Impaired Regulation of Redox Transcriptome during the Differentiation of iPSCs into Induced Cardiomyocytes (iCMs)

Abstract

Background: Reprogramming of somatic cells into pluripotent stem cells (iPSC) and subsequent differentiation into iPSC-derived cardiomyocytes (iCM) seems to be a promising strategy for cardiac regenerative therapy. However, recent failure or poor outcomes in cardiac cell therapy warrants further investigation focusing on the infarction/wound environment (site of healing) to improve the cardiac regenerative medicine. Here, using next generation sequencing (NGS), we analyzed the global transcriptome to discover the unidentified genes/pathways that are crucial for cell survival, cytoprotection and mitochondrial dynamics during the differentiation of iPSC into iCM.Methods: High throughput NGS was performed RNA from human iPSCs and iCMs (n=3/group) and analyzed the global changes in the transcriptome during differentiation. Furthermore, Ingenuity Pathway Analysis (IPA) and Gene Ontology (GO) for biological process were performed to understand the transcriptional networks that are involved during iCM differentiation. RNA-seq data were further validated by qRT-PCR analyses. Results:Global transcriptome analysis revealed that ~9,290 genes (log 2 FC >2) were significantly altered in human iCMs compared to the parent iPSCs, in which 4,784 transcripts were substantially upregulated and 4,506 transcripts were down-regulated during differentiation.GO enrichment and IPA analyses revealed the top 10 regulatory networks (i.e. hierarchical order) involved in differentiation of iCMs including cardiomyocyte remodeling, integrin-linked kinase signaling, Rho family of GTPases, etc. Surprisingly, none of the top 10 pathways listed the genes liable for redox signaling networks that are crucial for the basal cellular redox homeostasis, Nrf2-dependent antioxidant defense, mitochondrial functions and cell survival. Our deeper and unbiased analysis of this data revealed that the genes involved in above canonical signaling pathways are found in the middle of the inverted vertical cone. Of note, although these pathways are significantly altered during the differentiation (of iPS into cardiomyocytes), a majority of them are ranked low in the hierarchical list (>150). Validation of the randomly selected genes representing various pathways real-time qPCR confirmed the global transcriptome changes observed in NGS. Conclusion:We highlight the significance of Nrf2-redox and mitochondrial transcriptome during differentiation of iPSC into iCMs. Thus, targeting the redox signaling mechanisms in iCMs may enhance their efficiency for cell therapy and improved myocardial repair.180 genes were detected out of 230 genes known to constitute this pathway, whereas 58 genes were increased and 56 genes were decreased. From this list, the top 15 up and down-regulated genes were used to generate the heat-maps. A substantial list of genes were downregulated during cardiomyocyte differentiation including, MGST1, GSTM5, PRKCQ, PRKCB and PIK3R, while PIK3R5ACTA2, GSTM3, FMO1, ACTC1 and GSTA3 were increased during cardiomyocyte differentiation. RNA-seq data was valid...

Analysis of Protein-Protein Interaction Network Based on Transcriptome Profiling of Human Induced Pluripotent Stem Cells Identifies Candidate Genes

“…19 It is reported that RPS27A play a role in the regulation of cell cycle and promote cell proliferation. 20 This was consistent with the result of the present study that shows the expression Furthermore, KEGG pathway enrichment analysis showed that the five modules were mainly enriched in cell cycle, DNA replication, nucleotide excision repair, ribosome, ribosome biogenesis in eukaryotes, ErbB signaling pathway, EGFR tyrosine kinase inhibitor resistance, pancreatic cancer, breast cancer, prostate cancer, microRNAs in cancer, proteoglycans in cancer, pathways in cancer, endocytosis. Interestingly, it is notable that both DNA replication pathway and nucleotide excision repair pathway appeared twice in the KEGG pathway analysis of the five modules, accounting for 12 and 4 genes, respectively.…”