11 | Human immortalized myoblasts/induced pluripotent stem cell derived skeletal muscle model to investigate cystinosis myopathy

Jianhao Zhang¹, R. Mora De La Serna², M. Smits², V. Marini¹, A. Wang¹, M. Biglietto¹, M. Campaner Socias¹, L. Rinvenuto¹, Z. Ge¹, R. Gijsbers^2|3, M. Sampaolesi^1|4 | ¹Translational Cardiomyology Laboratory, Stem Cell and Developmental Biology, Department of Development and Regeneration, KU Leuven, Leuven, Belgium; ²Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium; ³Leuven Viral Vector Core, KU Leuven, Leuven, Belgium; ⁴Histology and Medical Embryology Unit, Department of Anatomy, Histology, Forensic Medicine and Orthopedics, Sapienza University of Rome, Rome, Italy.

Cystinosis myopathy develops in cystinosis patients with CTNS mutations as the disease progresses despite cysteamine therapy or kidney transplantation. It presents as muscle weakness in distal, swallowing and respiratory muscles. Muscle biopsy typically reveals autophagic vacuoles and cystine crystal deposition in muscle cells. Despite these observations, a model that accurately reproduces the pathological changes of cystinosis myopathy remains elusive, hindering investigations into its underlying mechanisms. Building up on our expertise in cell models to study severe myopathy, such as Duchenne muscular dystrophy, we established disease models using immortalized myoblasts/induced pluripotent stem cells (iPSCs) to simulate the cystinosis mild myopathy phenotype. We first differentiated the immortalized myoblasts to myotubes and evaluated their cystinosis-related mild myopathy phenotype. To further enhance the maturity of our model and promote the formation of skeletal muscle structures, we used a 14-day differentiation protocol combined with extracellular matrix coating. Proteomics and metabolomics analyses confirmed that the model recapitulated key aspects of the cystinosis myopathy features such as cystine accumulation and calcium-related protein alteration. Then, we generated isogenic human CTNS knock-out iPSCs lines via CRISPR/Cas9 gene editing. Subsequently, we applied protocols based on existing literature to differentiate iPSCs into skeletal muscle myotubes2. Proteomics, transcriptomics and metabolomics assays are planned to verify the cystinosis myopathy related characteristics of the iPSC-derived models. In conclusion, we successfully established human cell models for cystinosis- myopathy, which will serve as valuable tools to investigate the underlying molecular mechanisms and identify potential therapeutic targets of this poorly studied disease.