Engineering functional muscle tissues and modeling muscular diseases using myogenic cells differentiated from human pluripotent stem cells or human fibroblasts

Abstract

The advances in efficient generation of myogenic cells using human pluripotent stem cells (hPSCs) offer unlimited opportunities for translational applications, such as the study of muscle development and diseases, drug screening, and regenerative medicine. Functional muscle constructs tissue-engineered from these myogenic cells prove excellent tools for those applications. However, these myogenic cells are developmentally immature, and the protocol to derive them is time-consuming. In this thesis, we aim to model muscle diseases and to improve the maturity of muscles using hPSC-derived myogenic cells. We also develop transdifferentiation as an alternative method to obtain myogenic cells more quickly. We modeled Duchenne Muscular Dystrophy (DMD), a genetic disorder leading to muscle wasting and death. We fabricated nanogrooved substrate immobilized with muscle basement membrane mimicking materials and discovered that non-diseased and DMD muscles derived from hPSCs exhibit substantial differences in cell alignments on nanogrooved substrates when these substrates are functionalized with laminin. To improve the maturity of the muscles, we generated hPSC-derived muscle constructs in customized devices and discovered that addition of the endothelial growth medium-2 supplements in the first two weeks of differentiation leads to substantial increases in contractile forces. These constructs show wider myotubes and higher gene expression levels for skeletal muscle-specific myosin heavy chain isoforms, suggesting that a more mature differentiation stage of the cells. Those tissue-engineered constructs were also used to validate the screening of small molecules for enhancing the function and maturation during myogenic differentiation. We found a significant increase in contractile force generation when treated with a cocktail of four small molecules (SB431542, DAPT, Dexamethasone, and Forskolin). To explore an alternative approach to generating functional human muscles more quickly, we chose to transdifferentiate normal human dermal fibroblasts (NHDF) transduced with inducible MyoD. We demonstrated that myogenic transdifferentiation of NHDF could be enhanced by using small molecules CHIR99021 and DAPT when coupled with MyoD induction. We further proved that muscle constructs engineered from transdifferentiated NHDF can generate contractile forces in response to electrical stimuli after 2-week 3D culture. Temporal expression of MyoD in the first week boosts twitch and tetanic forces significantly, and small molecule (CHIR and DAPT) treatment could further improve force generation.