RNAseq Analysis of Isogenic Human Induced Pluripotent Stem Cell Lines Reveals Differential Gene Expression Patterns during Differentiation of Human Heart Organoids Due to a Patient-Specific TBX5 Mutation

Background: Human heart organoids (hHOs) develop by self-assembly from human induced pluripotent stem cells (iPSCs). Transcriptome, structure and cellular composition of hHOs are quite similar to fetal cardiac tissue. Thus, they represent an ideal model to study early cardiac development in congenital heart disease. We sought to analyze the consequences of a disease-causing TBX5 mutation on the development of hHOs using isogenic iPSC lines.

Methods: Established human iPSC line which carry a patient-specific TBX5 mutation (1456) and the CRISPR/Cas9 corrected iPSC line with the wild-type TBX5 (1456corr) were differentiated into human heart organoids (hHOs) in low-attachment round-bottom 96-well plates. Total RNA from hHOs was isolated at days 8, 10 and 15 and subjected to RNAseq analysis to identify differentially expressed genes (DEGs). Genes were considered as differentially regulated with an absolute log2 fold change > 2 and adjusted P < 0.05.

Results: Both human iPSC lines were equally potent to generate hHOs, which started to beat around day 6 and developed a stable contraction pattern by day 10 of differentiation. On day 8, 10 and 15 of hHO differentiation 1063, 866 and 773 DEGs were down-regulated and 294, 680 and 334 DEGs were up-regulated, respectively, in 1456-derived hHOs carrying the TBX5 mutation compared with hHOs which developed from 1456corr iPS cells with the wild-type TBX5 gene. Gene ontology analysis identified 335 GO terms in 1456 hHOs which were permanently down-regulated including extracellular matrix organization (P < 1.65 × 10^–19), muscle structure development (p < 3.54 × 10^–5) and TGFßR signaling pathway (P < 6.2x10–5). In contrast, DEGs associated with central nervous system development were consistently up-regulated (p < 2.44 × 10^–6) in 1456-derived hHOs.

Conclusion: Human iPSCs with a patient-specific TBX5 mutation or the wild-type TBX5 gene develop into beating hHOs with equal efficiency. Several essential pathways are apparently dysregulated in hHOs derived from 1456 iPSCs with the mutated TBX5 gene. This three-dimensional cellular system is a powerful tool to study the effects of a TBX5 mutation during early cardiac development and to identify dysregulated genes and signal pathways.