Gene expression primers.

<p>Gene expression primers.</p

miRNA and EZH2 mRNA degradation.

<p><b>A.</b> Relative gene expression (to <i>GAPDH</i>) of endogenous and exogenous <i>EZH2</i> (Total <i>EZH2</i>) daily during the transition from endoderm stage (day 4, endo_d4) to hepatoblast progenitor (day 8, HP_d8) in untreated (-) and EZH2 doxy induced cells (+). Data as mean ± SEM of n = 3 IEs. <b>B.</b> Relative expression of miR-101, miR-138, miR-214 and miR-124 during hepatocytes differentiation from hPSC-<i>i</i>EZH2 cell line doxy induced the first 8 days of differentiation. Relative gene expression to U6. Data as mean ± SEM of n = 3 IEs. <b>C.</b> Name, functions and reference papers of the miRNAs analyzed.</p

EZH2 regulation and fetal hepatocytes differentiation.

<p><b>A.</b> Upper part, directed differentiation of hPCS-<i>i</i>EZH2 toward fetal hepatocytes (FH_d16) with addition of 5μg/ml doxycycline (doxy) in the first 8 days of differentiation (red arrow). Bottom part, relative gene expression of the <i>EZH2</i> transgene (Exogenous <i>EZH2</i>) and both endogenous and exogenous EZH2 (Total <i>EZH2</i>) at endo_d4, HP_d8 and FH_d16 in untreated (-) and EZH2 doxy induced cells (+). Relative gene expression to GAPDH and same scale bar. Data as mean ± SEM of n ≥ 3 IEs. <b>B.</b> Western blot for EZH2 and GAPDH in untreated (-) and EZH2 doxy induced cells (+) at hPSC, endo_d4, HP_d8 and FH_d16. Positive control: HEK293T cells transiently transfected with pLVX-IRES-Hygro-hEZH2. The molecular weight size marker was cropped from the gel. Signals were quantified and indicated as % to loading control. <b>C.</b> Relative expression (to <i>GAPDH</i>) of hepatic markers <i>HNF1A</i>, <i>HNF3B</i>, <i>HNF6</i>, <i>GSTp</i> and transcription factors <i>CAR</i> and <i>CEBPA</i> in HP_d8 and FH_d16 untreated (-) and EZH2 doxy induced cells (+). Data as mean ± SEM of n ≥ 3 IEs. * p < 0.05 and ** p < 0.01. <b>D.</b> Relative gene expression (to <i>GAPDH</i>) of hepatic marker genes <i>AFP</i>, <i>ALBUMIN and AAT</i> in HP_d8 and FH_d16 untreated (-) and EZH2 doxy induced cells (+). Data as mean ± SEM of n ≥ 3 IEs. * p < 0.05, ** p < 0.01. <b>E.</b> Left part, relative gene expression (to <i>GAPDH</i>) of hepatic transcription factor HNF4A in HP_d8 and FH_d16 untreated (-) and EZH2 doxy induced cells (+). Right part, representative immunofluorescence images for HNF4A (red signal) on day 8 for cells treated without (untreated) or with doxy (doxy-treated). Nuclei are staining with DAPI (blue). On the right, more than 80% of HNF4A positive cells on HP_d8 EZH2 doxy induced cells were counted. Data as mean ± SEM of n ≥ 3 IEs. * p < 0.05. <b>F.</b> Immunofluorescence staining for ALBUMIN (red signal) at HP (day8) and FH (d16) in untreated (-) and EZH2 induced cells (+) (left part). Nuclei are staining with DAPI (blue). Data as representative images of n = 2 IES. <b>G.</b> ELISA for ALBUMIN secretion on FH_d16 of the hepatocyte differentiation protocol. EZH2 doxy induced cells (+) secrete significant amounts of albumin compared to untreated (-) cells. Data as mean ± SEM of n ≥ 3 IEs. ** p < 0.01. <b>H.</b> Immunofluorescence staining for AAT (red signal) at HP (day 8) and FH (day 16) showed abundant expression of the hepatocyte protein in doxy-treated cells compared to the untreated (left part). Nuclei are stained with DAPI (blue). Data as representative images of n = 2 IES. <b>I.</b> Intracellular flow cytometry analysis for AAT demonstrated that more then around 60% of EZH2 doxy induced cells (+) progeny were positive for AAT. Results represent the mean of three independent experiments ± SEM. ** p < 0.01.</p

Total H3 levels and H3K27me3 levels on endodermal and hepatocyte genes.

