Mutations in the DES gene, which encodes the intermediate filament desmin, lead to desminopathy, a rare disease characterized by skeletal muscle weakness and different forms of cardiomyopathies ...associated with cardiac conduction defects and arrhythmias. We generated human induced pluripotent stem cells (hiPSC) from a patient carrying the DES p.R406W mutation, and employed CRISPR/Cas9 to rectify the mutation in the patient's hiPSC line and introduced the mutation in an hiPSC line from a control individual unrelated to the patient. These hiPSC lines represent useful models for delving into the mechanisms of desminopathy and developing new therapeutic approaches.
Studies on animal models have shown that Irx5 is an important regulator of cardiac development and that it regulates ventricular electrical repolarization gradient in the adult heart. Mutations in ...IRX5 have also been linked in humans to cardiac conduction defects. In order to fully characterize the role of IRX5 during cardiac development and in cardiomyocyte function, we generated three genetically-modified human induced pluripotent stem cell lines: two knockout lines (heterozygous and homozygous) and a knockin HA-tagged line (homozygous).
Catecholamine-induced QT prolongation (CIQTP) is an inherited cardiac disease characterized by a normal baseline ECG and a risk of sudden cardiac death by ventricular arrhythmia due to a QT ...prolongation that only appears during catecholergic stimulation, especially mental stress. Induced pluripotent stem cells (hiPSCs) were generated from peripheral blood mononuclear cells collected from two CIQTP-affected patients from two different families. These two hiPSC lines are a valuable model to study biological alterations due to CIQTP.
Four human induced pluripotent stem cell (hiPSC) lines have been generated from healthy control European donors, and validated. This resource represents a useful tool for stem cell-based research, as ...references for developmental studies and disease modeling linked to any type of human tissue and organ, in an ethnical-, sex- and age-matched context. They providea reliable in-vitro model for single cell- and tissue-based investigations, and are also a valuable tool for genome editing-based studies.
Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an exercise and emotional stress-induced life-threatening inherited heart rhythm disorder, characterized by an abnormal cellular ...calcium homeostasis. Most reported cases have been linked to mutations in the gene encoding the type 2 ryanodine receptor gene, RYR2. We generated induced pluripotent stem cells (hiPSCs) from peripheral blood mononuclear cells (PBMC) from three CPVT-affected patients, two of them carrying p.R4959Q mutation and one carrying p.Y2476D mutation. These generated hiPSC lines are a useful model to study pathophysiological consequences of RYR2 dysfunction in humans and the molecular basis of CPVT.
This manuscript proposes an efficient and reproducible protocol for the generation of genetically modified human induced pluripotent stem cells (hiPSCs) by genome editing using CRISPR-Cas9 ...technology. Here, we describe the experimental strategy for generating knockout (KO) and knockin (KI) clonal populations of hiPSCs using single-cell sorting by flow cytometry. We efficiently achieved up to 15 kb deletions, molecular tag insertions, and single-nucleotide editing in hiPSCs. We emphasize the efficacy of this approach in terms of cell culture time.
For complete details on the use and execution of this protocol, please refer to Canac et al. (2022) and Bray et al. (2022).
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•Generation of knockout and knockin edits in hiPSCs using the CRISPR-Cas9 RNP system•FACS-assisted genome editing of hiPSCs•An optimized approach for culturing and genotyping hiPSC clones
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
This manuscript proposes an efficient and reproducible protocol for the generation of genetically modified human induced pluripotent stem cells (hiPSCs) by genome editing using CRISPR-Cas9 technology. Here, we describe the experimental strategy for generating knockout (KO) and knockin (KI) clonal populations of hiPSCs using single-cell sorting by flow cytometry. We efficiently achieved up to 15 kb deletions, molecular tag insertions, and single-nucleotide editing in hiPSCs. We emphasize the efficacy of this approach in terms of cell culture time.
Human heart development is governed by transcription factor (TF) networks controlling dynamic and temporal gene expression alterations. Therefore, to comprehensively characterize these ...transcriptional regulations, day-to-day transcriptomic profiles were generated throughout the directed cardiac differentiation, starting from three distinct human- induced pluripotent stem cell lines from healthy donors (32 days). We applied an expression-based correlation score to the chronological expression profiles of the TF genes, and clustered them into 12 sequential gene expression waves. We then identified a regulatory network of more than 23,000 activation and inhibition links between 216 TFs. Within this network, we observed previously unknown inferred transcriptional activations linking IRX3 and IRX5 TFs to three master cardiac TFs: GATA4, NKX2-5 and TBX5. Luciferase and co-immunoprecipitation assays demonstrated that these five TFs could (1) activate each other's expression; (2) interact physically as multiprotein complexes; and (3) together, finely regulate the expression of
, encoding the major cardiac sodium channel. Altogether, these results unveiled thousands of interactions between TFs, generating multiple robust hypotheses governing human cardiac development.
