Abstract：
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.
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.
摘要：
背景：源自人多能干细胞（hPSC）的三维（3D）类器官彻底改变了体外组织建模，提供了一个独特的机会来复制生理组织的组织和功能。本研究使用源自hPSC的创新的体外人类3D骨骼肌类器官（hSMO）模型研究了辐射对骨骼肌反应的影响。
方法：hSMOs模型是通过分化方案忠实地概括了通过hPSCs的近轴中胚层分化的胚胎肌生成和成熟而建立的。使用免疫荧光染色和RT-qPCR确认关键骨骼肌特征。随后，hSMO暴露于2Gy的临床相关辐射剂量，并使用免疫荧光染色和RNA-seq分析它们的反应。
结果：hSMO模型忠实地概括了胚胎的肌生成和成熟，保持关键的骨骼肌特征。暴露于2Gy辐射后，组织病理学分析显示hSMO扩张缺陷，分化,并在辐射后的早期（30分钟）和中间（18小时）时间点修复各种细胞类型的反应。针对γH2AX和53BP1的免疫荧光染色显示每个细胞的病灶水平升高，特别是在PAX7+细胞中，在早期和中间时间点，具有不同的动力学模式，显示在72小时时降低。RNA-seq数据提供了对hSMO内DNA损伤反应的全面了解。
结论：我们的发现强调了扩张中的缺陷，分化,以及辐射暴露后hSMO的修复反应，增强我们对辐射对骨骼肌的影响的理解，并有助于在这种情况下减轻辐射引起的损伤。
方法：hSMOs模型是通过分化方案忠实地概括了通过hPSCs的近轴中胚层分化的胚胎肌生成和成熟而建立的。使用免疫荧光染色和RT-qPCR确认关键骨骼肌特征。随后，hSMO暴露于2Gy的临床相关辐射剂量，并使用免疫荧光染色和RNA-seq分析它们的反应。
结果：hSMO模型忠实地概括了胚胎的肌生成和成熟，保持关键的骨骼肌特征。暴露于2Gy辐射后，组织病理学分析显示hSMO扩张缺陷，分化,并在辐射后的早期（30分钟）和中间（18小时）时间点修复各种细胞类型的反应。针对γH2AX和53BP1的免疫荧光染色显示每个细胞的病灶水平升高，特别是在PAX7+细胞中，在早期和中间时间点，具有不同的动力学模式，显示在72小时时降低。RNA-seq数据提供了对hSMO内DNA损伤反应的全面了解。
结论：我们的发现强调了扩张中的缺陷，分化,以及辐射暴露后hSMO的修复反应，增强我们对辐射对骨骼肌的影响的理解，并有助于在这种情况下减轻辐射引起的损伤。