<p><b>A.</b> Left, representative immunofluorescence image for H3K27me3 (green signal) of three different differentiations (n = 1, 2 and 3) at endo_d4, HP_d8 and FH_d16 stages in untreated and doxy-treated cells. Right, the fluorescence of H3K27me3 signal was digitally quantified (additional details in the Supplemental Information). Results represent the mean of three independent experiments ± SEM and displayed as arbitrary units (A.U.). ** p < 0.01. <b>B.</b> Histone modification levels of H3K27me3 at SOX17, CXCR4 and FOXA2 (left part) and GAPDH, MYOD1 and HOXD11 promoter (right part) at endo_d4 in untreated and doxy-treated cells. Results represent the mean of three independent experiments ± SEM. * p < 0.05. <b>C.</b> Histone modification levels of H3K27me3 at ALBUMIN and AAT (left part) and GAPDH, MYOD1 and HOXD11 promoter (right part) at FH_d16 in untreated and doxy-treated cells. Results represent the mean of three independent experiments ± SEM. * p < 0.05, ** p < 0.01.</p

hESC-inducible EZH2 (hPSC- <i>i</i>EZH2) cell line and definitive endoderm formation.

<p><b>A.</b> The original master cell line flanked by heterotypic FRT sequences and resulting RMCE line (Recombinase-Mediated Cassette Exchange, hPSC-<i>i</i>EZH2) are depicted. RMCE donor vector <i>pZ</i>:<i>F3-P TetOn hEZH2</i> for inducible expression was introduced by flippase (additional details in the Supplemental Information). <b>B.</b> Expression of pluripotency markers (TRA-1-60 and OCT4) of a representative hPSC-<i>i</i>EZH2 clone and wild-type hPSCs by immunocytochemistry. Right: isotype control of TRA-1-60 and OCT4. <b>C.</b> Upper part, directed differentiation of hPCS-<i>i</i>EZH2 toward endoderm (endo_d4) with addition of 5μg/ml doxycycline (doxy) in the first 4 days of differentiation (red arrow). Bottom part, protein analysis of untreated and doxy treated EZH2 induced cells (doxy -/+) using EZH2, GATA4 and GAPDH antibodies. GAPDH was used as loading control. Signals were quantified and indicated as % to loading control. <b>D.</b> Double extracellular staining for CXCR4 (PE channel) and cKIT (APC channel) measured by FACS in untreated and doxy treated EZH2 induced cells (doxy-treated) at definitive endoderm stage on day 4. Right: the percentage of cells co-expressing CXCR4 and cKIT. Data as mean ± SEM of n ≥ 3 IEs. ** p < 0.01 by Student’s t test. <b>E.</b> Representative immunofluorescence images for SOX17 (red signal) in untreated and EZH2 induced cells (doxy-treated) at day 4 of differentiation. EZH2 induced cells showed significantly high levels of SOX17 expression. Around 58% of positive cells were counted in EZH2 doxy induced cells, right part. Nuclei are staining with DAPI (blue). Data as mean ± SEM of n ≥ 3 IEs. *** p < 0.001 by Student’s t test. <b>F.</b> mRNA expression profile of the endoderm marker <i>SOX17</i> in untreated (-) and EZH2 doxy induced cells (+) at endo_d4. <b>G.</b> mRNA expression profile of untreated (-) and EZH2 doxy induced cells (+) at endo_d4 and HP_d8 of endoderm markers <i>FOXA2</i>, <i>EOMES</i>, <i>MIXL1</i>, <i>GOOSECOID</i> and <i>CXCR4</i>. Relative gene expression to GAPDH. Data as mean ± SEM of n ≥ 3 IEs.</p

EZH2 expression during hepatocyte differentiation.

