Investigating Adipogenic-Myogenic Signaling in Engineered Human Muscle Grafts to Treat Volumetric Muscle Loss

Introduction: Volumetric Muscle Loss (VML) is the loss of a large portion of skeletal muscle that overwhelms the natural repair mechanisms and results in functional impairment. Tissue engineered muscle grafts (TEMGs) provide a promising therapeutic strategy. We have previously developed grafts comprised of electrospun fibrin microfiber bundles with C2C12s or stem cells to create TEMGs. Human primary myogenic progenitors (hMPs) are myogenic and can be patient specific, making them a promising myogenic cell source for TEMGs. However, skeletal muscle is a complex tissue and contains a diversity of cells that are non-myogenic. Due to their great potential for clinical translation, we investigate human adipose-derived stem cells (hASCs) as a non-myogenic support population for our TEMGs. Our objectives were to (i) assess the effectiveness of hMP-seeded fibrin microfiber bundles as TEMGs (hMP TEMGs) for the treatment of VML, and (ii) assess the effectiveness of hMPs co-cultured with hASCs i.e. (hMP-hASC TEMGs) for treating VML.

Materials and

Methods: To accomplish the first objective, 900,000 hMPs (passage 3; Lonza) were seeded on electrospun fibrin microfiber bundles to create hMP TEMGs. The hMP TEMGs were implanted into critical-sized VML defects in which ~30–50% of the tibialis anterior (TA) of Male NOD-SCID IL2rγnull (NSG) mice aged 8–10 weeks was removed. We harvested at 28 days post-injury (DPI) and compared histologic sections with ‘No Treatment’ and contralateral ‘Uninjured’ groups. To accomplish the second objective, Passage 2 hASCs were seeded onto hMP TEMGs 7 days post-hMP seeding at 0.5% (4,500 hASCs), 5% (45,000 hASCs), and 50% (450,000 hASCs) of the hMPs concentration and compared to hMP TEMGs without hASCs to assess the effects of dosing at 21 days post-hMP seeding. Subsequently, hASCs were seeded (at 5% the concentration of hMPs) at 0 (6 hours), 3, and 7 days post-hMP seeding (D0, D3, D7) to assess the effects of co-culture timing. To assess the treatment potential of hMP-hASC TEMGs, grafts were implanted into the NSG TA VML model and compared to hMP TEMG treatment and no treatment histologically at 28 DPI. All animal and surgical procedures were performed in accordance with the Institutional Animal Care and Use Committee. All experiments have n = 4 except for qPCR which had n = 3-4. Statistical significance was determined using GraphPad Prism 5 software by either a one-way ANOVA with multiple comparisons and Bonferroni post-test or for qPCR unpaired two-tailed t-tests. Error bars represent SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Results, Conclusions, and Discussions: TAs treated with hMP TEMGs were harvested, sectioned, and stained with DAPI/Myosin Heavy Chain (MHC)/Desmin or H&E (Fig. 1A,B). Cross-sectional areas showed a significant increase between no treatment and hMP TEMG-treated VML, and no significant difference between hMP TEMG treated VML and uninjured (Fig. 1C). There was a slight increase in new or regenerating myofibers in hMP TEMG treated VML compared to no treatment VML, and both had significantly more than uninjured (Fig. 1D). During in vitro co-culture studies, the normalized MHC+ area increased up to 5% (hASCs), then decreased at 50% indicating a threshold in which hASCs exhibit pro-myogenic effects on hMPs TEMGs (Fig. 2A). Similarly, the normalized Desmin+ area was highest at 0.5% and 5% (Fig. 2B). Delaying the seeding of hASCs until Day 7 significantly increased the MHC+ and Desmin+ staining (Fig. 2C, D). To assess the impact of co-culture, we assessed gene expression of hMP TEMGs co-cultured with hASCs. Co-cultured hMP TEMG expressed decreased MYOG (as well as MYF5, MYF6, and MYOD1 (p>0.05)), decreased MYH1, and increased MYH7 (Fig. 2E). The hMP TEMGs co-cultured with hASCs also expressed higher MKI67 (Fig. 2F; p>0.05). Following VML treatment, TAs were weighed (Fig. 3B), sectioned, and stained with Masson’s Trichrome (Fig. 3A) to visualize collagen (scar tissue) area. The groups with hASCs showed significant overgrowth of the TAs. The normalized Col IV+ area increased when treated with hMP-hASC TEMGs (Fig. 3C). Quantification of hLamin AC+ cells showed an increase in survival of human cells when treated with hMP-hASC TEMGs (Fig. 3D). In sections stained with F4/80, iNOS, CD206, and DAPI, F4/80+/CD206+ signal was classified as M2 and F4/80+/iNOS+ signal was classified as M1 (Fig. 3E).
Dosing and timing of co-culture play critical roles in promoting myogenesis, however, hASCs promote a shift towards a more mature myogenic phenotype with higher proliferation. In vivo, hMP-hASC TEMGs were seen to increase ECM deposition, cell survival, and skewing towards an M2 phenotype. These benefits were accompanied by a large tumor-like growth which was not observed in prior studies with hASCs only or with hMPs only suggesting an interesting interplay in the cell-cell signaling.

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