Wnt7a stimulates myogenic stem cell motility and engraftment resulting in improved muscle strength

Abstract

Wnt7a/Fzd7 signaling stimulates skeletal muscle growth and repair by inducing the symmetric expansion of satellite stem cells through the planar cell polarity pathway and by activating the Akt/mTOR growth pathway in muscle fibers. Here we describe a third level of activity where Wnt7a/Fzd7 increases the polarity and directional migration of mouse satellite cells and human myogenic progenitors through activation of Dvl2 and the small GTPase Rac1. Importantly, these effects can be exploited to potentiate the outcome of myogenic cell transplantation into dystrophic muscles. We observed that a short Wnt7a treatment markedly stimulated tissue dispersal and engraftment, leading to significantly improved muscle function. Moreover, myofibers at distal sites that fused with Wnt7a-treated cells were hypertrophic, suggesting that the transplanted cells deliver activated Wnt7a/Fzd7 signaling complexes to recipient myofibers. Taken together, we describe a viable and effective ex vivo cell modulation process that profoundly enhances the efficacy of stem cell therapy for skeletal muscle.

Figure 1.

Wnt7a and Fzd7 induce the polarization and migration of myogenic cells. (A) Morphological quantification of triangular polarized C2C12 cells upon Wnt7a stimulation and Fzd7 overexpression. Vehicle (Veh.)-treated cells expressing YFP were set to 100%. Error bars represent means ± SEM; n ≥ 4. *, P < 0.05; **, P < 0.01. n.s., no significant difference. (B) Confocal images showing the localization of Fzd7-YFP and the tubulin cytoskeleton of a C2C12 cell. Bar, 4 µm. (C) Sequences derived from live imaging of C2C12 cells that were transfected with Fzd7-YFP or YFP at the given time points. The arrowheads show peripheral Fzd7-YFP that is dynamically rearranged during cell migration. Bar, 10 µm. (D) Frequency of peripheral Fzd7-YFP accumulation in C2C12 cells when compared with Fzd3-YFP. Fzd7-YFP was set to 100%. Error bars represent means ± SEM; n = 3. **, P < 0.01. (E) Representative images from scratch assays with C2C12 cells. The dashed line represents the border of the scratch wound. Cells that were treated with Wnt7a migrate farther than vehicle-treated cells. Bar, 100 µm. (F) Quantification of C2C12 migration in scratch assays as shown in E. Wnt7a significantly increases migration compared with vehicle. Error bars represent means ± SEM; n = 3. **, P < 0.01. (G) Overexpression of Fzd7-Flag also increases the migration of C2C12 cells in scratch assays when compared with empty vector (EV). Error bars represent means ± SEM; n = 3. **, P < 0.01.

Figure 2.

Wnt7a and Fzd7 facilitate directed cell migration. (A) Scratch migration assay with mouse primary myoblasts that were stimulated with Wnt7a or vehicle. Error bars represent means ± SEM; n = 3. *, P < 0.05. (B) Quantification of scratch assays using primary myoblasts overexpressing EV or Fzd7-Flag. Error bars represent means ± SEM; n = 3. ***, P < 0.001. (C) Primary myoblasts derived from Fzd7 knockout mice (Fzd7^−/−) do not respond to Wnt7a stimulation. Error bars represent means ± SEM; n ≥ 3. n.s., no significant difference. (D) Scratch migration assay showing that Fzd^−/− primary myoblasts migrate significantly less than heterozygous cells (Fzd7^+/−). Genetic Fzd7 knockout can be rescued by expression of Fzd7-Flag. Error bars represent means ± SEM; n = 3. ***, P < 0.001. (E) Quantification showing that canonical Wnt3a does not affect cell migration in scratch wound assays. Error bars represent means ± SEM; n = 3. (F) Mean velocity of satellite cells on single cultured myofibers as determined by live imaging. Error bars represent means ± SEM; n ≥ 27. *, P < 0.05. (G) The mean maximal speed of Wnt7a-stimulated satellite cells is not significantly different from the vehicle control. Error bars represent means ± SEM; n ≥ 27. (H) Quantification of the mean change in direction of Wnt7a-treated satellite cells. In the presence of Wnt7a the cells migrate with increased directional persistence when compared to vehicle. Error bars represent means ± SEM; n ≥ 27. ***, P < 0.001. (I) Representative tracks of satellite cells on single cultured myofibers. The green “x” represents the start of imaging, and the blue “x” is the stop. Fewer changes in directional motility can be observed for the Wnt7a-treated satellite cell.

