Human Pluripotent Stem Cell-Derived Hepatocytes Show Higher Transcriptional Correlation with Adult Liver Tissue than with Fetal Liver Tissue

Abstract

Human pluripotent stem cell-derived hepatocytes (hPSC-HEP) display many properties of mature hepatocytes, including expression of important genes of the drug metabolizing machinery, glycogen storage, and production of multiple serum proteins. To this date, hPSC-HEP do not, however, fully recapitulate the complete functionality of in vivo mature hepatocytes. In this study, we applied versatile bioinformatic algorithms, including functional annotation and pathway enrichment analyses, transcription factor binding-site enrichment, and similarity and correlation analyses, to datasets collected from different stages during hPSC-HEP differentiation and compared these to developmental stages and tissues from fetal and adult human liver. Our results demonstrate a high level of similarity between the in vitro differentiation of hPSC-HEP and in vivo hepatogenesis. Importantly, the transcriptional correlation of hPSC-HEP with adult liver (AL) tissues was higher than with fetal liver (FL) tissues (0.83 and 0.70, respectively). Functional data revealed mature features of hPSC-HEP including cytochrome P450 enzymes activities and albumin secretion. Moreover, hPSC-HEP showed expression of many genes involved in drug absorption, distribution, metabolism, and excretion. Despite the high similarities observed, we identified differences of specific pathways and regulatory players by analyzing the gene expression between hPSC-HEP and AL. These findings will aid future intervention and improvement of in vitro hepatocyte differentiation protocol in order to generate hepatocytes displaying the complete functionality of mature hepatocytes. Finally, on the transcriptional level, our results show stronger correlation and higher similarity of hPSC-HEP to AL than to FL. In addition, potential targets for further functional improvement of hPSC-HEP were also identified.

Figure 1

Hierarchical clustering of the merged dataset grouped data into distinct clusters of hPSC (d0), DE (d5), hepatoblasts (d14), hPSC-HEP (d25), human FL tissues, and human AL tissues. AL_26y and AL_29y in the red marked cluster of AL samples are from the E-MTAB-5367 dataset, and the rest of the AL samples are from the GSE61279 dataset. Despite different platforms used, all nine samples from the adult liver clustered tight together.

Figure 2

(A) Spearman’s correlation results for hPSC, DE, hepatoblasts, and hPSC-HEP day 25 with FL week 8–9, FL week 11–12, FL week 21, and AL (confidence interval 95%). (B) Similarity analysis results for hPSC-HEP day 25 to FL week 8–9, FL week 11–12, FL week 21, and AL for FC < 1.5 between mean values of mentioned comparisons and coefficient of variance < 30% across replicated samples.

Figure 3

Venn diagram showing the number of differentially expressed genes from the following comparisons: hPSC vs DE (d0vsd5), DE vs hepatoblasts (d5vsd14), hepatoblasts vs hPSC-HEP day 25 (d14vsd25), and hPSC-HEP day 25 vs AL (d25vsAL).

Figure 4

GO enrichment analysis for biological processes identified using the EnrichR software and visualized by the Revigo software for the following comparisons: (A) differentially expressed genes between hPSCs and DE cells, (B) differentially expressed genes between DE and hepatoblasts, (C) differentially expressed genes between hepatoblasts and hPSC-HEP day 25, and (D) differentially expressed genes between hPSC-HEP day 25 and AL.

Figure 5

Venn diagram constructed for TFs with significantly enriched binding sites among the differentially expressed genes in the following transitions: hPSC–DE (d0vsd5), DE–hepatoblasts (d5vsd14), hepatoblasts–hPSC-HEP day 25 (d14vsd25), and hPSC-HEP day 25–AL (d25vsAL). See Table 1 for a list of the TFs.

Figure 6

Cytochrome P450 (CYP) activities in hPSC-HEP (N = 6 cell lines) on day 29 of the differentiation and cryopreserved human primary hepatocyte (hphep) cultured for 20 h post-thaw/plating (N = 4 donors). The concentrations of the metabolites, paracetamol (A; CYP1A), 3-OH-midazolam (B; CYP3A), 4-OH-diclofenac (C; CYP2C9), 4-OH-bupropion (D; CYP2B6), 1-OH-bufuralol (E; CYP2D6), and 4-OH-mephenytoin (F; CYP2C19), were measured by liquid chromatography/mass spectrometry. The CYP activity is presented as pmol metabolite per mg protein per minute (mean ± SD). Statistical significance was determined by applying the t-test or Welch’s t-test. Y-axis is in the logarithmic scale. ns (non-significant), *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 7

Representative micrographs illustrating the expression of CYP3A4 in hPSC-HEP (derived from cell line ChiPSC6b) on day 29 (20× magnification, scale 50 μm). (A) 4′,6-Diamidino-2-phenylindole (DAPI) staining the nuclei, (B) staining for CYP3A4, and (C) merge of DAPI and CYP3A4.

Figure 8

Analysis of hPSC-HEP day 29 (N = 2 cell lines) and hphep 24 h post-thaw/plating (N = 4 donors). Functions measured after 24 h of incubation: (A) albumin secretion presented as μg albumin/mg protein/24 h (mean ± SD) and (B) urea production presented as mg urea/mg protein/24 h (mean ± SD, Y-axis in the logarithmic scale).

Figure 9

Schematic overview of the bioinformatic analysis performed on the datasets E-MTAB-5367 (ArrayExpress database) and GSE61279 (GEO database).