(B-47) Heterogeneity in collective migration of skeletal muscle cells in an injured muscle model

Introduction:: Skeletal muscle tissues contain undifferentiated myoblasts and satellite cells, which are sources for self-regeneration following injury. After muscle injury, quiescent satellite cells are activated, and myoblasts migrate to the injury site, align and differentiate into myotubes. Collective motions in skeletal muscle regeneration are influenced by cellular heterogeneity due to the presence of undifferentiated cells such as satellite cells and myoblasts. This collective behavior of skeletal muscle cells is crucial for regeneration because it serves as targeted migration toward the injured region, maintaining homeostasis and aligning motion for improved differentiation. Despite the importance of the collective behavior of muscle cells, most studies on muscle cell motility during skeletal muscle regeneration involve single-cell behavior, and little is known about the collective behavior. In this study, we constructed an in vitro cell-based platform to model skeletal muscles with specific biomolecules released from the skeletal muscle cells using conditioned media from the normal versus injured muscle conditions. We identified the heterogeneous traits of the muscle cell clusters during collective migration under specific conditioned media environments.

Materials and Methods:: C2C12 cells (ATCC, CRL-1772) were cultured in Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at normal culture conditions. To mimic in vivo normal skeletal muscle and regeneration environment, we manually made Normal muscle-conditioned media (NM) and Damaged muscle-conditioned media (DM). NM and DM-conditioned media were obtained by fully differentiated and scratch-damaged C2C12 skeletal muscles cultured with 2% horse serum-based media (base media, BM), respectively (Fig. 1). We prepared 11kPa polyacrylamide gel (PA gel) and collagen type I for mimicking in vivo environment. Collagen type I was only coated on a restricted circular region (diameter = 700μm) of PA gel, patterned with a PDMS stencil. After culturing the skeletal muscle cells in a circular region, when the confluency of the cells in the circular island reached 90-100%, the PDMS stencil was then removed, and cells were allowed to migrate to free space to induce collective skeletal muscle cell migration. Traction force and intercellular stress were calculated and measured based on the movement of fluorescent beads embedded in the PA gel.

Results, Conclusions, and Discussions:: For 24 hours, we observed how the cell clusters migrated in three different media conditions (DM, NM, and BM). Under each condition, distinct patterns in the spreading area, velocity, and directionality were shown. By using immunofluorescence imaging, we were able to measure the reserve cell (in vivo satellite cell) ratio and the differentiation ratio as well. The heterogeneity of skeletal muscle cells at each media condition, which is composed of migration, differentiation, and homeostasis, was determined by these findings (Fig. 2). Additionally, the traction force and intercellular stress of migrating cells displayed distinctively different trends and were closely associated with migrating behavior and cellular heterogeneity. We identified that, when comparing the damaged muscle-mimicking to the normal muscle-mimicking condition, the heterogeneous population of skeletal muscle cell clusters displayed specific behaviors.
In this study, we constructed in vitro cell-based platform to model skeletal muscles with specific conditions that mimic the biochemical environment of normal and injured skeletal muscles. As a result, we identified distinct patterns of migratory behavior and the heterogeneous traits of the cell clusters under different media conditions. From these results, we suggest that the biochemical cues from the normal and injured skeletal muscles can induce the heterogeneity of skeletal muscle cells composed of migration, differentiation, and homeostasis in the migrating clusters leading to distinct migrating patterns and mechanical characteristics. We expect that our study will provide insight into the underlying mechanisms of collective skeletal muscle migration.


Acknowledgements (Optional): : This research was supported by National Research Foundation of Korea (NRF) grants funded by the Korean Government (NRF-2020M3A9E4039658)


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