Figure 3.

Dvl2 and Rac1 are required for Wnt7a-induced cell migration. (A) Fzd7-tdTomato colocalizes with GFP-Rac1 (arrowhead) in the periphery of C2C12 cells but not in cytoplasmic vesicles. Bar, 5 µm. (B) Rac1 activation assay of mouse primary myoblasts transduced with Wnt7a-HA (Wnt7a) retrovirus or an empty control virus (EV). In addition, all cells were either treated with an siRNA SMARTpool targeting Dvl2 (siDvl2) or with a scrambled control (siSCR). Total Rac1 is shown as a loading control. (C) Coimmunoprecipitation of Rac1 with Dvl2 in primary myoblasts that were infected with Wnt7a or EV. More Rac1 associates with Dvl2 in Wnt7a-expressing cells. (D) Scratch assay with mouse primary myoblasts that were Wnt7a or vehicle treated. The cells were also transfected with either siSCR or siDvl2. Error bars represent means ± SEM; n = 3. *, P < 0.05. n.s., no significant difference. (E) Scratch assay with mouse primary myoblasts that overexpress EV or Fzd7-Flag and that were treated with siDvl2 or siSCR. Error bars represent means ± SEM; n = 3. *, P < 0.05. (F) Dominant-negative Rac1 (Rac1-DN) prevents Wnt7a-induced mouse primary myoblast migration in scratch assays. Error bars represent means ± SEM; n = 3. **, P < 0.01. (G) Rac1-DN prevents Fzd7-Flag–induced mouse primary myoblast migration in scratch assays. Error bars represent means ± SEM; n = 3. *, P < 0.05.

Figure 4.

The Fzd7/Wnt7a signal is strictly noncanonical. (A) Wnt3a induces a dose-dependent increase in TOP-flash luciferase reporter activity in C2C12 cells. Error bars represent means ± SEM; n = 3. *, P < 0.05. n.s., no significant difference. (B) Wnt7a does not activate the TOP-flash reporter at any tested concentration. Error bars represent means ± SEM; n = 3. (C) Wnt7a treatment decreases the abundance of peripheral Fzd7-YFP and leads to its accumulation in intracellular clusters (arrowheads). Bar, 5 µm. (D) Inhibition of clathrin-dependent endocytosis with monodansylcadaverine (MDC) prevents Wnt7a-induced migration of primary myoblasts in scratch assays. Error bars represent means ± SEM; n = 3. ***, P < 0.001. (E) Substantial amounts of Wnt7a-HA are present in intracellular stores (arrowhead) 72 h after a 3-h exposure to conditioned supernatants produced in COS-1 cells (top). In primary myoblasts from Fzd7^−/− mice, Wnt7a does not show such intracellular accumulation (bottom). Bars, 2 µm.

Figure 5.

Wnt7a loading increases myoblast dispersal in muscle tissue. (A) Experimental scheme for the in vivo myoblast dispersal assay. Cells expressing tdTomato were treated with Wnt7a or vehicle for 3 h, washed, and transplanted into C57BL/6 mice. Blue fluorescent microspheres were co-injected to mark the injection site. After 7 d, the distance from the closest microsphere to myofibers expressing high (tdT+++), medium (tdT++), or low (tdT+) level of tdTomato was enumerated in muscle cross sections. (B) The total number of tdTomato-expressing myofibers generated by fusion with donor myoblasts was increased by Wnt7a treatment compared to vehicle. Error bars represent means ± SEM; n = 3. *, P < 0.05. (C) Representative images showing the abundance and fluorescent intensity of myofibers expressing tdTomato with respect to the injection site (outlined by a dashed oval). The injection site is marked by a high concentration of blue fluorescent microspheres. In the case of the cells that were treated with Wnt7a, the myofibers are more spread out and generally express reduced levels of tdTomato. Bar, 50 µm. (D–F) The minimal distance of tdT+++, tdT++, and tdT+ myofibers to the microspheres was measured in muscle cross-section according to the false-color image shown in the respective insets. Asterisks in the false-color images indicate the tdT fluorescence intensity of the fibers that were quantified. The data for the minimal distance from microspheres of the myofiber types was grouped into 200-µm bins. In the vehicle-treated condition, a large fraction of myofibers proximal to the injection site is tdT+++, whereas few distal tdT+ myofibers are present. The Wnt7a condition shows the opposite trend. Error bars represent means ± SEM; n ≥ 3. *, P < 0.05; **, P < 0.01. n.s., no significant difference.

