Novel integrated workflow allows production and in-depth quality assessment of multifactorial reprogrammed skeletal muscle cells from human stem cells

Abstract

Skeletal muscle tissue engineering aims at generating biological substitutes that restore, maintain or improve normal muscle function; however, the quality of cells produced by current protocols remains insufficient. Here, we developed a multifactor-based protocol that combines adenovector (AdV)-mediated MYOD expression, small molecule inhibitor and growth factor treatment, and electrical pulse stimulation (EPS) to efficiently reprogram different types of human-derived multipotent stem cells into physiologically functional skeletal muscle cells (SMCs). The protocol was complemented through a novel in silico workflow that allows for in-depth estimation and potentially optimization of the quality of generated muscle tissue, based on the transcriptomes of transdifferentiated cells. We additionally patch-clamped phenotypic SMCs to associate their bioelectrical characteristics with their transcriptome reprogramming. Overall, we set up a comprehensive and dynamic approach at the nexus of viral vector-based technology, bioinformatics, and electrophysiology that facilitates production of high-quality skeletal muscle cells and can guide iterative cycles to improve myo-differentiation protocols.

Distinct human skeletal muscle-derived CD90 progenitor subsets for myo-fibro-adipogenic disease modeling and treatment in multiplexed conditions

“…For example, continuous induction of T cells in mixed culture conditions revealed a continuum of T cell fate with mixed phenotype and altered cytokine expression patterns ( Antebi et al, 2013 ). Such experimental systems contribute to validation of theoretical models aimed to predict cell fates and facilitate the development of improved theoretical models of cell differentiation network and identification of intervention targets in silico ( Zanudo and Albert, 2015 ; Faustino et al, 2022 ).…”

Novel Viral Vectors for Gene Therapy

“…On the other hand, the use of viral vectors as a vaccination tool during the COVID-19 crisis has taught us the lesson that there are still some issues to be solved to avoid unwanted serious side effects and make this next-generation gene delivery technology, proven successful in many approaches, clinically usable. The development of novel viral vectors that meet the requirements of future patients warrants their individual optimization and adaptation for different applications in gene therapy, cancer treatment, vaccine development, and cell reprogramming [ 1 ]. Since its approval as the first gene therapy product, a plethora of strategies using adenovectors emerged, including conditionally replicative oncolytic viruses [ 2 ], less-immunogenic, genetically stable high-capacity adenovirus-derived vehicles, which allow long-term episomal persistence of transgenes in non-dividing cells [ 3 ], or customized adenovectors for targeted transduction in vivo.…”