability of the hydrogel is essential for MuSC proliferation.

    (A) Representative z-projections of MuSCs cultured in RGD-functionalized hydrogels synthesized with protease-sensitive and protease-insensitive cross-linkers (day 4). Scale bars, 100 μm. TdTom., TdTomato. (B) Quantification of myogenic colonies. Median ± interquartile range. n = 20. (C) Representative myogenic colony formation over time in protease-sensitive hydrogel and in protease-insensitive hydrogels over 5 days. Scale bars, 50 μm (main images) and 10 μm (insets). (D) Storage modulus and (E) mesh size of protease-sensitive and protease-insensitive hydrogels over 5 days. **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus time-matched protease-sensitive hydrogels via two-way ANOVA with Sidak’s test. n = 4 hydrogels per time point. Mean ± SD.

  • Fig. 4 Synthetic matrix supports higher MuSC proliferation potential than natural matrices.

    (A) Representative z-projections of myogenic colonies formed in 4% PEG-4MAL and collagen gels (2.7 mg ml−1; day 4). Scale bars, 100 μm. (B) Quantification of myogenic colony size. n = 14 to 19 colonies. ****P < 0.0001 via two-tailed Mann-Whitney U test. (C) Representative z-projections of EdU-labeled myogenic colonies formed in 4% PEG-4MAL and collagen gels (2.7 mg ml−1; day 4). Scale bars, 100 μm. (D) Quantification of EdU+ nuclei. n = 19 colonies. P = 0.11 via unpaired two-tailed t test. (E) Representative z-projections of GFP+ MuSCs 1 day after encapsulation in 4% PEG-4MAL and collagen gels (2.7 mg ml−1). BF, bright field. Scale bars, 100 μm. (F) Quantification of TOTO-3+ dead MuSCs 1 day after encapsulation. n = 7 hydrogels. ****P < 0.0001 via unpaired two-tailed t test. (G) Representative images of GFP+ MuSCs 4 days after encapsulation in 4% PEG-4MAL and Matrigel. Scale bars, 100 μm. (H) Quantification of GFP+ myogenic colony area 4 days after encapsulation. n = 16 to 19 colonies. ****P < 0.0001 via two-tailed Mann-Whitney U test.

  • Fig. 5 Synthetic matrix enhances engraftment in dystrophic muscle trauma.

    (A) Schematic of injury and cell delivery. We applied cryo-injury on the surface of TA muscle. We supramuscularly delivered freshly isolated MuSCs in media (hydrogel-free) or encapsulated in hydrogel. Gastroc., gastrocnemius muscles. (B) Representative IVIS images of mdx-4CV mouse hindlimbs treated with cell-laden 3% 20-kDa PEG-4MAL, 6% 20-kDa PEG-4MAL, or media (12,500 MuSCs per TA). (C) Quantification of bioluminescence over time. Mean ± SEM. n = 8 per condition. *P < 0.05, **P < 0.01, and ****P < 0.0001 versus hydrogel-free via Tukey’s test within time. ++P < 0.01 versus 6% PEG-4MAL via Tukey’s test within time. P < 0.05 for interactions, xxP < 0.01 for biomaterials effect, and ###P < 0.001 for time effect via two-way repeated-measures ANOVA. (D) Quantification of area under the bioluminescence curve (AUC). Mean ± SEM. n = 8 per condition. *P < 0.05 versus hydrogel-free via one-way ANOVA with Tukey’s test. (E) Representative cross sections of TA muscles treated with cell-laden 3% 20-kDa PEG-4MAL, 6% 20-kDa PEG-4MAL, or media on day 28 after transplantation. Scale bars, 50 μm. (F) Quantification of GFP+ fibers per square millimiter. n = 8 per condition. *P < 0.05 versus all groups via Kruskal-Wallis with Dunn’s test. (G) Representative cross sections of TA muscles treated with cell-laden 3% 20-kDa PEG-4MAL functionalized with RGD or RDG peptides at day 28 after transplantation (12,500 MuSCs per TA). Scale bars, 50 μm. (H) Quantification of GFP+ fibers per square millimeter. n = 6 per condition. *P < 0.05 via two-tailed Mann-Whitney U test. (I) Representative IVIS images of MuSC-transplanted mdx-4CV mice. TA muscles were cryo-injured, and 20,000 MuSCs were delivered to the injured TA muscles in either PEG-4MAL hydrogel (left) or collagen gel (2.7 mg ml−1; right). (J) Quantification of bioluminescence over time. Mean ± SEM. n = 5 per condition. ++P < 0.01 within time via Sidak’s test. *P < 0.05 for interactions and biomaterials effect via two-way repeated-measures ANOVA. ###P < 0.05 for time effect via two-way repeated-measures ANOVA. (K) Quantification of AUC. n = 8 per condition. *P < 0.05 via two-tailed paired t test. (L) Representative cross sections of TA muscles treated with 50,000 MuSCs in either PEG-4MAL or collagen gel (2.7 mg ml−1). Scale bars, 100 μm. (M) Quantification of engrafted GFP+ fibers per square millimeter. n = 8 per condition. **P < 0.01 via two-tailed Wilcoxon matched-pairs signed-rank test.

