PSC can differentiate into hepatocyte-like cells <i>in vitro</i>.

<p>PSC were expanded for 7 days in culture and subsequently cultured for additional 14 days in (<b>A</b>) IMDM supplemented with 10% FCS (control medium) or (<b>B</b>) IMDM containing cytokines (FGF₄, HGF), linoleic acid-albumin and ITS (hepatocyte differentiation medium). (<b>B</b>) Hepatocyte-like cells appeared in primary cultures of PSC during cytokine treatment. (<b>C</b>) Under control conditions primary cultured PSC did not express the hepatocyte marker cytokeratin 18 (CK18) as investigated by immunofluorescence (red), but (<b>D</b>) CK18 synthesis was induced after treatment with hepatocyte differentiation medium for 14 days. (<b>E</b>) The hepatocyte-associated bile salt export pump (BSEP; red) remained undetectable in vimentin-expressing PSC (green) of the control, whereas (<b>F</b>) BSEP and vimentin were detectable in PSC treated with hepatocyte differentiation medium. The cell nuclei were marked by DAPI (blue). (<b>G</b>) Molecular markers of stellate cells (GFAP, α-SMA) as well as of immature and mature hepatocytes (HHEX, CYP7A1, α-fetoprotein, albumin, CX32, HNF1α, HNF4α, HNF6, MRP2) were analyzed by RT-PCR in the control (IMDM and 10% FCS; left column) and cytokine treated PSC (FGF₄, HGF, linoleic acid-albumin and ITS; right column) after 14 days of culture. The induction of notch3 and genes typically expressed by liver parenchymal cells indicated cell differentiation. (<b>H</b>) This cell differentiation potential was also preserved by the PSC clones, which displayed a comparable expression pattern after cytokine treatment for 7 days (right column). In the control, clonally expanded PSC were cultured in medium without cytokines (IMDM and 10% FCS; left column). (<b>I</b>) The PSC clone displayed the morphology of myofibroblast-like cells (control), but (<b>J</b>) developed into hepatocyte-like cells after treatment with hepatocyte differentiation medium for 7 days. (<b>K</b>) In this experimental setup, the release of albumin was measured by a rat-specific albumin ELISA. High amounts of albumin were secreted by primary cultured PSC and the PSC clone 2F5 after treatment with hepatocyte differentiation medium, whereas rat albumin was not detected (n.d.) under control conditions or in experimental media without cells (n = 3).</p

Quantitative mRNA analysis of stellate cell and stem cell-related factors in PSC.

<p>The expression of the stellate cell markers α-SMA, GFAP, desmin and synemin as well as the stem/progenitor cell-associated proteins CD133, SOX9, GDF3 and slain1 was investigated by qPCR in one day-cultured PSC, PSC clones and UCBSC clones. PSC expressed comparable or higher levels of stem cell-associated factors than UCBSC clones. Three to five independent primary cultures of PSC were used for qPCR analysis. The PSC clones and the UCBSC clones were measured in triplicates. Significant differences and the standard error of mean (±SEM) are indicated [*P<0.05%].</p

Stellate cell markers in cultured PSC, UCBSC and muscle fibroblasts.

<p>(<b>A,D,G,J</b>) Freshly isolated PSC cultured for 1 day, (<b>B,E,H,K</b>) clonally expanded UCBSC and (<b>C,F,I,L</b>) muscle fibroblasts from rats were analyzed by phase contrast light microscopy and antibodies against the stellate cell markers (<b>D,E,F</b>) α-SMA, (<b>G,H,I</b>) desmin and (<b>J,K,L</b>) GFAP (red). The isolated PSC were found to be typical stellate cells with respect to their α-SMA, desmin and GFAP expression. The stellate cell markers α-SMA and GFAP were only weakly expressed by UCBSC clones, whereas desmin remained undetectable at protein level. The cell nuclei were stained by DAPI (blue).</p

Analysis of stem/progenitor cell-associated factors in PSC by Western blot.

