A human iPS cell myogenic differentiation system permitting high-throughput drug screening

Abstract

Muscular dystrophy is a disease characterized by progressive muscle weakness and degeneration. There are currently no available treatments for most muscular diseases, such as muscular dystrophy. Moreover, current therapeutics are focused on improving the quality of life of patients by relieving the symptoms or stress caused by the disease. Although the causative genes for many muscular diseases have been identified, the mechanisms underlying their pathogenesis remain unclear. Patient-derived induced pluripotent stem cells (iPSCs) have become a powerful tool for understanding the pathogenesis of intractable diseases, as well as for phenotype screening, which can serve as the basis for developing new drugs. However, it is necessary to develop an efficient and reproducible myogenic differentiation system. Previously, we reported a tetracycline-inducible MyoD overexpression model of myogenic differentiation using human iPSCs (hiPSCs). However, this model has certain disadvantages that limit its use in various applications, such as a drug screening. In this study, we developed an efficient and reproducible myogenic differentiation system by further modifying our previous protocol. The new protocol achieves efficient differentiation of feeder-free hiPSCs to myogenic cells via small-scale culture in six-well microplates to large-scale culture in 384-well microplates for high-throughput applications.

Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping

“…Differentiation of iPSCs to skeletal muscle cells. Dox-inducible MYOD1 expressing iPSCs were established and differentiated to skeletal muscle cells as previously described 46 . In brief on Day 1, 3 × 10 5 iPSCs were seeded onto a six-well plate coated with Matrigel in AK03N StemFit media.…”

Inhibition of cyclooxygenase-1 by nonsteroidal anti-inflammatory drugs demethylates MeR2 enhancer and promotes Mbnl1 transcription in myogenic cells

“…We next examined the effect of knockdown of COX-1 and COX-2 on MBNL1 expression in DM1 patient-derived induced pluripotent stem cells (MyoD-DM1-iPSCs), which were differentiated into myotubes by the induction of MyoD expression ( Fig. 3a,b) 32,33 . Similar to C2C12 myoblasts and myotubes ( Fig.…”

“…MyoD-DM1-iPSCs cell culture. DM1 patient-derived iPSCs (MyoD-DM1-iPSCs) were cultured essentially as described elsewhere with minor modifications 32,33 . Briefly, MyoD-DM1-iPSCs were cultured on a plate coated with iMatrix-511 (Takara, 892011) in StemFit (Takara, AK02N) containing 125 ng/ml puromycin and 10 μM Y-27632 (Wako, 030-24021).…”

“…Immunofluorescence staining of MyoD-DM1-iPSCs. Myotubes differentiated from MyoD-DM1-iPSCs were immunostained essentially as described elsewhere with minor modifications 33 . Briefly, MyoD-DM1-iPSCs were fixed with 2% PFA/PBS on 5 days after replating.…”

A multiplexed in vitro assay system for evaluating human skeletal muscle functionality in response to drug treatment

“…Several functional systems that use rodent‐derived skeletal muscle tissue in two‐dimensional (2D) or hybrid three‐dimensional (A. S. Smith, Long, Pirozzi, & Hickman, ; Uchimura, Otomo, Sato, & Sakurai, ; Wilson, Das, Wahl, Colton, & Hickman, ; Wilson, Molnar, & Hickman, ) and three‐dimensional (3D) (Agrawal, Aung, & Varghese, ; Vandenburgh, ; Vandenburgh et al, ; Vandenburgh et al, ) models have been developed for tissue engineering and drug screening applications. Three‐dimensional hydrogel constructs tend to more accurately represent the hierarchical nature of native skeletal muscle tissue, but accurately measuring their contractile ability is challenging, as many models rely on video recordings or other visual interrogation methods to infer force output (Madden, Juhas, Kraus, Truskey, & Bursac, ; Sakar et al, ; Vandenburgh, ).…”