Figure 6.

Wnt7a-loaded satellite cells have an enhanced engraftment potential. (A) Schematic of the isolation, ex vivo treatment, and transplantation of satellite cells from Pax7-zsGreen mice. (B) Representative pictures showing engraftment of zsGreen- and Pax7-expressing donor-derived cells that were either Wnt7a or vehicle treated. Bar, 20 µm. (C) Quantification of engraftment of Wnt7a- or vehicle-treated donor satellite cells (Pax7⁺/zsGreen⁺, arrowheads in B). Error bars represent means ± SEM; n = 3. **, P < 0.01. (D) Representative images showing dystrophin-expressing myofibers (asterisks) derived from transplanted Wnt7a or vehicle-treated satellite cells. Bar, 20 µm. (E) Quantification of myofibers expressing dystrophin after transplantation of Wnt7a- or vehicle-treated satellite cells. Error bars represent means ± SEM; n = 3. **, P < 0.01. (F) Engrafted satellite cells form clusters of dystrophin-expressing myofibers in host mdx muscles. The photographs show the tibialis anterior muscle that was injected with Wnt7a- or vehicle-treated cells. The distance between the two maximally spaced clusters of dystrophin-expressing myofibers (highlighted in red) is indicated with a magenta line. (G) Quantification of the maximal cluster distance in muscles injected with Wnt7a- or vehicle-treated cells. Error bars represent means ± SEM; n ≥ 4. **, P < 0.01. (H) Minimal feret measurements of dystrophin-expressing myofibers in host muscles. Myofibers that had fused with a Wnt7a-treated cell become hypertrophic. Error bars represent means ± SEM; n = 3. *, P < 0.05. (I) Quantification of the twitch force tension of mdx muscles injected with Wnt7a- or vehicle-treated cells. Error bars represent means ± SEM; n ≥ 7. **, P < 0.01. (J) Maximal specific force generated by mdx muscles injected with Wnt7a- or vehicle-treated cells. Error bars represent means ± SEM; n ≥ 7. *, P < 0.05.

Figure 7.

Wnt7a stimulates dispersal of human primary myoblasts. (A) Scratch assay with human myoblasts. Wnt7a treatment significantly increases cell migration into the scar and expression of Rac1-DN prevents this effect. Error bars represent means ± SEM; n = 3. **, P < 0.01. (B) Fzd7-Flag increases human myoblast migration and Rac1-DN prevents this effect. Error bars represent means ± SEM; n = 3. **, P < 0.01. n.s., no significant difference. (C) Strategy used for Wnt7a or vehicle treatment and subsequent transplantation of human primary myoblasts into mdx mice. (D) Number of dystrophin-expressing myofibers after transplantation of Wnt7a- or vehicle-treated cells. Error bars represent means ± SEM; n ≥ 4. ***, P < 0.001. (E) Minimal myofiber Feret of dystrophin-expressing myofibers generated from fusion with Wnt7a- or vehicle-treated human primary myoblasts. Error bars represent means ± SEM; n = 3. *, P < 0.05. (F) Mean maximum cluster difference in muscles transplanted with Wnt7a- or vehicle-treated human primary myoblasts. Error bars represent means ± SEM; n ≥ 4. **, P < 0.01.

Figure 8.

Molecular mechanisms of ex vivo Wnt7a modulation. Upon stimulation, Wnt7a induces the symmetric proliferation of Myf5-independent satellite cells in conjunction with fibronectin (FN1), syndecan-4 (SDC4) and Vangl2 through the planar cell polarity pathway. In myogenic progenitors, Wnt7a also facilitates Rac1-mediated cell polarization and migration. Fusion of Wnt7a-treated cells activates the AKT–mTOR pathway, leading to myofiber hypertrophy. Therefore, Wnt7a acts on three levels to facilitate the outcomes of cell therapy: (1) it boosts stem cell number, (2) facilitates their dispersion in the host tissue, and (3) leads to muscle growth.