<p>The expression of CD133 was analyzed in cell membrane fractions of PSC and UCBSC. PSC of three different cell isolations, which were cultured for 7 days (7d), and UCBSC of three different clones were taken for the immunoblot. To indicate cell membrane protein fractions, annexin II served as a control. The notch1 receptor was detected predominantly in freshly isolated PSC cultured for 1 day (1d) and to a lesser extent also in culture-activated PSC (7d) as well as UCBSC. Nuclear localization of β-catenin in PSC and UCBSC indicated active β-catenin-dependet Wnt signaling as investigated by Western blot of nuclear protein fractions. In line with this, the Wnt target gene <i>PITX2</i> was also detected in both cell types. The stem/progenitor cell-associated numb1/3 isoforms were detected in PSC and UCBSC, whereas numb2/4 remained undetectable in both cell types. Numb2/4 isoforms were found in lysates of whole liver.</p

Analysis of stem/progenitor cell-associated factors in PSC by immunofluoresecence.

<p>(<b>A,D,G,J</b>) Freshly isolated PSC, (<b>B,E,H,K</b>) the UCBSC clone 1G11 and (<b>C,F,I,L</b>) muscle fibroblasts from rats were analyzed with antibodies against (<b>A,B,C</b>) CD133, (<b>D,E,F</b>) β-catenin, (<b>G,H,I</b>) PITX2 and (<b>J,K,L</b>) numb by immunofluorescence staining (red). The stem/progenitor cell-associated proteins CD133, PITX2 and numb were restricted to PSC and UCBSC, whereas β-catenin occurred in all cell types. In (<b>D</b>) PSC and (<b>E</b>) UCBSC β-catenin was detected in the cell nucleus, indicating active β-catenin-dependent WNT signaling, (<b>F</b>) whereas fibroblasts displayed β-catenin mainly in the cell membrane. The cell nuclei were stained by DAPI (blue).</p

Estimation of PSC-derived cells in the wild type host liver.

<p>(<b>A</b>) In order to quantify the contribution of transplanted eGFP⁺ PSC to liver regeneration, qPCR of the eGFP DNA was performed 14 days after cell transplantation. Livers from eGFP-expressing and wild type rats served as positive and negative controls for the eGFP gene, respectively. Ten samples of different animals were measured to assess the eGFP expression of liver after PSC transplantation. (<b>B</b>) The engraftment of stellate cells was further verified through qPCR of male-specific SRY DNA after gender-mismatched transplantation of male PSC into female recipient rats (n = 4). In contrast to PSC, muscle fibroblasts were unable to survive in the host liver (n = 4).</p

DNA methylation analysis in repetitive DNA elements.

<p>The DNA methylation was measured within consensus sequences of different repetitive elements by qAMP (n = 5). DNA methylation of freshly isolated HSC (0d) was set to 100%. There were no significant changes in the DNA methylation level between freshly isolated and 3d cultured HSC detectable.</p

DNA methylation and expression analysis of genes identified by EpiQuest sequencing.

<p>(A) EpiQuest sequencing result of Mmrn2 promoter region displayed in UCSC genome browser. DNA methylation at individual CpG dinucleotides within the sequence are specified in percent. (B+D) DNA methylation analysis of two hyper- and hypomethylated genes by direct bisulfite sequencing (n = 3 independent experiments). The position of the analyzed region is indicated in the figure (* p<0.05). (C+E) Corresponding quantitative gene expression analysis to determine the effect of DNA methylation changes on the gene expression in quiescent (cultured overnight) and early activated (cultured for 3 days) HSC (n = 5 independent experiments).</p

Epigenetic Changes during Hepatic Stellate Cell Activation

<div><p>Background and Aims</p><p>Hepatic stellate cells (HSC), which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during <i>in vitro</i> activation of HSC.</p><p>Methods and Results</p><p>The analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism.</p><p>Conclusions</p><p>In summary, <i>in vitro</i> activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism.</p></div

Global DNA demethylation during HSC activation.

<p>(A) Global DNA methylation determined by a 5meC ELISA showed a significant reduction to approximately 40% of the initial DNA methylation of freshly isolated HSC within 3 days of culture (* p<0.05 compared to HSC 0d, n = 4 independent experiments). (B) Global DNA methylation in hepatocytes was not altered during culture. (C) IF staining of 5meC (red) during early HSC activation. The nuclei were stained with DAPI (blue). All pictures were taken with the same exposure time and adjustments to enable direct comparison of the 5meC amount. Shown are representative images from one of 4 independent experiments.</p