1,721,059 research outputs found
Subtype-Directed Differentiation of Human iPSCs into Atrial and Ventricular Cardiomyocytes
No full textThe generation of homogeneous populations of subtype-specific cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) is crucial in cardiovascular disease modeling as well as in drug discovery and cardiotoxicity screenings. This protocol describes a simple, robust, and efficient monolayer-based differentiation of hiPSCs into defined atrial and ventricular cardiomyocytes. For complete details on the use and execution of this protocol, please refer to Cyganek et al., 2018
Generation of a genetically-modified induced pluripotent stem cell line harboring an oncogenic gene variant KRAS p.G12V
No full texthttp://dx.doi.org/10.13039/100010447 Deutsches Zentrum für Herz-Kreislaufforschunghttp://dx.doi.org/10.13039/501100001659 Deutsche Forschungsgemeinschafthttp://dx.doi.org/10.13039/501100002347 Bundesministerium für Bildung und Forschunghttp://dx.doi.org/10.13039/501100003042 Else Kröner-Fresenius-Stiftun
Generation of a genetically-modified induced pluripotent stem cell line harboring a Noonan syndrome-associated gene variant MRAS p.G23V
No full textPatients harboring causative gene variants in RAS GTPase MRAS develop Noonan syndrome and early-onset hypertrophic cardiomyopathy. Here, we describe the generation of a human iPSC line harboring the Noonan syndrome-associated MRAS p.G23V variant by using CRISPR/Cas9 technology. The established MRASG23V iPSC line allows to study MRAS-specific pathomechanisms and to test novel therapeutic strategies in various disease-relevant cell types and tissues
Cardiac Progenitor Cells and their Therapeutic Application for Cardiac Repair
No full textHeart disease is the principal cause of death in humans. Stem cell-based therapy for heart regeneration has long been seen as a potential application since the heart lacks adequate intrinsic regenerative potential. In the cardiovascular field, clinical trials have already been carried out by implantation of both bone marrow-derived stem cells and cardiac resident progenitor cells derived from the adult heart tissue into the injured myocardium to restore the functionality of the heart after damage. However, before a robust stem and progenitor cell-based therapy for cardiovascular diseases can be applied in the clinical setting, more research is necessary to generate sufficient quantities of functional cardiomyocytes from stem cells and to understand behavior of cardiomyocytes upon transplantation. A comprehensive understanding of the developmental processes involved in cardiogenesis might support further investigations in more efficient cell-based regeneration therapies. This review discusses the molecular aspects of cardiogenesis during early development and links the insights with the in vitro generation of cardiac progenitor cells as well as functional cardiomyocytes. Furthermore, we discuss the advantages of cardiac progenitor cells and cardiomyocytes derived from pluripotent stem cells, cardiac resident stem cells in regenerative applications to cope with the damaged heart.Open-Access-Publikationsfonds 201
Establishment of two homozygous CRISPR interference (CRISPRi) knock-in human induced pluripotent stem cell (hiPSC) lines for titratable endogenous gene repression
No full textUsing nuclease-deficient dead (d)Cas9 without enzymatic activity fused to transcriptional inhibitors (CRISPRi) allows for transcriptional interference and results in a powerful tool for the elucidation of developmental, homeostatic and disease mechanisms. We inserted dCas9KRAB (CRISPRi) cassette into the AAVS1 locus of hiPSC lines, which resulted in homozygous knock-in with an otherwise unaltered genome. Expression of dCas9KRAB protein, pluripotency and the ability to differentiate into all three embryonic germ layers were validated. Furthermore, functional cardiomyocyte generation was tested. The hiPSC-CRISPRi cell lines offer a valuable tool for studying endogenous transcriptional repression with single and multiplexed possibilities in all human cell types
Establishment of a second generation homozygous CRISPRa human induced pluripotent stem cell (hiPSC) line for enhanced levels of endogenous gene activation
No full textCRISPR/Cas9 technology based on nuclease inactive dCas9 and fused to the heterotrimeric VPR transcriptional activator is a powerful tool to enhance endogenous transcription by targeting defined genomic loci. We generated homozygous human induced pluripotent stem cell (hiPSC) lines carrying dCas9 fused to VPR along with a WPRE element at the AAVS1 locus (CRISPRa2). We demonstrated pluripotency, genomic integrity and differentiation potential into all three germ layers. CRISPRa2 cells showed increased transgene expression and higher transcriptional induction in hiPSC-derived cardiomyocytes compared to a previously described CRISPRa line. Both lines allow studying endogenous transcriptional modulation with lower and higher transcript abundance
Combining a tetracycline (Tet)-inducible gRNA system and CRISPRa for titratable and timely controlled enhancement of endogenous SHISA3 activation in human induced pluripotent stem cells (hiPSC)
No full textTowards increasing the possibility for temporal control of gene expression using CRISPR activation (a) systems, we generated homozygous human induced pluripotent stem cell (hiPSC) lines carrying a doxycycline (dox)-inducible guide(g)-RNA construct targeting the SHISA3 transciptional start site, as proof-of-principle, or a non targeting gRNA as a control. The dox-inducible gRNA cassette was inserted into the human ROSA26 locus in a line with dCas9VPR integrated at the AAVS1 locus (CRISPRa/Tet-iSHISA3). Pluripotency, genomic integrity and differentiation potential into all three germ layers were maintained. Dox-dependent gene induction was validated in hiPSCs as well as derived fibroblasts. These lines provide an attractive tool for cellular reprogramming in hiPSC-derived cells in a timely controlled manner
P1235Acetylcholine-activated inward-rectifier potassium currents in atrial cardiomyocytes derived from induced pluripotent stem cells and atrial engineered heart muscle preparations
No full textSecreted-Frizzled-Related Protein 5 Modulates Calcium Handling in Human Ipsc-Cardiomyocytes
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