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Depletion of IRX3 promotes early cardiac commitment and maturation of human Ipsc-derived cardiomyocytes

ABSTRACT: The limited regenerative capacity of the adult human heart makes human induced pluripotent stem cell (hiPSC)-based therapies a promising approach for treating myocardial infarction. However, hiPSC-derived cardiomyocytes (hiPSC-CM) display immature features. In this study, we demonstrate that depletion of the transcription factor IRX3 promotes early cardiac commitment and enhances the maturation of hiPSC-CMs. We first established the temporal expression of IRX3 during hiPSC-CM differentiation and subsequently investigated its role by generating two IRX3 mutant hiPSC lines. IRX3-depleted cardiac progenitors exhibited increased expression of GATA4, NKX2-5, TBX5 and Wnt signaling-related genes, along with increased proliferation. Differentiation of IRX3-depleted cells yielded more cardiomyocytes with enhanced expression of TNNI1 and CX43. Notably, IRX3-depleted cardiomyocytes showed improved sarcomere alignment, enhanced cell-to-cell communication, and electrophysiological maturation. Furthermore, in silico analysis suggests that IRX3 regulates cardiomyocyte differentiation by modulating TBX5, GATA4 and NKX2-5 transcription along with their targets. Collectively, our findings underscore the potential of IRX3 depletion in facilitating the molecular and functional development of human cardiomyocytes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE262776 | GEO | 2026/03/28

REPOSITORIES: GEO

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Project description:Genome-wide occupancy analysis of TBX5, NKX2-5 and GATA4 in differentiating WT, Nkx2-5KO (NKO), Tbx5KO (TKO) and Nkx2-5;Tbx5KO (DKO) cells at the cardiac precursor (CP) and cardiomyocyte (CM) differentiation stages. Analysis of TBX5, NKX2-5 and GATA4 occupancy a and gene expression in WT, Tbx5KO, Nkx2-5KO and DoubleKO precursor (CP) and mature (CM) in vitro differentiated cardiomyocytes. We performed ChIP-exo experiments for NKX2-5, TBX5 and GATA4 in differentiating WT, Nkx2-5KO (NKO), Tbx5KO (TKO) and Nkx2-5;Tbx5KO (DKO) cells at the CP and CM stages. ChIP-exo was performed according to methods published previously (Boyer et al., 2005; Serandour et al., 2013; Wamstad et al., 2012) using specific antisera to TBX5 (sc-17866 XS, lot no. B1213), NKX2-5 (sc-8697 XS, lot no. B1213), and GATA4 (sc-1237 XS, lot no. B1213) on chromatin isolated from 10 mill. cells. Re-ChIP-exo was performed from 40 mill cells, combining methods published previously (Serandour et al., 2013; Shankaranarayanan et al., 2011). ChIP-exo Analysis: Barcoded libraries were single-end 50bp sequenced on an Illumina HiSeq 2500 instrument. Reads were trimmed using the fastq-mcf program (Aronesty, 2011), and then aligned to the mm9 mouse genome assembly using bowtie2 (ChIP-seq, ChIP-exo) (Langmead and Salzberg, 2012). Samtools was then used to retain reads that had a mapq score of 30 or greater (Li et al., 2009), thereby ensuring that each mapped uniquely to the genome assembly. The 5' -most position of reads that mapped to the reference strand and the 3Ã¢??-most position of reads that mapped to the non-reference strand were identified for each read as the actual edges of each exonuclease-treated fragment. The genome was divided into 20bp genomic bins and a normalized tag density was calculated for each genomic bin 'i' as follows: tag density(i)=([#tags within 75bp of i] X [total# genomic bins])/(total #of tags) To identify broad regions of binding, bins with tag densities of greater than 100 were merged to generate a peak list for each sample. Within 1kb of each region, strand-specific single-base-resolution tag densities were calculated for each dataset by dividing each region into 1bp bins, then counting the number of tags within 5bp of each bin. For each region of binding, the footprint for each bound region was defined as the span from the peak position of '+' strand binding to the peak position of '-' strand binding as seen from the high-resolution tag densities. For subsequent analyses, any overlapping footprint for replica/factor/stage/genotype was merged to generate an average footprint list. Only average footprints present in at least two replicas of the same factor, stage and genotype were considered (Final Average Footprints).

