Cell fate decisions of human iPSC-derived bipotential hepatoblasts depend on cell density

<div><p>During embryonic development bipotential hepatoblasts differentiate into hepatocytes and cholangiocytes- the two main cell types within the liver. Cell fate decision depends on elaborate interactions between distinct signalling pathways, namely Notch, WNT, TGFβ, and Hedgehog. Several <i>in vitro</i> protocols have been established to differentiate human pluripotent stem cells into either hepatocyte or cholangiocyte like cells (HLC/CLC) to enable disease modelling or drug screening. During HLC differentiation we observed the occurrence of epithelial cells with a phenotype divergent from the typical hepatic polygonal shape- we refer to these as endoderm derived epithelial cells (EDECs). These cells do not express the mature hepatocyte marker ALB or the progenitor marker AFP. However they express the cholangiocyte markers SOX9, OPN, CFTR as well as HNF4α, CK18 and CK19. Interestingly, they express both E Cadherin and Vimentin, two markers that are mutually exclusive, except for cancer cells. EDECs grow spontaneously under low density cell culture conditions and their occurrence was unaffected by interfering with the above mentioned signalling pathways.</p></div

Interference with various signalling pathways does not change cell fate.

<p>iPSCs were differentiated into EDECs. Directly after low-density splitting, small molecules were applied in order to interfere with signalling pathways important for differentiation into hepatocytes or cholangiocytes. (A-G) Immunocytochemistry for ALB (red) and CK19 (green). (A) DMSO control, (B) activation of WNT signalling with Chir99021, (C) inhibition of WNT signaling with PKF118-310, (D) activation of Hh signalling with Purmorphamine, (E) inhibition of Hh signalling with Cyclopamine-KAAD, (F) inhibition of TGFβ signalling with SB431242, (G) inhibition of TGFβ signalling with A-83-01. Scale bar: 100 μm.</p

Differentiation of hPSCs into hepatocyte like cells (HLCs) and endoderm derived epithelial cells (EDECs).

<p>hPSCs were differentiated into hepatic endoderm (HE) which consists of bipotential hepatoblasts. Afterwards, cultures were either continued unperturbed in order to obtain HLCs, or split and replated at low density to obtain EDECs. Morphological changes were documented for each stage.</p

Inhibition of Notch signalling does not prevent EDEC development.

<p>hPSCs were differentiated into EDECs while treating them with γ-secretase inhibitors. (A) HFF derived cells treated with Compound E (left) or H1 derived cells treated with γ-secretase inhibitor I (right) adopted the EDEC morphology. (B) Immunocytochemistry for CK19, SOX9, ECAD, and OPN. (C,D) qRT-PCR for hepatocyte or cholangiocyte determining genes. Gene expression was normalized to RPS16 and fold change was calculated relative to untreated cells. Mean values of technical triplicates of biological duplicates (C) or of one sample (D) are show.</p

Expression of characteristic cholangiocyte markers in HLCs and EDECs.

<p>Two iPSC lines and one ESC line were differentiated into either HLCs (A-C) or EDECs (D-F) and stained for the expression of characteristic cholangiocyte markers.</p

Temporal expression of markers during differentiation.

<p>Two iPSC lines and one ESC line were differentiated into either HLCs or EDECs and qRT-PCRs for expression of characteristic hepatocyte and cholangiocyte markers was performed. Gene expression was normalized to RPS16 and fold change was calculated relative to HE cells. Mean values of technical triplicates of biological duplicates are show. Error bars represent SEM. P-Values were calculated with two-tailed student’s t-tests (*** = p-value < 0.001, ** = p-value < 0.01, * = p-value < 0.05).</p

EDECs express a unique combination of markers.

<p>Two iPSC lines and one ESC line were differentiated into either HLCs (A) or EDECs (B-D) and marker expression was analysed. (A,B) Immunocytochemistry for ECAD. (C) Endpoint RT-PCR for <i>VIM</i> and <i>CDX2</i>. cDNA derived from mesenchymal stem cells (MSCs) and from the colon cancer line DLD1 served as positive controls for <i>VIM</i> and <i>CDX2</i> expression, respectively. (D) Immunocytochemistry for GFAP.</p

Expression of characteristic hepatocyte markers in HLCs and EDECs.

<p>Two iPSC lines and one ESC line were differentiated into either HLCs (A-C) or EDECs (D-F) and stained for the expression of characteristic hepatocyte markers.</p

Gene expression analysis unravels differences and similarities between H1 derived HLCs and EDECs.

<p>(A) Heatmap representation of key genes involved in hepatic cell fate decision making or characteristic for either hepatocytes (yellow) or cholangiocytes (orange). (B) Venn diagram illustrating the numbers of genes which were expressed either by only one cell type or shared between the cell types. (C) Gene Ontology (GO) analysis of genes expressed only in HLCs (yellow) or in EDECs (with or without Notch inhibition, orange) Shown are pre-selected, significant GO-Terms, for full data set see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200416#pone.0200416.s008" target="_blank">S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200416#pone.0200416.s009" target="_blank">S5</a> Tables.</p

DataSheet1_Forskolin induces FXR expression and enhances maturation of iPSC-derived hepatocyte-like cells.PDF

The generation of iPSC-derived hepatocyte-like cells (HLCs) is a powerful tool for studying liver diseases, their therapy as well as drug development. iPSC-derived disease models benefit from their diverse origin of patients, enabling the study of disease-associated mutations and, when considering more than one iPSC line to reflect a more diverse genetic background compared to immortalized cell lines. Unfortunately, the use of iPSC-derived HLCs is limited due to their lack of maturity and a rather fetal phenotype. Commercial kits and complicated 3D-protocols are cost- and time-intensive and hardly useable for smaller working groups. In this study, we optimized our previously published protocol by fine-tuning the initial cell number, exchanging antibiotics and basal medium composition and introducing the small molecule forskolin during the HLC maturation step. We thereby contribute to the liver research field by providing a simple, cost- and time-effective 2D differentiation protocol. We generate functional HLCs with significantly increased HLC hallmark gene (ALB, HNF4α, and CYP3A4) and protein (ALB) expression, as well as significantly elevated inducible CYP3A4 activity.</p