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

miR-27 Negatively Regulates Pluripotency-Associated Genes in Human Embryonal Carcinoma Cells

<div><p>Human embryonic stem cells and human embryonal carcinoma cells have been studied extensively with respect to the transcription factors (OCT4, SOX2 and NANOG), epigenetic modulators and associated signalling pathways that either promote self-renewal or induce differentiation in these cells. The ACTIVIN/NODAL axis (SMAD2/3) of the TGFß signalling pathway coupled with FGF signalling maintains self-renewal in these cells, whilst the BMP (SMAD1,5,8) axis promotes differentiation. Here we show that miR-27, a somatic-enriched miRNA, is activated upon RNAi-mediated suppression of OCT4 function in human embryonic stem cells. We further demonstrate that miR-27 negatively regulates the expression of the pluripotency-associated ACTIVIN/NODAL axis (SMAD2/3) of the TGFß signalling pathway by targeting <i>ACVR2A</i>, <i>TGFßR1</i> and <i>SMAD2</i>. Additionally, we have identified a number of pluripotency-associated genes such as <i>NANOG</i>, <i>LIN28</i>, <i>POLR3G</i> and <i>NR5A2</i> as novel miR-27 targets. Transcriptome analysis revealed that miR-27 over-expression in human embryonal carcinoma cells leads indeed to a significant up-regulation of genes involved in developmental pathways such as TGFß- and WNT-signalling.</p></div

List of pathways and associated genes significantly up-regulated 72 h after post-transfection of NCCIT with miR27.

<p>List of pathways and associated genes significantly up-regulated 72 h after post-transfection of NCCIT with miR27.</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

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

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

Primers that have been used to generate GFP- miRNA target gene constructs. Restriction sites are highlighted in bold letters.

<p>Primers that have been used to generate GFP- miRNA target gene constructs. Restriction sites are highlighted in bold letters.</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

OCT4 knockdown in the hESC line H1 leads to activation of miR-27a and miR-27b expression.

<p>Successful OCT4 knockdown in hESC cells transfected twice with siRNA targeting either OCT4 or EGFP 72 h post transfection and confirmed by real-time PCR (A) and Western Blotting (B). (A) Relative <i>OCT4</i> and <i>NANOG</i> expression of three biological OCT4 knockdown samples (siOCT4#1-3) normalized to the siGFP knockdown control. (B) Western Blot analysis of OCT4 protein levels carried out for sample (siOCT4#1) and siGFP control sample with densitometric quantification (OCT4/GAPDH) (C) Relative expression of pluripotency-associated genes validated by real-time PCR for sample siOCT4#1 normalized to the siGFP knockdown control. (D) miR-27 expression was carried out using TaqMan-based PCR for all three biological siOCT4 samples and normalized to the siGFP control sample.</p

miR-27 directly inhibits a number of genes of the TGFß signalling pathway that promote self-renewal in undifferentiated embryonic stem cells.

<p> (A) Table shows putative miR-27 target genes associated with TGFß-signalling as predicted by Diana Micro-T (DT), MiRanda (miRa), MirWalk (miRW) and TargetScan. (B) Normalized GFP expression (48 hours post transfection) of HEK293 cells co-transfected with EGFP-sensors bearing parts of the 3′-UTR of <i>TGFßR1</i>, <i>ACVR2a</i>, <i>SMAD2</i> or the 3′-UTR of the empty eGFP-vector (lane 1)together with either miR-27 mimics or a scrambled negative control mimic (neg. con.). All transfections were performed twice in biological triplicates (n = 6). An unpaired two tailed t-test was performed to reveal significant differences (*p<0.05, ** p<0.01, *** p<0.001). (C) Schematic representation of the TGFß-signalling cascade adopted from the KEGGs pathway database (<a href="http://www.genome.jp/kegg/pathway.html" target="_blank">www.genome.jp/kegg/pathway.html</a>).</p