Human iPSC gene signatures and X chromosome dosage impact response to WNT inhibition and cardiac differentiation fate

Abstract

Non-genetic variability in human induced pluripotent stem cell (iPSC) lines impacts their differentiation outcome, limiting their utility for genetic studies and clinical applications. Despite the importance of understanding how non-genetic molecular variability influences iPSC differentiation outcome, large-scale studies capable of addressing this question have not yet been conducted. Here, we performed 258 directed differentiations of 191 iPSC lines using established protocols to generate iPSC-derived cardiovascular progenitor cells (iPSC-CVPCs). We observed cellular heterogeneity across the iPSC-CVPC samples due to varying fractions of two cell types: cardiomyocytes (CMs) and epicardium-derived cells (EPDCs). Analyzing the transcriptomes of CM-fated and EPDC-fated iPSCs discovered that 91 signature genes and X chromosome dosage differences influence WNT inhibition response during differentiation and are associated with cardiac fate. Analysis of an independent set of 39 iPSCs differentiated to the cardiac lineage confirmed shared sex and transcriptional differences that impact cardiac fate outcome. The scale and systematic approach of our study enabled novel insights into how iPSC transcriptional and X chromosome gene dosage differences influence WNT signaling during differentiation and hence cardiac cell fate.

Allele-specific NKX2-5 binding underlies multiple genetic associations with human electrocardiographic traits

“…Of note, the iPSC-CMs showed slightly greater variation (i.e. lower correlation values) than the iPSCs, likely due to cellular heterogeneity [24][25][26] .…”

“…6b), we followed up three loci previously associated with heart rate (rs7612445, NC_000003.11:g.179172979G>T; rs8044595, NC_000016.9:g.15906130A>G; and rs6606689, NC_000012.11:g. 110975675T>C) and a potential novel locus (rs176107, NC_000005.9:g.89392662A>G) with additional experimental data (Supplementary Note). These data included Hi-C chromatin conformation maps from the same iPSC-CM samples 46 ( Supplementary Table 2a), and RNA-seq data from iPSC-CMs from an additional 128 whole-genome-sequenced subjects 26 , to examine associations between the putative causal NKX2-5 ASE-SNVs and expression of nearby or distal candidate target genes. For rs7612445 (98% PPA), which altered a T-box motif in the GNB4 locus, we validated that the two alleles have differential binding using EMSA, and that it is associated with differential expression in iPSC-CMs of several genes, including GNB4 (heart specific eQTL in GTEx) and MFN1 (influencing heart rate in zebrafish and Drosophila 15 ; Supplementary Fig.…”

“…We used RNA-seq of iPSC-CMs from 128 different individuals 26 . Subjects included 43 males and 85 females, between 9 and 88 years of age, of diverse ethnicities (Europeans, n = 78, and Asians, n = 23).…”

“…iPSCs show variation in molecular phenotypes associated with their genetic background [18][19][20] , making them a suitable model to perform expression QTL (eQTL) studies [19][20][21][22][23][24] . However, there are only a few studies showing similar utility of iPSC-derived cardiomyocytes (iPSC-CMs) to study regulatory variations 22 , with potential limitations being cell-type heterogeneity that arises from directed differentiation [24][25][26] and the functional immaturity of iPSC-CMs 27 . Thus, while human iPSC-CMs are a promising model system, it has yet to be shown that they could enable the identification and characterization of regulatory variants that play important roles in cardiac traits.…”

Practical adoption of state-of-the-art hiPSC-cardiomyocyte differentiation techniques

“…It is unsurprising that, though we observed similar trends across three hiPSC lines, differences in differentiation outcomes were present. Recent work has shown that different hiPSC lines possess distinct transcription profiles and that these profiles are correlated with more or less cardiogenic hiPSC lines [21,22]. This may explain why some lines show more sensitivity to Wnt activation via Chiron compared to others.…”