Search

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.

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 IRX5mutations, have defects in various organs including the heart and the limbs. Interestingly, heart and limbs defects are also found in Holt-Oram syndrome patients (TBX5mutations), 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 SCN5Aexpression: 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.

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 vitrocardiac 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.