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RNA sequencing of cells treated with DMSO or Retinoic acid during cardiac differentiation

ABSTRACT: Analysis of transcriptional differences between control and RA-treated cells during cardiac differentiation. The hypothesis tested in these samples is that addition of RA during differentiation towards atrial-like cardiomyocytes while control cells treated with DMSO result in ventricular-like cardiomyocytes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE67866 | GEO | 2015/05/28

SECONDARY ACCESSION(S): PRJNA281093

REPOSITORIES: GEO

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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.

Project description:Central questions like cardiomyocyte subtype emergence during cardiogenesis or availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, characterization and implementation of pure cardiomyocyte subtypes is still challenging due to technical limitations. Our aim was to identify surface markers enabling the selective detection and purification of atrial and ventricular cardiomyocytes from mouse hearts. In a surface marker screen we found differential expression of CD49f in atrial and ventricular embryonic cardiomyocytes (E13.5). By flow cytometry we could correlate a high CD49f expression with MLC-2a on the single cell level; a low CD49f expression corresponded to MLC-2v. Based on the persisting differential CD49f expression we developed purification protocols for cardiomyocytes subtypes from the developing mouse heart. Flow sorting of E15.5 hearts into ErbB-2+/CD49flow and ErbB-2+/CD49fhigh cells led to a selective depletion (CD49flow) or enrichment of MLC-2a+ cells (CD49fhigh). We found a corresponding CD49f-dependent distribution of MLC-2a when pre-enriched neonatal cardiomyocytes (P2) were flow-sorted into CD49flow and CD49fhigh. Atrial and ventricular identity was confirmed by expression profiling and patch clamp analysis of sorted embryonic hearts, which unequivocally demonstrated that the sorted cells were viable and functional. For the first time, we introduce a non-genetic, antibody-based approach to specifically isolate atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This newly gained capability of obtaining highly pure, viable cells will facilitate in-depths characterization of the individual cellular subsets and will aid translational research and therapeutic applications. The dataset comprises four different cardiomyocytes subtypes from the developing mouse heart. Embryonic (E15.5) hearts were dissociated and flow-sorted into ErbB-2+/CD49flow and ErbB-2+/CD49fhigh cardiomyocytes. Neonatal (P2) hearts were dissociated, contaminating non-myocytes were removed by MACS depletion, and the purified cardiomyocytes were flow-sorted into CD49flow and CD49fhigh cardiomyocytes. Four biological replicates were available for each sample groups. Microarray analysis was conducted on the Agilent Whole Mouse Genome Oligo Microarray 8x60K platform.

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RNA sequencing of cells treated with DMSO or Retinoic acid during cardiac differentiation

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