<p><b>A.</b> Upper part, differentiation protocol of hPSCs into hepatocytes-like cells. hESC were harvested and plated at ±7 x 10⁴ cell/cm² and differentiated with a combination of growth factors: 50ng/ml of Activin-A and Wnt3a for the first 2 days and 50ng/ml Activin-A until day 4, followed by BMP4 50 ng/ml from day 4 until day 8, FGF1 50 ng/ml from day 8 until day 12, and HGF 20 ng/ml from day 12 until day 16. Differentiated progeny of the hESC line were harvested on day 4 (endo_d4, definitive endoderm cells), on day 8 (HP_d8, hepatoblast progenitor) and on day 16 (FH_d16, fetal hepatocyte). Bottom part, transcript expression levels of <i>EZH2</i> and <i>RING1B</i> in hPSCs, endo, HP and FH stages. <i>GAPDH</i> was used as a control. Data as mean ± SEM of n ≥ 3 IEs. *** p < 0.001 by Student’s t test. <b>B.</b> Western blot analysis of EZH2, RING1B and GAPDH on hPSCs, endo, HP and FH stages. GAPDH was used as loading control. HEK293T transiently transfected with pLVX-IRES-Hygro-hEZH2 cells were used as positive control. Signals were quantified and indicated as % to loading control. <b>C.</b> Schematic representation of the <i>EZH2</i> regulatory region. Approximately 270bp (arrow in front of the TTS) in a CpG island were bisulfite sequenced in undifferentiated hPSC, HP and FH. Percentages of the results of bisulfite sequencing of the regions are at right side of the figure for each stage.</p

miRNAs expression primers.

<p>miRNAs expression primers.</p

14TF iENDO cells differentiate into hepatocyte like cells.

<p>A) Protocol time-line for hepatocyte differentiation in 3D from iENDO cells protocol A with or without DMSO. B) Morphology of iENDO differentiated organoids at day 20 with and without DMSO by bright field and H&E staining (N = 3). C) Relative gene expression (to <i>PPIG</i>, log scale) in day 20 iENDO-HLCs ± DMSO represented as a heat-map for hepatocyte markers compared with hESC-HLCs organoids (d30) and PHHs (N = 3). D-F) Immunostaining for AFP, ALB and AAT on day 20 iENDO organoids ± DMSO with respective isotype controls. Scale bar 25 μm (representative example of N = 3). G) Albumin secretion in supernatants of day 20 iENDO organoids ± DMSO, hESC day 30 organoids and primary hepatocytes (PHH) (N = 3). Error bars represents standard deviation of three independent experiments. *p<0.05, **p<0.01 and ***p<0.001 determined by unpaired 2-tailed Student’s t-test. NS- Not significant.</p

14TF iENDO cells are expandable long-term <i>in vitro</i>.

<p>A) Expansion curve of 14TF iENDO cells shown as days in culture (horizontal axis) vs. the number of population doublings (PDs) (vertical axis). B) Immunostaining analysis of CXCR4 (C) SOX17 and CK18 (D) and protein expression of 14TF iENDO cells, at passage 4 (magnification 20x, scale bar: 50μm and 40x, scale bar: 100μm), representative sample of n = 3. (E-G) Relative gene expression (to <i>PPIG</i>, log scale) in 14TF iENDO cells at 6–8 and 15–20 passages represented as a heat-map for mesendoderm (E), definitive endoderm and epithelial marker genes (F) and late endoderm marker genes (G) compared with day 20 iENDO cells. H) Relative gene expression (to PPIG, log scale) represented as a heat-map of transgene expression in 14TF iENDO cells on day 20, P6-P8, and P15-P20. All data represent mean of three independent experiments. *p<0.05, **p<0.01 and ***p<0.001 determined by unpaired 2-tailed Student’s t-test.</p

Generation of 14TF iENDO cells from hMAPC.

<p>A) Selected 14TFs for induction of iENDO cells from hMAPC B) Protocol for iENDO generation from hMAPC. C) Morphological changes of 14TF transduced hMAPC from day 0 to day 20 after transduction (scale bar: 100μm). (D-F) Relative gene expression (to <i>PPIG</i>, log scale) in day 20 14TF iENDO cells represented as a heat-map for mesendoderm (D), definitive endoderm and epithelial marker genes (E) and late endoderm marker genes (F) compared with untransduced hMAPCs, and hESC derived endodermal progenitors (d4) or mature endodermal cells (d20). All data represent mean of three independent experiments. *p<0.05, **p<0.01 and ***p<0.001 determined by unpaired 2-tailed Student’s t-test.</p