A defective Krab-domain zinc-finger transcription factor contributes to altered myogenesis in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is an RNA-mediated disorder caused by a non-coding CTG repeat expansion that, in particular, provokes functional alteration of CUG-binding proteins. As a consequence, several genes with misregulated alternative splicing have been linked to clinical symptoms. In our search for additional molecular mechanisms that would trigger functional defects in DM1, we took advantage of mutant gene-carrying human embryonic stem cell lines to identify differentially expressed genes. Among the different genes found to be misregulated by DM1 mutation, one strongly downregulated gene encodes a transcription factor, ZNF37A. In this paper, we show that this defect in expression, which derives from a loss of RNA stability, is controlled by the RNA-binding protein, CUGBP1, and is associated with impaired myogenesis—a functional defect reminiscent of that observed in DM1. Loss of the ZNF37A protein results in changes in the expression of the subunit α1 of the receptor for the interleukin 13. This suggests that the pathological molecular mechanisms linking ZNF37A and myogenesis may involve the signaling pathway that is known to promote myoblast recruitment during development and regeneratio

ACVR1, a Therapeutic Target of Fibrodysplasia Ossificans Progressiva, Is Negatively Regulated by miR-148a

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder of skeletal malformations and progressive extraskeletal ossification. There is still no effective treatment for FOP. All FOP individuals harbor conserved point mutations in ACVR1 gene that are thought to cause ACVR1 constitutive activation and activate BMP signal pathway. The constitutively active ACVR1 is also found to be able to cause endothelial-to-mesenchymal transition (EndMT) in endothelial cells, which may cause the formation of FOP lesions. MicroRNAs (miRNAs) play an essential role in regulating cell differentiation. Here, we verified that miR-148a directly targeted the 3′ UTR of ACVR1 mRNA by reporter gene assays and mutational analysis at the miRNA binding sites, and inhibited ACVR1 both at the protein level and mRNA level. Further, we verified that miR-148a could inhibit the mRNA expression of the Inhibitor of DNA binding (Id) gene family thereby suppressing the BMP signaling pathway. This study suggests miR-148a is an important mediator of ACVR1, thus offering a new potential target for the development of therapeutic agents against FOP

CRISPR/Cas9-mediated generation of human embryonic stem cell sub-lines with HPRT1 gene knockout to model Lesch Nyhan disease

Lesch-Nyhan disease (LND) is a X-linked genetic disease affecting boys characterized by complex neurological and neuropsychiatric symptoms. LND is caused by loss of function mutations in the HPRT1 gene leading to decrease activity of hypoxanthine-guanine phosphoribosyl transferase enzyme (HGPRT) and altered purine salvage pathway (Lesch and Nyhan, 1964). This study describes the generation of isogenic clones with deletions in HPRT1 produced from one male human embryonic stem cell line using CRISPR/Cas9 strategy. Differentiation of these cells into different neuronal subtypes will help elucidating the neurodevelopmental events leading to LND and develop therapeutic strategies for this devastating neurodevelopmental disorder

Global transcriptional profiling of neural and mesenchymal progenitors derived from human embryonic stem cells reveals alternative developmental signaling pathways.

International audienceHuman embryonic stem cells can be differentiated along different lineages, providing the possibility of a precise analysis of genes profiles associated with specific commitments. Subtractive gene expression profiling between differentiated and undifferentiated cells provides lists of potential actors in this commitment. This combines, however, genes that are specifically associated with development and others that are over expressed because of nonlineage-specific differentiation systems. As a way to establish gene profiles associated with the neural and/or to the mesodermal commitments of human embryonic stem cells more precisely, we have carried out a 2-step analysis. We first performed a subtractive analysis of gene profiles of each of these lineages as compared to the undifferentiated stage. Then, we extended the analysis by comparing the 2 sets of results with each other. This strategy has allowed us to eliminate large numbers of genes that were over expressed in both sets of results and to uniquely associate different gene networks with either the neural or the mesodermal commitments

Establishment of heterozygous and homozygous SHANK3 knockout clonal pluripotent stem cells from the parental hESC line SA001 using CRISPR/Cas9

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Generation of a heterozygous SCN5A knockout human induced pluripotent stem cell line by CRISPR/Cas9 edition

International audienceMutations leading to haploinsufficiency in SCN5A, the gene encoding the cardiac sodium channel Na v 1.5 α-subunit, are involved in life-threatening cardiac disorders. Using CRISPR/Cas9-mediated genome edition, we generated here a human induced-pluripotent stem cell (hiPSC) line carrying a heterozygous mutation in exon 2 of SCN5A, which leads to apparition of a premature stop codon. SCN5A-clone 5 line maintained normal karyotype, morphology and pluripotency and differentiated into three germ layers. Cardiomyocytes derived from these hiPSCs would be a useful model for investigating channelopathies related to SCN5A heterozygous deficiency

Semi-automated optimized method to isolate CRISPR/Cas9 edited human pluripotent stem cell clones

International audienceAbstract Background CRISPR/Cas9 editing systems are currently used to generate mutations in a particular gene to mimic a genetic disorder in vitro. Such “disease in a dish” models based on human pluripotent stem cells (hPSCs) offer the opportunity to have access to virtually all cell types of the human body. However, the generation of mutated hPSCs remains fastidious. Current CRISPR/Cas9 editing approaches lead to a mixed cell population containing simultaneously non-edited and a variety of edited cells. These edited hPSCs need therefore to be isolated through manual dilution cloning, which is time-consuming, labor intensive and tedious. Methods Following CRISPR/Cas9 edition, we obtained a mixed cell population with various edited cells. We then used a semi-automated robotic platform to isolate single cell-derived clones. Results We optimized CRISPR/Cas9 editing to knock out a representative gene and developed a semi-automated method for the clonal isolation of edited hPSCs. This method is faster and more reliable than current manual approaches. Conclusions This novel method of hPSC clonal isolation will greatly improve and upscale the generation of edited hPSCs required for downstream applications including disease modeling and drug screening. Graphical Abstrac

Pathological modeling of glycogen storage disease type III with CRISPR/Cas9 edited human pluripotent stem cells

International audienceIntroduction: Glycogen storage disease type III (GSDIII) is a rare genetic disease caused by mutations in the AGL gene encoding the glycogen debranching enzyme (GDE). The deficiency of this enzyme, involved in cytosolic glycogen degradation, leads to pathological glycogen accumulation in liver, skeletal muscles and heart. Although the disease manifests with hypoglycemia and liver metabolism impairment, the progressive myopathy is the major disease burden in adult GSDIII patients, without any curative treatment currently available. Methods: Here, we combined the self-renewal and differentiation capabilities of human induced pluripotent stem cells (hiPSCs) with cutting edge CRISPR/Cas9 gene editing technology to establish a stable AGL knockout cell line and to explore glycogen metabolism in GSDIII. Results: Following skeletal muscle cells differentiation of the edited and control hiPSC lines, our study reports that the insertion of a frameshift mutation in AGL gene results in the loss of GDE expression and persistent glycogen accumulation under glucose starvation conditions. Phenotypically, we demonstrated that the edited skeletal muscle cells faithfully recapitulate the phenotype of differentiated skeletal muscle cells of hiPSCs derived from a GSDIII patient. We also demonstrated that treatment with recombinant AAV vectors expressing the human GDE cleared the accumulated glycogen. Discussion: This study describes the first skeletal muscle cell model of GSDIII derived from hiPSCs and establishes a platform to study the mechanisms that contribute to muscle impairments in GSDIII and to assess the therapeutic potential of pharmacological inducers of glycogen degradation or gene therapy approaches