Global Gene Expression Profiling Reveals Isorhamnetin Induces Hepatic-Lineage Specific Differentiation in Human Amniotic Epithelial Cells

Abstract

Human amnion epithelial cells (hAECs), derived from discarded term placenta, is anticipated as a new stem cell resource because of their advantages over embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), such as no risk of tumorigenicity and minimal ethical issue. hAECs have been reported to differentiate into hepatic-like cells (HLCs) with variable functionalities suitable for cell-based therapy of end-stage liver diseases, drug screening, and drug toxicity tests. On the other hand, a new research stream has been evolving to use natural compounds as stimulants of stem cell differentiation because of their high availability and minimum side effects. Isorhamnetin is a naturally occurring flavonoid commonly found in fruits and vegetables and has been reported to improve hepatic fibrosis and steatosis. In this present study, we have screened the differentiation potential of isorhamnetin in hAECs. The cells were grown on 3D cell culture and were treated with 20 μM of synthesized isorhamnetin for 10 days without adding any additional growth factors. DNA microarray global gene expression analysis was conducted for differentially expressed genes between isorhamnetin-treated and untreated control cells, gene expression validation was carried out using RT-qPCR method, and finally, several hepatic functions were assessed. Microarray analysis showed that isorhamnetin could activate essential biological processes, molecular functions, and signaling pathways for hepatic differentiation. Hepatic progenitor markers, EPCAM and DLK1, were upregulated in the isorhamnetin-treated hAECs. AFP was downregulated, while ALB was upregulated on Day 10. Furthermore, isorhamnetin-treated cells could show increased CYP enzyme mRNA levels, ICG uptake and release, glycogen storage activity, and urea secretion. Additionally, isorhamnetin-treated cells did not show any trace of transdifferentiation evident by significant downregulation of several colon- and cholangiocyte-specific markers. However, longer treatment with isorhamnetin did not promote hepatic maturation. Altogether, our findings indicate that isorhamnetin has a promising effect on directing the hepatic-lineage specific differentiation in hAECs.

Keywords: hepatic-lineage-specific differentiation; human amnion epithelial cell; isorhamnetin; microarray and bioinformatics; natural compound.

FIGURE 1

Volcano plots showing DEGs (fold change >2, p value < 0.05) between (A) D10 isorhamnetin-treated and D0 control hAECs, and (B) D10 isorhamnetin-treated and D10 control hAECs. The Y-axis corresponds to –log10 p-value, and the X-axis displays linear fold change. The red dots represent the upregulated genes; the green dots represent the downregulated genes. (C) Bar graph showing number of DEGs in each treatment pair. (D) Venn diagram showing common and unique sets of DEGs between each exposure. Blue circle denotes DEGs between D10 and D0 controls; red circle denotes DEGs between D10 isorhamnetin-treated and D0 control hAECs. Iso, Isorhamnetin.

FIGURE 2

Bar graph showing significantly enriched biological process gene ontology (p < 0.05; Modified Fisher’s Exact Test). Values are presented in logarithm (10 base) scale. Fold enrichment is calculated as the ratio of the two proportions: the proportion of genes associated with the GO category in a list of DEGs of a study and the proportion of genes associated with that GO in the human genome (DAVID online tool: https://david.ncifcrf.gov/home.jsp). Iso, Isorhamnetin; Cont, Control.

FIGURE 3

Pie charts showing top significantly enriched (p < 0.05; Modified Fisher’s Exact Test) (A) Molecular function GOs by the DEGs between D10 isorhamnetin-treated and D0 control hAECs, and (B) Gene functional categories of the DEGs between D10 isorhamnetin-treated and D10 control hAECs. Fold enrichment values are presented in each corresponding pie. (C) Bar graph showing significantly enriched (p < 0.05; Modified Fisher’s Exact Test) top KEGG pathways by DEGs between D10 isorhamnetin-treated and D10 control hAECs. Values are presented in logarithm (10 base) scale.

FIGURE 4

(A) Relative gene expression levels of hepatic progenitor markers. Heatmaps showing signal intensities (log2) of genes (from microarray analysis) related to (B) Undefined placenta, (C) Pluripotency, (D) Hepatoblast, and (E) Hepatocyte. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 5

(A) Bar graph showing significantly downregulated GO in D10 isorhamnetin-treated hAECs compared to D10 control. Heatmaps showing signal intensities (log2) of genes (from microarray analysis) related to (B) Transdifferentiation, (C) Cholangiocyte, and (D) Colon. (E) Relative expression levels of genes to trace transdifferentiation. *P < 0.05, **P < 0.01, ns: not significant.

FIGURE 6

(A) Heatmap showing signal intensities (log2) of CYP enzymes (from microarray analysis), (B) Relative gene expression of CYP enzymes. (C) Heatmap showing signal intensities (log2) of APO genes (from microarray analysis), (D) Relative expression levels of APO genes. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.

FIGURE 7

(A) ICG uptake and release, (B) PAS staining, (C) Urea secretion assay. *P < 0.05.