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

Phelan-McDermid syndrome (PMS) is a rare genetic disease characterized by a global developmental delay with autism spectrum disorder. PMS is caused by loss of function mutations in the SHANK3 gene leading to SHANK3 protein haploinsufficiency. This study describes the generation of isogenic clones produced from one male human embryonic stem cell line with deletions in SHANK3, in a heterozygous or homozygous manner, using CRISPR/Cas9 indel methodology. Differentiation of these clones into different neuronal lineages will help understanding PMS etiology and find treatments for PMD patients. (85/100 words).

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.

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

Introduction: 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.

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

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.

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

Mutations leading to haploinsufficiency in SCN5A, the gene encoding the cardiac sodium channel Nav1.5 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.

Cytostatic Effect of Repeated Exposure to Simvastatin: A Mechanism for Chronic Myotoxicity Revealed by the Use of Mesodermal Progenitors Derived from Human Pluripotent Stem Cells.

Statin treatment of hypercholesterolemia can lead to chronic myotoxicity which is, in most cases, alleviated by drug withdrawal. Cellular and molecular mechanisms of this adverse effect have been elusive, in particular because of the lack of in vitro models suitable for long-term exposures. We have taken advantage of the properties of human pluripotent stem cell-derived mesodermal precursors, that can be maintained unaltered in vitro for a long period of time, to develop a model of repeated exposures to simvastatin during more than 2 weeks. This approach unveiled major differences, both in functional and molecular terms, in response to single versus repeated-dose exposures to simvastatin. The main functional effect of the in vitro simvastatin-induced long-term toxicity was a loss of proliferative capacity in the absence of concomitant cell death, revealing that cytostatic effect could be a major contributor to statin-induced myotoxicity. Comparative analysis of molecular modifications induced by simvastatin short-term versus prolonged exposures demonstrated powerful adaptive cell responses, as illustrated by the dramatic decrease in the number of differentially expressed genes, distinct biological pathway enrichments, and distinct patterns of nutrient transporters expressed at the cell surface. This study underlines the potential of derivatives of human pluripotent stem cells for developing new approaches in toxicology, in particular for chronic toxicity testing.

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 regeneration.

mTOR-dependent proliferation defect in human ES-derived neural stem cells affected by myotonic dystrophy type 1.

Patients with myotonic dystrophy type 1 exhibit a diversity of symptoms that affect many different organs. Among these are cognitive dysfunctions, the origin of which has remained elusive, partly because of the difficulty in accessing neural cells. Here, we have taken advantage of pluripotent stem cell lines derived from embryos identified during a pre-implantation genetic diagnosis for mutant-gene carriers, to produce early neuronal cells. Functional characterization of these cells revealed reduced proliferative capacity and increased autophagy linked to mTOR signaling pathway alterations. Interestingly, loss of function of MBNL1, an RNA-binding protein whose function is defective in DM1 patients, resulted in alteration of mTOR signaling, whereas gain-of-function experiments rescued the phenotype. Collectively, these results provide a mechanism by which DM1 mutation might affect a major signaling pathway and highlight the pertinence of using pluripotent stem cells to study neuronal defects.

miR-125 potentiates early neural specification of human embryonic stem cells.

The role of microRNAs (miRNAs) as coordinators of stem cell fate has emerged over the last decade. We have used human embryonic stem cells to identify miRNAs involved in neural lineage commitment induced by the inhibition of TGFß-like molecule-mediated pathways. Among several candidate miRNAs expressed in the fetal brain, the two isoforms of miR-125 alone were detected in a time window compatible with a role in neural commitment in vitro. Functional analysis indicated that miR-125 isoforms were actively involved in the promotion of pluripotent cell conversion into SOX1-positive neural precursors. miR-125 promotes neural conversion by avoiding the persistence of non-differentiated stem cells and repressing alternative fate choices. This was associated with the regulation by miR-125 of SMAD4, a key regulator of pluripotent stem cell lineage commitment. Activation of miR-125 was directly responsive to the levels of TGFß-like molecules, placing miR-125 at the core of mechanisms that lead to the irreversible neural lineage commitment of pluripotent stem cells in response to external stimuli.

Combined mRNA and microRNA profiling reveals that miR-148a and miR-20b control human mesenchymal stem cell phenotype via EPAS1.

Mesenchymal stem cells (MSCs) are present in a wide variety of tissues during development of the human embryo starting as early as the first trimester. Gene expression profiling of these cells has focused primarily on the molecular signs characterizing their potential heterogeneity and their differentiation potential. In contrast, molecular mechanisms participating in the emergence of MSC identity in embryo are still poorly understood. In this study, human embryonic stem cells (hESs) were differentiated toward MSCs (ES-MSCs) to compare the genetic patterns between pluripotent hESs and multipotent MSCs by a large genomewide expression profiling of mRNAs and microRNAs (miRNAs). After whole genome differential transcriptomic analysis, a stringent protocol was used to search for genes differentially expressed between hESs and ES-MSCs, followed by several validation steps to identify the genes most specifically linked to the MSC phenotype. A network was obtained that encompassed 74 genes in 13 interconnected transcriptional systems that are likely to contribute to MSC identity. Pairs of negatively correlated miRNAs and mRNAs, which suggest miRNA-target relationships, were then extracted and validation was sought with the use of Pre-miRs. We report here that underexpression of miR-148a and miR-20b in ES-MSCs, compared with ESs, allows an increase in expression of the EPAS1 (Endothelial PAS domain 1) transcription factor that results in the expression of markers of the MSC phenotype specification.

Comparison of gene expression pattern in SP cell populations from four tissues to define common "stemness functions".

The goal of our study was to identify a subset of genes commonly expressed in Side Populations (SP), isolated by Hoechst staining followed by flow cytometry, from adult mouse bone marrow, male adult germinal cells, muscle primary culture, and mesenchymal cells. These SP cells have been proposed to be a "stem-like" population and are used here as a "model" that may reveal mechanisms which would be relevant for a better understanding of stem cell properties. Transcriptional profiles for SP and the more differentiated non-SP cells isolated from the four tissues were compared by hybridization on microarray using a common external reference. Among the 503 genes differentially expressed, which discriminate SP and non-SP cells in all the tissues, the genes upregulated in SP cells are implicated in the quiescent status of the cells, the maintenance of their pluripotency and the capacity to undergo asymmetric division. These genes may be responsible for the decision for self-renewal of these cells, whereas the repression of lineage-affiliated genes in SP cells could be responsible for their undifferentiated state. These genes, acting in concert, may be the key players that mediate the mechanisms that control stem cell functions, and our results suggest that we have identified common "stemness functions" of these "stem-like" cells.