Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

Abstract

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.

Dual Gene Expression in Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells Using Episomal Vectors

“…Previous studies have employed FuGENE HD, lipofectamine PLUS, and lipofectamine 2000 reagents for transfection. 17,21,22 For the first time, we compared FuGENE, LTX, X-tremeGENE, and TransIT-2020 reagents to identify a method that would achieve higher transfection efficiency in iPS cells. Transfections are typically performed on cells that are growing and attaching onto plates or dishes.…”

“…In the suspension method, cells were harvested with Accutase, suspended, and incubated in the transfection reagent for 5 min at room temperature in a hood. 17 The transfection reagents, FuGENE HD Transfection Reagent (Promega, Madison, WI), Lipofectamine LTX (Life Technologies, Grand Island, NY), X-tremeGENE Transfection Reagent (Roche, Basel, Switzerland), and TransIT-2020 Transfection Reagent (Mirus Bio, Madison, WI), were used following the manufacturer's instructions.…”

“…17,18 Introduction of multiple transcription factors to iPS cells promote differentiation to target somatic cells. 19 Transcription factors are introduced into iPS cells through plasmids or viral vectors.…”

Regeneration: making muscle from hPSCs

“…Therefore, supplementation of functional myogenic progenitors to repair damaged tissues through transplantation may represent an effective treatment against MDs. Successful generation of myogenic progenitors from hPSCs has been achieved by exogenous expression of transcription factors PAX7 or MYOD1 [1][2][3][4][5] (Table 1). Upon ectopic expression of PAX7 in hPSCs-derived embryoid bodies (EBs), the derived myogenic progenitors can differentiate to multinucleated myofibers in vitro, and develop into myocytes after transplantation into mdx mice (a mouse model of MDs) [2].…”

“…Moreover, PAX7+ progenitors can improve muscle contractility in the mdx mouse and supplement satellite cells. As an alternative approach, exogenous expression of MYOD1 in hPSCs differentiates them into terminal multinucleated myogenic cells [1,[3][4][5]. Myogenic cells generated using MYOD1 usually fail to maintain a progenitor status under current culture conditions, and thus are not an ideal cell type for transplantation.…”