CRISPR gene editing in pluripotent stem cells reveals the function of MBNL proteins during humanin vitromyogenesis

Abstract

Alternative splicing has emerged as a fundamental mechanism for the spatiotemporal control of development. A better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are mis-regulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of MBNL proteins, a family of tissue-specific splicing regulators whose loss of function is associated with Myotonic Dystrophy type 1 (DM1), an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the consequences of their losses on the generation of skeletal muscle cells. Our results suggested that MBNL proteins are required for the late myogenic maturation. In addition, loss of MBNL1 and MBNL2 recapitulated the main features of DM1 observed in hiPSC-derived skeletal muscle cells. Comparative transcriptomic analyses also revealed the muscle-related processes regulated by these proteins that are commonly mis-regulated in DM1. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope with the loss of function of these MBNL proteins.

Establishment of quantitative and consistent in vitro skeletal muscle pathological models of myotonic dystrophy type 1 using patient-derived iPSCs

“…The new modified method not involving replating and EFS led to robust myogenic differentiation on day 17 and clear detection of splicing defects in the DM1-MyoD-hiPSC-derived myotubes. For basic research on DM1, a variety of human in vitro models have been reported over the years, established using DM1 patient-derived cells, e.g., primary dermal fibroblasts 4,36,37 , myoblasts 12,13,38 , urine-derived cells 39 , and hiPSCs [40][41][42] . Mondragon-Gonzalez et al 40 reported an in vitro DM1 model established using patient-derived iPSCs as a tool for drug discovery.…”

Optogenetically controlled human functional motor endplate for testing botulinum neurotoxins

“…The WTSli020 hiPSC line from fibroblasts of dermis of a healthy female that was provided by EBiSC (European Bank for induced pluripotent Stem Cells) was cultured in feeder-free conditions using Vitronectin-coated culture vessels (VTN-N; Thermo Fisher Scientific, Waltham, MA, USA) and Essential 8 Flex medium (Gibco, Grand-Island, NY, USA) supplemented with Penicillin/Streptomycin (1:1000 PenStrep; Gibco). Another hiPSC line derived from a healthy female was also used as previously described [ 38 ]. Briefly, cells were thawed and manually expanded over five supplementary passages.…”

NF-YAl drives EMT in Claudinlow tumours

“…We retrieved the FASTQ files associated to 51 BRCA cell lines from CCLE, and to six SUM159 cell line samples from Yang et al [ 60 ]. For RBPs analyses, we acquired expression data from KD, OE and KO experiments [ 54 , 61 – 64 ]. All FASTQ files were downloaded using the SRA Explorer website ( https://sra-explorer.info/ ), and the accession codes are in Supplementary Table S4 .…”