In vitro modeling of skeletal muscle ischemia-reperfusion injury based on sphere differentiation culture from human pluripotent stem cells.

Skeletal muscle ischemia-reperfusion (IR) injury poses significant challenges due to its local and systemic complications. Traditional studies relying on two-dimensional (2D) cell culture or animal models often fall short of faithfully replicating the human in vivo environment, thereby impeding the translational process from animal research to clinical applications. Three-dimensional (3D) constructs, such as skeletal muscle spheroids with enhanced cell-cell interactions from human pluripotent stem cells (hPSCs) offer a promising alternative by partially mimicking human physiological cellular environment in vivo processes. This study aims to establish an innovative in vitro model, human skeletal muscle spheroids based on sphere differentiation from hPSCs, to investigate human skeletal muscle developmental processes and IR mechanisms within a controlled laboratory setting. By eticulously recapitulating embryonic myogenesis through paraxial mesodermal differentiation of neuro-mesodermal progenitors, we successfully established 3D skeletal muscle spheroids that mirror the dynamic colonization observed during human skeletal muscle development. Co-culturing human skeletal muscle spheroids with spinal cord spheroids facilitated the formation of neuromuscular junctions, providing functional relevance to skeletal muscle spheroids. Furthermore, through oxygen-glucose deprivation/re-oxygenation treatment, 3D skeletal muscle spheroids provide insights into the molecular events and pathogenesis of IR injury. The findings presented in this study significantly contribute to our understanding of skeletal muscle development and offer a robust platform for in vitro studies on skeletal muscle IR injury, holding potential applications in drug testing, therapeutic development, and personalized medicine within the realm of skeletal muscle-related pathologies.

Unraveling radiation-induced skeletal muscle damage: Insights from a 3D human skeletal muscle organoid model.

BACKGROUND: Three-dimensional (3D) organoids derived from human pluripotent stem cells (hPSCs) have revolutionized in vitro tissue modeling, offering a unique opportunity to replicate physiological tissue organization and functionality. This study investigates the impact of radiation on skeletal muscle response using an innovative in vitro human 3D skeletal muscle organoids (hSMOs) model derived from hPSCs. METHODS: The hSMOs model was established through a differentiation protocol faithfully recapitulating embryonic myogenesis and maturation via paraxial mesodermal differentiation of hPSCs. Key skeletal muscle characteristics were confirmed using immunofluorescent staining and RT-qPCR. Subsequently, the hSMOs were exposed to a clinically relevant dose of 2 Gy of radiation, and their response was analyzed using immunofluorescent staining and RNA-seq. RESULTS: The hSMO model faithfully recapitulated embryonic myogenesis and maturation, maintaining key skeletal muscle characteristics. Following exposure to 2 Gy of radiation, histopathological analysis revealed deficits in hSMOs expansion, differentiation, and repair response across various cell types at early (30 min) and intermediate (18 h) time points post-radiation. Immunofluorescent staining targeting γH2AX and 53BP1 demonstrated elevated levels of foci per cell, particularly in PAX7+ cells, during early and intermediate time points, with a distinct kinetic pattern showing a decrease at 72 h. RNA-seq data provided comprehensive insights into the DNA damage response within the hSMOs. CONCLUSIONS: Our findings highlight deficits in expansion, differentiation, and repair response in hSMOs following radiation exposure, enhancing our understanding of radiation effects on skeletal muscle and contributing to strategies for mitigating radiation-induced damage in this context.