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Human pluripotent stem cell (hPSC)-derived progenies (ChIP-Seq)

ABSTRACT: The in vitro culture of human cardiac progenitor cells remains a major challenge in biomedicine. The molecular identity of hPSC-derived cardiac progenies and mechanisms controlling their proliferation and differentiation remain unclear. Here, we show that chromatin remodeling and WNT pathway modulation by chemical inhibitors (IQ-1 and CHIR, respectively) synergistically enables stabilization of human cardiac progenitors (SCPs), in a process of transcriptional uncoupling. SCPs are characterized by ISL1pos/KI-67pos/NKX2-5neg expression and are maintained in a quiescent state in the presence of the inhibitors. Upon compound removal, cell autonomous NKX2-5 upregulation hallmarks the recoupling to the cardiomyogenic program, whilst directed differentiation generates endothelial and smooth muscle cells. Chip-sequencing revealed a narrowly defined open chromatin state in SCPs and, when combined with single cell transcriptome analyses, showed a yet unreported Cardiac Neural Crest Cells (CNCCs) footprint. Enforced expression of the oncogene c-MYC could notably not overcome SCPs proliferative quiescence but disrupted the cell autonomous cardiomyogenic potential instead. In contrast, Retinoic Acid (RA) stimulates SCPs proliferation and further supports cells’ phenotypic homogeneity. We consequently show that our in vitro established treatment with IQ1 can also retain ISL1pos cardiac progenies in vivo in a dose and stage specific manner during zebrafish heart development. Due to its chemically defined and reversible nature, our approach provides an unprecedented opportunity to dissect the key mechanisms in cardiac progenitor cell biology, providing a new tool for the advancement of human heart regeneration.

ORGANISM(S): Homo sapiens

PROVIDER: GSE165588 | GEO | 2025/01/27

REPOSITORIES: GEO

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Project description:Aims: MiRNAs are post-transcriptional regulatory molecules with recognized roles in human heart development and disease. The purpose of this study was to define human miRNA expression profile in cardiac progenitors and early-differentiated cardiomyocytes and to determine if these miRNAs that are governing cardiogenesis is dependent on NKX2-5, a highly conserved pan-cardiac transcription factor. Methods: MiRNA expression profiles of pre-NKX2-5 mesoderm, cardiac progenitors and early cardiomyocytes derived from heterozygous HES3 NKX2-5eGFP/w and null NKX2-5eGFP/eGFP hESC lines were generated by small RNA sequencing in triplicates, using Illumina HiSeq. Reads between 17-28 base pairs were selected for sequence alignment to the human genome (hg19) using Bowtie2. FeatureCounts (Subread, R package) was used to count the number of reads mapping to miRNAs and analysis of statistically differentially expressed miRNA was carried out using Limma (Bioconductor) through the statistical language R. qRT–PCR validation was performed using Taqman MicroRNA Assays. Results: We identified 11 miRNAs that were differentially expressed between pre-NKX2-5 mesoderm and cardiac progenitors, and 112 differentially expressed miRNAs between cardiac progenitors and early cardiomyocytes. Four of which were validated with qRT–PCR, including canonical myogenic miRNAs such as MIR-1-1, -133A1 and 208A that were enriched in both the cardiac progenitor and early cardiomyocyte populations. Strikingly, deletion of NKX2-5 did not result in gross changes in cardiac miRNA profile either at progenitor or cardiomyocyte stage. Instead, non-hierarchical clustering and principal component analysis demonstrated that the different stages of differentiation (day 6 vs day 10) was a larger discriminator between samples than NKX2-5 genotype. Conclusion: Our study demonstrates the application of human embryonic stem cells as an in vitro model to investigate the role of miRNAs in human cardiac development. We conclude that while specific miRNAs have been identified in our study to have a role in the early stages cardiac cell fate specification, the majority of cardiac myogenic miRNA program is regulated separately from the highly conserved NKX2-5 -dependent gene network. This study provides a framework for further investigation of other key transcription factors of cardiac muscle development that might drive cardiomyogenic expression of miRNAs.

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Human pluripotent stem cell (hPSC)-derived progenies (ChIP-Seq)

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