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

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

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

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

miR-27 inhibits OCT4 and LIN28 expression at both the transcriptional and translational level in embryonal carcinoma cells (NCCIT).

<p>(A) Analysis of miR-27 expression was carried out for miR-27a and miR-27b using TaqMan-based PCR on total RNA samples isolated from NCCIT cells undergoing RA stimulated neuronal differentiation for seven days or by blocking TGFßR2 with SB431542 for seven days and normalized to the DMSO-treated control. (B) qRT-PCR of selected genes (log2-fold change relative to the negative control) was validated for NCCIT cells transfected once with miR-27 or treated with SB431542 for 48 h. NCCIT cells transfected with a scrambled miRNA mimic was used for normalization. (C) Relative <i>OCT4</i> and <i>LIN28</i> expression in NCCIT cells transfected with scrambled negative control miRNA mimics, let-7a, miR-125b, miR-27a, miR-200c or treated with SB431542 for 72 h and validated by qRT-PCR. (D) Western Blot analysis of OCT4 and LIN28 expression in NCCIT cells treated as described in (C). (E) Normalized densitometric-derived ratios of Western Blot presented in (D).</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

Schematic overview of our proposed regulatory network between miR-27 and pluripotency-associated genes.

<p>Genes highlighted in bold, black letters are those validated experimentally to be direct targets of miR-27.</p

Transcriptome analysis of human embryonal carcinoma cells (NCCIT) post transfection with miR-27, let-7, miR-125 or miR-200.

<p>(A) Hierarchical clustering of NCCIT cells transfected either with miRNAs (miR-27, let-7, miR-125, miR-200 or neg. control mimic) or treated with the TGFßR2 inhibitor SB431542 (B) Heat map representing the expression of selected genes relative to the negative control transfection (Detection P-Value <0.01) (C) Venn diagrams representing the overlap of up- and down-regulated genes by let-7 and miR-27 (Detection P-Value <0.01) in comparison to the negative control transfection. (D) Venn diagram representing the overlap of down-regulated genes by miR-27 in comparison to miR-27 target genes predicted by TargetScan (human) V6.2.</p