(A) Photomicrographs illustrate control and NAFLD iPSC-Heps
on day 22 of differentiation. Panels depict phase contrast morphology and
fluorescent images documenting lipid content (Bodipy 493/503). Graph
demonstrates the triglyceride content of iPSC-Heps at day 22. *
P < 0.05. (B) Heatmap illustrates
unsupervised clustering of RNA sequencing data and the accompanying principal
component analysis from control and NAFLD iPSC-Heps (367 genes, |FC| >
1.5, FDR < 0.05). Beneath the heatmap is the SNP genotyping profile of
each subject for PNPLA3 (rs738409), TM6SF2
(rs58542926), MBOAT7 (rs641738), GCKR
(rs780094), HSD17B13 (rs72613567) and MARC1
(rs2642438). Black = homozygous for risk or protective allele; gray =
heterozygous; white = no risk or protective allele. ● denotes subjects
with histologic hepatic steatosis but not NASH. (C) Volcano plot
illustrates 235 upregulated and 331 downregulated genes in NAFLD iPSC-Heps vs.
controls (|FC| > 1.5, P < 0.01). Select genes are
noted by name. (D) Histogram illustrates select Gene Ontology
biological processes that were significantly dysregulated in NAFLD vs. control
iPSC-Heps (|FC| > 1.5, FDR < 0.25). (E) Heatmap
illustrates differential regulation of select candidate genes in NAFLD vs.
control iPSC-Heps, organized by function. (F) Heatmap demonstrates
the top 25 differentially expressed upstream regulators in NAFLD vs. control
iPSC-Heps, compared to data from 5 publicly available datasets of liver tissue
from humans with NAFLD as noted by accession number (via Ingenuity Pathway
Analysis; see Methods).