Abstract
ObjectivesMyoblast transfer therapy (MTT) is a technique to replace muscle satellite cells with genetically repaired or healthy myoblasts, to treat muscular dystrophies. However, clinical trials with human myoblasts were ineffective, showing almost no benefit with MTT. One important obstacle is the rapid senescence of human myoblasts. The main purpose of our study was to compare the various methods for scalable generation of proliferative human myoblasts.MethodsWe compared the immortalization of primary myoblasts with hTERT, cyclin D1 and CDK4R24C, two chemically defined methods for deriving myoblasts from pluripotent human embryonic stem cells (hESCs), and introduction of viral MyoD into hESC‐myoblasts.ResultsOur results show that, while all the strategies above are suboptimal at generating bona fide human myoblasts that can both proliferate and differentiate robustly, chemically defined hESC‐monolayer‐myoblasts show the most promise in differentiation potential.ConclusionsFurther efforts to optimize the chemically defined differentiation of hESC‐monolayer‐myoblasts would be the most promising strategy for the scalable generation of human myoblasts, for applications in MTT and high‐throughput drug screening.
Highlights
Skeletal muscle stem cells exist as satellite cells in adult mammalian skeletal muscles
When the monolayer‐derived human embryonic stem cells (hESCs)‐myoblasts were allowed to differentiate into myotubes under standard myogenic differen‐ tiation conditions,[25,26] we found that most of the cells adopted an elongated morphology typical of myocytes (Figure 6H), but only a minor fraction (21.3 ± 5.3%) of these myocytes fused and differen‐ tiated into myotubes (Figure 6H‐K)
The encouraging results obtained by grafting mouse myoblasts into the mdx mouse model,[5] translated into several clinical trial failures with Duchenne muscular dystrophy (DMD) patients.[7-9]
Summary
Skeletal muscle stem cells exist as satellite cells in adult mammalian skeletal muscles. Mouse models bearing mutations similar to those described in human muscular dystrophies, such as the dystrophin mutation in DMD, have been employed to develop myoblast transfer therapy (MTT) against muscular dystrophies. This is a technique to replace muscle satellite cells with genetically repaired or healthy myoblasts, to treat the muscular dystrophy.[5,6]. This limitation is manifested as progressively compromised differentiation and proliferation po‐ tential, during in vitro culture.[10,11] This limitation prevents us from achieving MTT for muscular dystrophy patients, and limits our ability to conduct high‐throughput drug screening and carry out molecular characterization in human myoblasts with high reproducibility.[12]. Our results further sug‐ gest that hESC‐myoblasts show more promise in differentiation potential, and that further efforts to optimize the directed differen‐ tiation of hESC‐myoblasts would be useful
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