Project description:The cardiomyocytes (CMs) differentiation of 12 validated iPSC lines were induced using the Gibco PSC Cardiomyocyte Differentiation Kit and following manufacturer method with minor modifications (Thermo Fisher Scientific, Waltham, MA, USA). The differentiation kit consists of a set of serum-free and xeno-free media that enable efficient differentiation of human iPSCs into spontaneously contracting functional cardiomyocytes in 14 days. On day 14 of the differentiation, spontaneously contracting CMs were characterized by immunocytochemistry (ICC) analysis of cardiac mesoderm marker NKX2-5, and mature cardiomyocyte marker TNNT2 and genome-wide mRNA sequencing of the 12 iPSC lines and generated 12 CM lines were performed. The CMs differentiated from all the 12 iPSC lines expressed NKX2-5 and TNNT2 cardiomyocyte markers. Based on the criteria normalized read count (NRC) ≥ 20 in all 12 CM samples, a total of 12280 genes were found expressed. The average correlation coefficient at 95% CI between 12 CM samples, calculated based on all expressed genes, was 0.92 ± 0.02, suggesting a uniform differentiation of generated CMs across 12 samples. The expression of the well-known CMs genes/markers MYH6, MYL4, MYL3, MYL7, NPPA, NPPB, MYH7, NKX2-5, TBX5, TNNT2, ACTN2, TNNC1 and MB, was significantly up-regulated across all 12 CM samples (FDR corrected p-value ≤ 0.05 and fold change absolute (FC-abs) ≥ 2.0); whereas the expression of pluripotency markers and the ectodermal markers was significantly down regulated. The endodermal markers SOX17 and CLDN6 were below the expression threshold of NRC ≥ 20. A differential gene expression analysis of iPSC’s and CM’s expressed transcription identified 4,191 genes that were significantly differentially expressed (DE) between iPSCs and differentiated CMs. The 2,116 DE genes that were significantly up regulated in differentiated CMs showed significantly high enrichment in cardiovascular system development and functions (571 mRNAs; FDR adjusted p-value range: 1.75x10-66 – 1.61x10-14) and predicted activation of the functions associated with development of human heart and cardiovascular system.

Project description:Background: Cardiomyocytes (CMs) induced from human induced pluripotent stem cells (hiPSCs) by traditional methods are a mix of atrial and ventricular CMs and many other non-cardiomyocytes. Retinoic acid (RA) plays an important role in regulation of the spatiotemporal development of the embryonic heart and high concentrations of RA have been shown to steer differentiation towards atrial CMs, whereas lower concentrations of RA promote a more ventricular-like CM profile. Aim: Create engineered heart tissue (EHT) with left and right ventricular phenotype from hiPSCs by intervening with specific concentrations of retinoic acid (RA) during hiPSC differentiation towards CM. Methods: hiPSC were derived by reprogramming skin fibroblasts. Different concentrations of RA (Control group without RA, LRA group with 0.05 µM and HRA group with 0.1 µM) were administered during third to sixth days of the differentiation process. Engineered heart tissues (EHTs) were generated by assembling CMs derived from hiPSC (hiPSC-CM) at high cell density in a low collagen hydrogel. The maturation and growth of EHTs were induced in a customized biomimetic tissue culture system, that provides continuous electrical stimulation, medium agitation and stretch. The function of CMs and EHTs was analyzed under different conditions. Finally, RNA extraction and tissue fixation were performed on CMs and EHTs for RT-qPRC and immunofluorescence staining analysis. RNA sequencing was conducted on EHTs to examine how RA affects both the function and structure of EHT. Results: In the HRA group, hiPSC-CMs exhibited the first onset of beating and showed the highest expression of maturity genes MYH7 and cTnT. The expression of TBX5, NKX2.5 and CORIN, which are the marker genes for left ventricular CMs, was also the highest in the HRA group. The transcription factor MEF2C associated with RA and ventricular development genes, NPPA and MYH7, were highly expressed in the HRA group, while GATA4 was less expressed in the HRA group. In terms of EHT, the HRA group displayed the highest contraction force, the lowest beating frequency, and the highest sensitivity to hypoxia and isoprenaline, which means it was more functionally similar to the left ventricle. The expression of TBX5 and NKX2.5 was found to be the highest expression in the HRA group of EHT, while expression of TBX20 and ISL1 was found to be the highest expression in control group. When the electrical stimulation frequency of EHT in the HRA group was raised, it correspondingly increased its contractile force. The heightened contractility of EHT within the HRA group can be attributed to the promotion of augmented extracellular matrix strength by RA. Conclusion: By interfering with the differentiation process of hiPSC with a specific concentration of RA at a specific time, we were able to successfully induce CMs and EHT with a phenotype similar to that of the left ventricle or right ventricle. Our research paved the way for future studies on in vitro left ventricular or right ventricular function, personalized drug screening, and precision medicine.

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Depletion of IRX3 promotes early cardiac commitment and maturation of human Ipsc-derived cardiomyocytes

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