Abstract
Advancements in reprogramming somatic cells into induced pluripotent stem cells (iPSCs) have provided a strong framework for in vitro disease modeling, gene correction and stem cell-based regenerative medicine. In cases of skeletal muscle disorders, iPSCs can be used for the generation of skeletal muscle progenitors to study disease mechanisms, or implementation for the treatment of muscle disorders. We have recently developed an improved directed differentiation method for the derivation of skeletal myogenic progenitors from hiPSCs. This method allows for a short-term (2 weeks) and efficient skeletal myogenic induction (45–65% of the cells) in human pluripotent stem cells (ESCs/iPSCs) using small molecules to induce mesoderm and subsequently myotomal progenitors, without the need for any gene integration or modification. After initial differentiation, skeletal myogenic progenitors can be purified from unwanted cells using surface markers (CD10+CD24−). These myogenic progenitors have been extensively characterized using in vitro gene expression/differentiation profiling as well as in vivo engraftment studies in dystrophic (mdx) and muscle injury (VML) rodent models and have been proven to be able to engraft and form mature myofibers as well as seeding muscle stem cells. The current protocol describes a detailed, step-by-step guide for this method and outlines important experimental details and troubleshooting points for its application in any human pluripotent stem cells.
Highlights
Human induced pluripotent stem cells serve as an excellent model system to study disease phenotype and mechanism, identify therapeutic targets and develop stem cell-based therapies for many degenerative disorders [1,2]. Due to their ease of derivation and reprogramming from somatic cells, pluripotency and differentiation potential as well as autologous source, iPSCs are an excellent candidate for regenerative medicine [3,4]
The main purpose of these efforts is to provide safe cell prep techniques useful for translational medicine, such as integration-free methods for iPSC reprogramming to eliminate the risk of mutagenesis or the production of various tissue progenitors by mirroring natural developmental signaling through directed differentiation methods [4,6]
By the initial induction of presomitic mesoderm using WNT activation, followed by somite/myotome induction through TGF-β and BMP inhibition, skeletal myogenic progenitors can be isolated using surface markers (CD10+CD24−). This method allows for a short-term (2 weeks) and efficient induction of skeletal myogenic program (45–65% of the cells) in Human induced pluripotent stem cells (hiPSCs) [19]
Summary
Human induced pluripotent stem cells (hiPSCs) serve as an excellent model system to study disease phenotype and mechanism, identify therapeutic targets and develop stem cell-based therapies for many degenerative disorders [1,2]. The main purpose of these efforts is to provide safe cell prep techniques useful for translational medicine, such as integration-free methods for iPSC reprogramming to eliminate the risk of mutagenesis or the production of various tissue progenitors by mirroring natural developmental signaling through directed differentiation methods [4,6] In this regard, our group has a long-term interest in generation of safe and scalable skeletal muscle progenitors from iPSCs useful for disease modeling or regenerative applications [14,15,16]. By developing a knock-in double reporter human ESC cell line for PAX7 and MYF5, we were able to screen important governing pathways to develop a directed differentiation method for derivation of skeletal myogenic progenitors from human pluripotent stem cells (hPSCs) [17,18,19] These efforts led to the development of an efficient and short-term differentiation and purification strategy for the isolation of skeletal myogenic progenitors. Successful implementation of this protocol facilitates the application of hiPSCs for disease phenotyping, drug screening and the development of therapeutic strategies for skeletal muscle disorders and dystrophies by other investigators
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