Hepatocyte-like cells derived from human induced pluripotent stem cells using small molecules: implications of a transcriptomic study

Abstract

Background: Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (iPSCs) hold great promise in toxicological applications as well as in regenerative medicine. Previous efforts on hepatocyte differentiation have mostly relied on the use of growth factors (GFs) to recapitulate developmental signals under in vitro conditions. Recently, the use of small molecules (SMs) has emerged as an attractive tool to induce cell fate transition due to its superiority in terms of both quality and cost. However, HLCs derived using SMs have not been well characterized, especially on the transcriptome level.

Methods: HLCs were differentiated from human iPSCs using a protocol that only involves SMs and characterized by transcriptomic analysis using whole genome microarrays.

Results: HLCs derived using the SM protocol (HLC_SM) displayed specific hepatic marker expression and demonstrated key hepatic functions. Transcriptomic analysis of the SM-driven differentiation defined a hepatocyte differentiation track and characterized the expression of some key marker genes in major stages of hepatocyte differentiation. In addition, HLC_SM were scored with CellNet, a bioinformatics tool quantifying how closely engineered cell populations resemble their target cell type, and compared to primary human hepatocytes (PHHs), adult liver tissue, fetal liver tissue, HLCs differentiated using GFs (HLC_GF), and commercially available HLCs. Similar to HLC_GF, HLC_SM displayed a mixed phenotype of fetal and adult hepatocytes and had relatively low expression of metabolic enzymes, transporters, and nuclear receptors compared to PHHs. Finally, the differentially expressed genes in HLC_SM compared to HLC_GF and to PHHs were analyzed to identify pathways and upstream transcription regulators which could potentially be manipulated to improve the differentiation of HLCs.

Conclusions: Overall, the present study demonstrated the usefulness of the SM-based hepatocyte differentiation method, offered new insights into the molecular basis of hepatogenesis and associated gene regulation, and suggested ways for further improvements in hepatocyte differentiation in order to obtain more mature HLCs that could be used in toxicological studies.

Keywords: Hepatocyte differentiation; Hepatocyte-like cells; Induced pluripotent stem cells; Microarray; Small molecules; Transcriptomics.

Fig. 1

Small molecule-based protocol for the differentiation of hepatocytes from induced pluripotent stem cells. a Schematic diagram showing the three-phase differentiation process. b Representative images showing sequential morphological changes at each stage of the differentiation. iPSC, induced pluripotent stem cell; ME, mesendoderm; DE, definitive endoderm; HP, hepatic progenitor; HLC, hepatocyte-like cell. Magnification: × 10, Insert: × 40, Scale bar: 400 μm. c Representative immunocytochemistry images demonstrating the expression of key markers at each stage of the differentiation. The percentage of marker positive cells, presented as mean ± SD of three independent experiments, is listed at the bottom under each marker. Scale bar: 400 μm

Fig. 2

Functional analysis of hepatocyte-like cells derived using the small molecule-based protocol in comparison to those derived using the growth factor-based protocol. a Secretion of serum proteins (albumin and fibronectin). Wells without seeded cells were included as blank. b Urea synthesis in small molecule-derived hepatocyte-like cells upon challenging with ammonia. Wells without seeded cells were included as blank control. c Representative PAS staining images showing glycogen storage. d Basal level activity of major cytochrome P450 enzymes. HLC_SM, hepatocyte-like cells derived using small molecules; HLC_GF, hepatocyte-like cells derived using growth factors; CYP, cytochrome P450. All values are presented as mean ± SD. n = 4 for serum protein secretion, and n = 3 for urea synthesis and CYP activity. * p < 0.05, ** p < 0.01, *** p < 0.001 by unpaired two-tailed t-test

