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. 2022 Aug 12;11:e71591. doi: 10.7554/eLife.71591

Figure 4. Forward programming of human embryonic stem cells (hESCs) into hepatocytes with nuclear receptors.

(A) Phase contrast images and (B) immunofluorescence staining for Albumin (yellow) and (C) A1AT (green) in hESCs forward programmed for 20 days with 3TFs alone or in combination with the nuclear receptors RORc, ERɑ, and AR. Nuclei were counterstained with DAPI (blue). Scale bars, 200 µm. (D) mRNA levels of hepatocyte markers (ALB, SERPINA1, and AFP) in FoP-Heps generated with 3TFs alone or in combination with nuclear receptors for 20 and 30 days (n=4). Expression data was normalised to the average of two housekeeping genes (PBGD and RPLP0). (E) Protein secretion levels of Albumin, A1AT, and AFP in hESC-derived FoP-Heps generated with 3TFs alone or in combination with nuclear receptors for 20 days (n=4). Data was normalised per total cell number (millions). (F) CYP3A4 activity levels normalised per cell number (millions) in FoP-Heps targeted with 3TFs with or without nuclear receptors, after 20 and 30 days of forward programming (n=3–6). Statistical differences were calculated with one-way ANOVA, corrected for multiple comparisons compared to 3TFs (day 20). (G) CYP3A4 fold induction levels in FoP-Heps treated with 100 nM of the ligands as indicated from day 2. Data is normalised to untreated control at day 20 of forward programming (n=3). Statistical differences were calculated with paired t-test. In all plots, bars represent mean with SD, and individual datapoints are shown for all biological replicates. Hepatocyte-like cells (HLCs) generated by direct differentiation and primary human hepatocytes (PHHs) where plotted as controls for CYP3A4 activity and expression data. Significant p-values are shown at each comparison and indicated as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4—source data 1. Individual measurements and statistical tests related to Figure 4 and supplements.

Figure 4.

Figure 4—figure supplement 1. Validation of inducible overexpression (iOX) of combinations of 3TFs and nuclear receptors.

Figure 4—figure supplement 1.

(A) Schematic representation of the combinations of factors cloned into the AAVS1 locus. (B) Immunofluorescence staining of the 3TFs and nuclear receptors in human embryonic stem cells (hESCs) targeted with the constructs shown in (A) after 24 hr of iOX with doxycycline (dox) confirming transgene induction. (C) Immunofluorescence staining of nuclear receptors in hESCs targeted with 3TFs alone, as negative control. Scale bars, 200 µm. (D) Zoomed images of immunofluorescence staining of nuclear receptors showing nuclear localisation. Scale bars, 50 µm. Nuclei were counterstained with DAPI (blue). (E) Immunofluorescence staining for AFP (cyan) in hESCs forward programmed for 20 days with 3TFs alone or in combination with the nuclear receptors RORc, ERɑ, and AR. Nuclei were counterstained with DAPI (blue). Scale bars, 200 µm.
Figure 4—figure supplement 2. Forward programming of human induced pluripotent stem cells (hiPSCs) into hepatocytes with 4TFs.

Figure 4—figure supplement 2.

(A) Immunofluorescence staining of the 3TFs in hiPSCs targeted with 3TFs alone or with RORc after 24 hr of inducible overexpression (iOX) with doxycycline (dox) confirming transgene induction. Scale bars, 200 µm. (B) Phase contrast images and (C) immunofluorescence staining for Albumin (yellow), (D) A1AT (green), and (E) AFP (cyan) in hiPSCs forward programmed for 20 days with 3TFs alone or with RORc. Nuclei were counterstained with DAPI (blue). Scale bars, 200 µm. (F) mRNA levels of hepatocyte markers (ALB, SERPINA1, and AFP) in hiPSC-derived FoP-Heps generated with 3TFs alone or with RORc for 20 and 30 days (n=4). Statistical differences were calculated with unpaired t-test. All expression data was normalised to the average of two housekeeping genes (PBGD and RPLP0). (G) Protein secretion levels of Albumin, A1AT, and AFP in hiPSC-derived FoP-Heps generated with 3TFs alone or with RORc for 20 days (n=4). Data was normalised per total cell number (millions). (H) CYP3A4 activity levels normalised per cell number (millions) in hiPSC FoP-Heps targeted with 3TFs with or without RORc after 20 days of forward programming (n=6). Statistical differences were calculated with one-way ANOVA, corrected for multiple comparisons compared to 3TFs (day 20). In all plots, bars represent mean with SD, and individual datapoints are shown for all biological replicates. Hepatocyte-like cells (HLCs) generated by direct differentiation and primary human hepatocytes (PHHs) where plotted as controls for CYP3A4 activity and expression data. Significant p-values are shown at each comparison and indicated as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.