Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction

DNA input in ddPCR reaction. The figure shows copy number of Chr 8 (crosses) and Y (squares) measured by ddPCR with various input quantities of genomic DNA template. Vertical bars are Standard Errors. The experiment demonstrates linearity across the range of concentrations relevant to the DNA preparations assayed. This is a key point for the robustness of the screen, as gDNA preparations are challenging to standardize due to the disparity of growth rates between ES cell clones. (PDF 26 kb

Pipeline for the Generation and Characterization of Transgenic Human Pluripotent Stem Cells Using the CRISPR/Cas9 Technology

International audienceRecent advances in genome engineering based on the CRISPR/Cas9 technology have revolutionized our ability to manipulate genomic DNA. Its use in human pluripotent stem cells (hPSCs) has allowed a wide range of mutant cell lines to be obtained at an unprecedented rate. The combination of these two groundbreaking technologies has tremendous potential, from disease modeling to stem cell-based therapies. However, the generation, screening and molecular characterization of these cell lines remain a cumbersome and multi-step endeavor. Here, we propose a pipeline of strategies to efficiently generate, sub-clone, and characterize CRISPR/Cas9-edited hPSC lines in the function of the introduced mutation (indels, point mutations, insertion of large constructs, deletions)

Induced Pluripotent Stem Cells for Primary Ciliary Dyskinesia Modeling and Personalized Medicine

International audiencePrimary ciliary dyskinesia (PCD) is a rare and heterogeneous genetic disorder that affects the structure and function of motile cilia. In the airway epithelium, impaired ciliary motion results in reduced or absent mucociliary clearance that leads to the appearance of chronic airway infection, sinusitis and bronchiectasis. Currently, there is no effective treatment for PCD, and research is limited by the lack of convenient models to study this disease and investigate innovative therapies. Furthermore, the high heterogeneity of PCD genotypes is likely to hinder the development of a single therapy for all patients. The generation of patient-derived induced pluripotent stem cells (iPSC) and their differentiation into airway epithelium as well as genome editing technologies could represent major tools for in vitro PCD modelling and for developing personalized therapies. Here, we review PCD pathogenesis, and then discuss how human iPSC could be used to model this disease for the development of innovative patient-specific biotherapies

Generation of four severe early-onset chronic obstructive pulmonary disease (COPD) patient-derived induced pluripotent stem cell lines from peripheral blood mononuclear cells

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Generation of the induced pluripotent stem cell line UHOMi002-A from peripheral blood mononuclear cells of a healthy male donor

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Generation of the induced pluripotent stem cell line UHOMi001-A from a patient with mutations in CCDC40 gene causing Primary Ciliary Dyskinesia (PCD)

Primary Ciliary Dyskinesia (PCD) is a rare heterogeneous genetic disorder affecting motile cilia structure and function leading to lung disease. We generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of a female PCD patient carrying disease-causing variants in the CCDC40 gene. Reprogramming was performed with the human OSKM transcription factors using the Sendai-virus delivery system. The resulting transgene free iPSCs had normal karyotype, expressed pluripotency markers, could differentiate into the three germ layers in vivo and retained the disease-causing CCDC40 mutations. This iPSC line could be useful to model PCD disease and test gene therapy strategies.Resource TableUnlabelled TableUnique stem cell line identifierUHOMi001-AAlternative name(s) of stem cell lineiPCD02.30InstitutionInstitute for Regenerative Medicine & Biotherapy (IRMB), Montpellier, FRANCEContact information of distributorJohn De Vos [email protected] of cell lineiPSCOriginhumanAdditional origin infoAge: 34Sex: FemaleCell SourceDermal fibroblastsClonalityClonalMethod of reprogramminghOCT4, hSOX2, hC-MYC, hKLF4 (CytoTune™-iPS 2.0 Sendai Reprogramming Kit - Invitrogen, Thermo Fisher Scientific Inc.)Genetic ModificationYESType of ModificationSpontaneous mutationAssociated diseasePrimary Ciliary DyskinesiaGene/locusCompound Heterozygous mutations the Coiled-Coil Domain Containing 40 Gene (CCDC40): c.1116_1117delCT (Exon 7) and c.3180 + 1G > A (Intron 19)Method of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock date2018-02-12Cell line repository/bankN/AEthical approvalThe study was approved by the regional scientific ethical committee (CPP Sud Med IV) under the number ID-RCB: 2013-A00892–43/CILIPS, Promoter University Hospital Of Montpellier) and informed consent was obtained from the patient

Les organoïdes pulmonaires

International audienceAs burden of chronic respiratory diseases is constantly increasing, improving in vitro lung models is essential in order to reproduce as closely as possible the complex pulmonary architecture, responsible for oxygen uptake and carbon dioxide clearance. The study of diseases that affect the respiratory system has benefited from in vitro reconstructions of the respiratory epithelium with inserts in air/liquid interface (2D) or in organoids able to mimic up to the arborescence of the respiratory tree (3D). Recent development in the fields of pluripotent stem cells-derived organoids and genome editing technologies has provided new insights to better understand pulmonary diseases and to find new therapeutic perspectives

Generation of a healthy heavy smoker patient-derived induced pluripotent stem cell line UHOMi007-A from peripheral blood mononuclear cells

International audienceHuman pluripotent stem cells (hiPSC) represent a unique opportunity to model lung development and chronic bronchial diseases. We generated a hiPSC line from a highly characterized healthy heavy smoker male donor free from emphysema or tobacco related disease. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai virus. The cell line had normal karyotype, expressed pluripotency hallmarks, and differentiated into the three primary germ layers. The reported UHOMi007-A iPSC line may be used as a control to model lung development, study human chronic bronchial diseases and drug testing