To the Editor: Brugada syndrome (BrS) is an inherited arrhythmic disease predisposing to sudden cardiac death (SCD), characterized by a typical electrocardiogram pattern that includes a J point ...elevation with a coved type ST segment.1 BrS is a complex genetic disease in which ∼20% of patients carry rare variants in SCN5A gene, whereas the others remain genetically unresolved.2 Despite this genetic complexity, we hypothesize that a common cellular phenotypic trait is at the root of this specific BrS ECG pattern. Furthermore, the steady-state activation and inactivation gating properties were not modified in BrS hiPSC-CMs (Figure S3A; Table S4). ...INa reduction is not a common trait of BrS hiPSC-CMs and appears to be solely associated with the presence of variants affecting SCN5A expression or function. SEE PDF The occurrence of EADs may be linked to an abnormally high density of depolarizing late sodium current (INa,L) during APs repolarizing phase.8 Accordingly, BrS hiPSC-CMs presented with a higher density of INa,L as compared to Ctrl and non-BrS hiPSC-CMs (Figure 3C,D). ...an increase in INa,L density was observed only in 6% and 12% of Ctrl and non-BrS hiPSC-CMs respectively, in accordance with their low EAD occurrence, whereas increased INa,L density was present in 50–85% of all BrS ventricular-like hiPSC-CMs, reminiscent of the high EAD occurrence (Figure 3B,E). ...the authors are grateful to the patients and families who agreed to participate in our research.
Abstract only
Introduction:
Recent genetic data suggest that abnormal cardiac development participate to the pathogenesis of Brugada Syndrome (BrS), a rare inherited arrhythmia responsible for sudden ...cardiac death in young adults.
In vitro
cardiac differentiation of human induced pluripotent stem cells (hiPSCs) mimics cardiac development at the cellular level up to a prenatal stage.
Objective:
This study aims at defining whether BrS impairs cardiac differentiation of hiPSCs.
Methods & Results:
Transcriptomic kinetics (daily bulk 3’RNA-seq from day 0 to day 30 of in vitro cardiac differentiation) were generated in triplicate for 2 control hiPSC lines and 2 BrS-patient hiPSC lines. First, global analysis unveiled that BrS and control kinetics start to diverge as early as day 8, coinciding with the emergence of beating cells. The 500 most differentially expressed genes between BrS and control kinetics revealed 7 main distinct expression profiles. Interestingly, in one of the clusters (Cluster 2), enriched in genes involved in ventricular development (
e.g. IRX4
,
NKX2-5
), the expression levels were higher in BrS as compared to control, starting at day 8. Inversely, another cluster (Cluster 4), enriched in genes involved in atrial development (
e.g. TBX18
,
PITX2
), displayed an opposite expression profile. Cell-type annotation of single-cell RNA-seq data obtained at day 30 of cardiac differentiation for 1 control (n=2; 11,499 cells) and 1 BrS hiPSCs line (n=2; 12,142 cells) confirmed this ventricular-to-atrial imbalance with an average ventricular-to-atrial cell number ratio of 0.97 and 8.27 for control and BrS lines, respectively.
Conclusion:
This first transcriptomic kinetic study supports the hypothesis of an early developmental defect in BrS. Altogether, our data show that BrS hiPSCs are more prone to ventricular specification as compared to control cells. This suggests that an abnormal cell fate during cardiac differentiation may participate to BrS pathogeny.
Abstract only
Introduction:
The transcription factor (TF) Iroquois homeobox 5 (Irx5) is a key patterning and differentiation regulator. In humans, IRX5 regulates the expression of the main cardiac ...sodium ion channel gene,
SCN5A
. However, its global mechanistic role in human cardiomyocytes is still to be fully understood. Hamamy syndrome patients, carrying
IRX5
mutations, have defects in various organs including the heart and the limbs. Interestingly, heart and limbs defects are also found in Holt-Oram syndrome patients (
TBX5
mutations), suggesting a functional link between TBX5 and IRX5. Moreover, a cooperation of TBX5 with GATA4 and NKX2-5 in cardiac development and functions has already been shown.
Hypothesis:
We hypothesized that IRX5 may cooperate with the TF complex TBX5/GATA4/NKX2-5 to regulate key cardiac functions in humans.
Methods & Results:
First, immunoprecipitations in HEK293 cells showed that IRX5 can bind to TBX5, GATA4 and NKX2-5, individually and together as a complex. Using five truncated forms of IRX5 protein, its homeodomain was identified as being the essential protein region for these interactions. Second, we showed by luciferase assays, that each combination of these TFs impacted differently
SCN5A
expression:
e.g.
by itself, NKX2-5 has a strong activator effect (increased activity by 10 fold
vs
. no TF) that is potentialized by IRX5 (12 fold
vs
. no TF) but inhibited by TBX5 (8 fold
vs
. no TF). Third, genes bound by IRX5 (n=2253) were identified by ChIP-Seq on cardiomyocytes derived from induced pluripotent stem cells generated from 2 healthy individuals. Analyzing published ChIP-Seq datasets for TBX5, GATA4 or NKX2-5 in the same cellular model, we identified 2990 genes that were bound by these 3 TFs, and associated with cardiac biological pathways, such as muscle contraction. Among these genes, 848 were also bound by IRX5 and were associated with the function of electrical activity and with the fibrosis signaling, suggesting a specific role for IRX5 in these cardiac processes.
Conclusions:
Overall, our data show new physical and functional interactions between IRX5 and 3 key cardiac TFs (TBX5, GATA4 and NKX2-5), and suggest an unexpected regulatory role for IRX5 in specific human heart functions.
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