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

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

Generating Functional and Highly Proliferative Melanocytes Derived from Human Pluripotent Stem Cells: A Promising Tool for Biotherapeutic Approaches to Treat Skin Pigmentation Disorders

Melanocytes are essential for skin homeostasis and protection, and their loss or misfunction leads to a wide spectrum of diseases. Cell therapy utilizing autologous melanocytes has been used for years as an adjunct treatment for hypopigmentary disorders such as vitiligo. However, these approaches are hindered by the poor proliferative capacity of melanocytes obtained from skin biopsies. Recent advances in the field of human pluripotent stem cells have fueled the prospect of generating melanocytes. Here, we have developed a well-characterized method to produce a pure and homogenous population of functional and proliferative melanocytes. The genetic stability and potential transformation of melanocytes from pluripotent stem cells have been evaluated over time during the in vitro culture process. Thanks to transcriptomic analysis, the molecular signatures all along the differentiation protocol have been characterized, providing a solid basis for standardizing the protocol. Altogether, our results promise meaningful, broadly applicable, and longer-lasting advances for pigmentation disorders and open perspectives for innovative biotherapies for pigment disorders

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

DataSheet1_Generation of heterozygous and homozygous NF1 lines from human-induced pluripotent stem cells using CRISPR/Cas9 to investigate bone defects associated with neurofibromatosis type 1.pdf

Neurofibromatosis type 1 (NF1) is one of the most common genetic disorders caused by heterozygous germline NF1 mutations. NF1 affects many systems, including the skeletal system. To date, no curative therapies are available for skeletal manifestations such as scoliosis and tibial dysplasia, mainly due to the lack of knowledge about the mechanisms that underlie this process. By using CRISPR/Cas9-mediated gene editing in human-induced pluripotent stem cells (hiPSCs) to minimize the variability due to genetic background and epigenetic factors, we generated isogenic heterozygous and homozygous NF1-deficient hiPSC lines to investigate the consequences of neurofibromin inactivation on osteoblastic differentiation. Here, we demonstrate that loss of one or both copies of NF1 does not alter the potential of isogenic hiPSCs to differentiate into mesenchymal stem cells (hiPSC-MSCs). However, NF1 (+/−) and NF1 (−/−) hiPSC-MSCs show a defect in osteogenic differentiation and mineralization. In addition, we show that a mono-allelic deletion in NF1 in an isogenic context is sufficient to impair cell differentiation into osteoblasts. Overall, this study highlights the relevance of generating isogenic lines, which may help in genotype–phenotype correlation and provide a human cellular model to understand the molecular mechanisms underlying NF1 and, thus, discover new therapeutic strategies.</p