Abstract
Direct generation of skeletal muscle cells from human pluripotent stem cells (hPSCs) would be beneficial for drug testing, drug discovery, and disease modelling in vitro. Here we show a rapid and robust method to induce myogenic differentiation of hPSCs by introducing mRNA encoding MYOD1 together with siRNA-mediated knockdown of POU5F1 (also known as OCT4 or OCT3/4). This integration-free approach generates functional skeletal myotubes with sarcomere-like structure and a fusion capacity in several days. The POU5F1 silencing facilitates MYOD1 recruitment to the target promoters, which results in the significant activation of myogenic genes in hPSCs. Furthermore, deep sequencing transcriptome analyses demonstrated that POU5F1-knockdown upregulates the genes associated with IGF- and FGF-signaling and extracellular matrix that may also support myogenic differentiation. This rapid and direct differentiation method may have potential applications in regenerative medicine and disease therapeutics for muscle disorders such as muscular dystrophy.
Highlights
Human pluripotent stem cells such as human embryonic stem cells and induced pluripotent stem cells have the potential to differentiate into essentially all the cell types in our body including skeletal muscle cells. human pluripotent stem cells (hPSCs)-derived skeletal muscle cells would be an unlimited cell source for potential clinical and research applications such as disease modeling, platform for drug screening, and cell transplantation
We hypothesized that the pluripotency-specific factors such as POU5F1 and NANOG inhibit the MYOD1-mediated conversion of hPSCs because these genes are essential for maintenance of the undifferentiated state[18]
We examined the expression states of POU5F1 and NANOG in synMYOD1-transfected human embryonic stem cells (hESCs)
Summary
Human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have the potential to differentiate into essentially all the cell types in our body including skeletal muscle cells. hPSC-derived skeletal muscle cells would be an unlimited cell source for potential clinical and research applications such as disease modeling, platform for drug screening, and cell transplantation. Previous studies have developed in vitro culture conditions of hPSCs using media supplemented with suitable growth factors and cytokines to follow the steps of embryonic development[1,2] Those protocols mimic the process of developmental stages, in most cases, they require long-term, complicated steps; yet, the efficiency of differentiation is rather low. When the combination of a piggyBac transposon and drug-inducible expression system induces the high expression of MYOD1, direct myogenic conversion of hiPSCs can be successfully achieved[8], suggesting that stable and robust expression of MYOD1 proteins is required to activate skeletal myogenesis in hPSCs. recent studies have shown that additional expression of epigenetic modifying factors such as JMJD3 and BAF60C is required to initiate the myogenic program in hPSCs6,9. Myogenic differentiation hardly occurs when synMYOD1 is introduced in undifferentiated hPSCs9, which corresponds to the results using DNA-based methods
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