Fig. 3

Principal component analysis (PCA) plot showing the differentiation track (dotted line) and similarities/dissimilarities among the samples. The two axes PC1 and PC2 represent the first two principal components identified by the analysis. The percentage contribution of each component to the overall source of variation is included in the parentheses following the component name. Color codes of different cell types are shown at the top-right corner. The timing of the small molecule-driven and growth factor-driven differentiation is denoted by D (day) followed by the number of the day. iPSC, induced pluripotent stem cell; DE, definitive endoderm; HP, hepatic progenitor; HLC, hepatocyte-like cell; PHHs, primary human hepatocytes. HLC_Wruck and HLC_Yang are reference datasets

Fig. 4

Expression of key marker genes at each stage of the hepatocyte differentiation. a Definitive endoderm markers. b Hepatic progenitor markers. c Hepatocyte markers. Left panel, heat maps. The expression data are the average of three replicates in log2 scale and color-coded as shown in the scheme at the top. Right panel, bar graphs. The expression values are relative to the starting induced pluripotent stem cells (average = 1). DE_SM, definitive endoderm derived using small molecules; DE_GF, definitive endoderm derived using growth factors; HP_SM, hepatic progenitor derived using small molecules; HP_GF, hepatic progenitor derived using growth factors; HLC_SM_D17, hepatocyte-like cells derived using small molecules at day 17; HLC_SM_D24, hepatocyte-like cells derived using small molecules at day 24; HLC_GF, hepatocyte-like cells derived using growth factors

Fig. 5

Hierarchical clustering analysis (HCA) of all the samples. The clustering was based on 520 probesets representing hepatotoxicity-related genes, drug-metabolizing enzymes, transporters, and nuclear receptors (see main text). The list of genes is provided in Supplemental Table 1. The clustering was performed through Ward’s minimum variance linkage on normalized expression data. The dendrogram on the right of the image shows clusters of genes (names not shown), while that on the top of the image shows clusters of samples with names shown. SM, small molecule; GF, growth factor; D, day of differentiation; Liver_F, fetal liver; Liver_A, adult liver; HLC_iCell, iCell hepatocyte-like cells (FCDI); PHHs, primary human hepatocytes

Fig. 6

Relative gene expression of major cytochromes P450 enzymes (top), functional transporters (middle), and nuclear receptors (bottom). Gene expression value for each enzyme, transporter, or nuclear receptor was the average of the samples in each group and is in log2 scale and color-coded as shown in the scheme located at the top of the figure. HLC_SM, hepatocyte-like cells derived using small molecules; HLC_GF, hepatocyte-like cells derived using growth factors; HLC_iCell, iCell hepatocyte-like cells (FCDI); Liver_F, fetal liver; Liver_A, adult liver; PHHs, primary human hepatocytes

Fig. 7

Cell and tissue type classification using CellNet. Liver classification average score for each sample is shown on top of the bar. The “esc” average score for the iPSC samples is also shown. iPSC, induced pluripotent stem cells. HLC_SM, hepatocyte-like cells derived using small molecules; HLC_GF, hepatocyte-like cells derived using growth factors; HLC_iCell, iCell hepatocyte-like cells (FCDI); Liver_F, fetal liver; Liver_A, adult liver; PHH, primary human hepatocytes. HLC_Wruck and HLC_Yang are reference datasets. esc, embryonic stem cell; bcell, B cell; rand, random (unknown); hspc, hematopoietic stem and progenitor cell; muscle-skel, musculoskeletal tissue; tcell, T cell

Fig. 8

Volcano plots showing the differentially expressed genes (DEGs) between HLC_SM and PHH (left panel), between HLC_GF and PHH (middle panel), and between HLC_iCell and PHH (right panel). All DEGs are shown as red dots. Downregulated genes are on the top-left corners with numbers denoted by green arrows, and upregulated genes are on the top right corners with numbers denoted with red arrows. The total numbers of DEGs are included in the parentheses under the title

Fig. 9

A gene network in Ingenuity Pathway Analysis illustrating the large number of downstream differentially expressed genes (DEGs) under the regulation of HNF4A. The DEGs are arranged in a subcellular layout and color-coded by expression changes as shown in the legend. The predicted action (activation or inhibition) and relationship for each DEG are also color-coded as shown in the legend