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

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

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

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

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

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

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

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

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

Additional file 1: of The presence of human mesenchymal stem cells of renal origin in amniotic fluid increases with gestational time

Table S1. Antibodies used in this study Table S2. Primers used for semiquantitative and quantitative real-time PCR. (DOCX 18 kb