  • Fig. 6 Synthetic matrix enhances engraftment in aged muscle trauma.

    (A) Hematoxylin and eosin images of young (4 months) and aged (22 months) TA muscles. Muscles were cryo-injured, and endogenous regeneration was assessed on day 14 (D14). (B) Quantification of cross-sectional area (CSA). Box-and-whiskers plot with 10 to 90 percentile whiskers. We measured ≥138 fibers. p.i., postinjury. ****P < 0.0001 versus young uninjured and young D14 postinjury; ####P < 0.0001 versus aged uninjured via one-way ANOVA with Dunn’s test. (C) Representative IVIS images of aged mice (23 months). TA muscles were cryo-injured, and 20,000 MuSCs encapsulated in PEG-4MAL hydrogel (left) or suspended in media (right) were transplanted. (D) Quantification of bioluminescence over time. Mean ± SEM. n = 6 per condition. *P < 0.05 via two-tailed Wilcoxon matched-pairs signed-rank test within time. (E) Quantification of AUC. n = 6 per condition. *P < 0.05 via two-tailed Wilcoxon matched-pairs signed-rank test. (F) Cross sections of TA muscles at day 28 after transplantation. Scale bars, 50 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/8/eaar4008/DC1

    Fig. S1. Isolated primary MuSCs are Pax7+.

    Fig. S2. RGD-presenting hydrogels promote MuSC activation and proliferation.

    Fig. S3. RGD-presenting hydrogels promote MuSC differentiation.

    Fig. S4. Pax7/MyoD expression of MuSCs in 4 and 6% 20-kDa PEG-4MAL hydrogels.

    Fig. S5. 1D diffusion assay in PEG-4MAL hydrogels.

    Fig. S6. Synthetic matrix supports higher MuSC proliferation potential than collagen gel.

    Table S1. List of cell-adhesive synthetic peptides and their targets.

    Movie S1. Differentiated myotubes in RGD hydrogels contract in vitro.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Isolated primary MuSCs are Pax7+.
    • Fig. S2. RGD-presenting hydrogels promote MuSC activation and proliferation.
    • Fig. S3. RGD-presenting hydrogels promote MuSC differentiation.
    • Fig. S4. Pax7/MyoD expression of MuSCs in 4 and 6% 20-kDa PEG-4MAL hydrogels.
    • Fig. S5. 1D diffusion assay in PEG-4MAL hydrogels.
    • Fig. S6. Synthetic matrix supports higher MuSC proliferation potential than collagen gel.
    • Table S1. List of cell-adhesive synthetic peptides and their targets.

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Differentiated myotubes in RGD hydrogels contract in vitro.

    Files in this Data Supplement:

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