Enhance myogenic transdifferentiation efficiency using engineering approaches

Project Summary Myogenic direct reprogramming from non-muscle somatic cells has become an important strategy to produce abundant, patient-specific, and disease-specific human myogenic cells, which are highly desirable for therapeutic applications and disease modeling. As compared to deriving myogenic cells from hiPSCs, direct reprogramming is substantially quicker, and reprogrammed cells avoid the risk of teratoma formation and retain aging- and disease-associated epigenetic signatures, which is particularly important for modeling aging-related muscle diseases. Despite these advantages, applications of directly reprogrammed myogenic cells are hampered by low reprogramming efficiency and their immature nature. It is challenging to improve reprogramming through rational design, as molecular mechanisms underlying the process remain largely unknown; therefore, high-throughput screening (HTS) is an important strategy to expedite discovery of more efficient direct reprogramming technologies. A major hurdle to effective HTS for direct reprogramming technologies is the difficulty to establish a simple, low-cost phenotypic readout that truly represents an integrative biological endpoint defining the target lineage. We recently discovered that myogenic cells cultured on surfaces patterned with parallel nanogrooves/ridges and functionalized with Matrigel form myotubes aligning nearly perpendicular to the nanogrooves, and this phenotype is unique and universal for all non-diseased myogenic cells, regardless of their origin and species. Quantitative analysis of myotube orientations reveals a single peak near 90°; furthermore, when normal myogenic cells are mixed with diseased cells that do not exhibit this phenotype, myotube orientation angle decreases with the percentage of the normal myogenic cells. We hypothesize that when cultured on nanogrooved, Matrigel-functionalized surface, myotubes derived from reprogrammed cells will exhibit increased orientation angles relative to the nanogrooves with increasing reprogramming efficiency, and this highly reproducible and quantifiable phenotype will provide a simple, low- cost, and physiologically relevant readout for effective HTS to discover efficient myogenic reprogramming technologies. We plan to test our hypothesis by (1) developing a high-throughput screening platform using myotube orientation relative to nanogrooves as a physiologically relevant readout and use this platform to discover novel small compounds capable of enhancing myogenic reprogramming efficiency, (2) characterizing the myotubes at molecular, structural, and functional levels, and (3) dissecting transcriptional and epigenetic mechanisms underlying the positive effects of the novel compounds. The proposed study will result in new technologies to generate directly reprogrammed human myogenic cells exhibiting more similarities to true myogenic cells. The established HTS platform can be used to discover other types of enhancers for myogenic reprogramming (transcription factors, microRNAs) and the enriched pathways and motifs identified in the cells reprogrammed with the novel compounds will indicate novel targets to further improve reprogramming efficiency.