Generation of functional hepatocytes by forward programming with nuclear receptors
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, United Kingdom
- Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, United Kingdom
- Wellcome Sanger Institute, Wellcome Genome Campus, United Kingdom
Figures
Figure 1 with 2 supplements
Forward programming of human pluripotent stem cells (hPSCs) into hepatocytes with four and three liver-enriched transcription factors (LETFs).
(A) Schematic representation of the two sequentially targeted loci. The human ROSA26 was targeted with a constitutively expressed reverse tetracycline transactivator (rtTA). The AAVS1 locus was …
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Figure 1—source data 1
Individual measurements and statistical tests related to Figure 1.
- https://cdn.elifesciences.org/articles/71591/elife-71591-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Validation of inducible overexpression (iOX).
(A) Schematic representation of the combinations of three liver-enriched transcription factors (LETFs) cloned into the AAVS1 safe harbour. (B) Immunofluorescence staining of all four LETFs in human …
Figure 1—figure supplement 2
Characterisation of the phenotype of human pluripotent stem cells (hPSCs) forward programmed into hepatocytes with three liver-enriched transcription factors (LETFs).
(A) Phase contrast images and (B) immunofluorescence staining of hepatocyte markers Albumin (yellow), A1AT (green), and AFP (cyan) in human embryonic stem cells (hESCs) forward programmed with …
Figure 2 with 1 supplement
Hepatocyte-like cells (HLCs) and primary human hepatocytes (PHHs) display transcriptomic differences associated with their state of maturation.
(A) Immunofluorescence staining of Albumin (yellow) and HNF4A (red) in HLCs differentiated for 30 days. Nuclei were counterstained with DAPI (blue). Scale bar, 100 µm. (B) CYP3A4 activity levels …
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Figure 2—source data 1
Individual measurements and statistical tests related to Figure 2 and corresponding supplements.
- https://cdn.elifesciences.org/articles/71591/elife-71591-fig2-data1-v1.xlsx
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Figure 2—source data 2
List of genes differentially expressed in the four clusters in Figure 2D.
- https://cdn.elifesciences.org/articles/71591/elife-71591-fig2-data2-v1.xlsx
Figure 2—figure supplement 1
Characterisation of transcriptional differences between hepatocyte-like cells (HLCs) and primary human hepatocytes (PHHs).
(A) mRNA levels measured by qPCR throughout 30 days of human induced pluripotent stem cell (hiPSC) differentiation into HLCs. Representative markers of each stage of differentiation are shown: …
Figure 3 with 1 supplement
Epigenetic status of regulatory regions differs between states of maturation in hepatocyte-like cells (HLCs) and primary human hepatocytes (PHHs).
(A) Principal component analysis (PCA) of the global enrichment profile of H3K27ac, H3K4me1, and H3K27me3 across two replicates of undifferentiated human induced pluripotent stem cells (hiPSCs), …
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Figure 3—source data 1
Peak annotation results for primary human hepatocyte (PHH)-unique H3K27ac regions.
- https://cdn.elifesciences.org/articles/71591/elife-71591-fig3-data1-v1.xlsx
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Figure 3—source data 2
Peak annotation results for hepatocyte-like cell (HLC)-unique H3K27ac regions.
- https://cdn.elifesciences.org/articles/71591/elife-71591-fig3-data2-v1.xlsx
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Figure 3—source data 3
Motif enrichment results for primary human hepatocyte (PHH)-unique H3K27ac regions.
- https://cdn.elifesciences.org/articles/71591/elife-71591-fig3-data3-v1.xlsx
Figure 3—figure supplement 1
Comparison of the epigenetic profile of hepatocyte-like cells (HLCs) and primary human hepatocytes (PHHs).
(A) Distance to TSS of H3K27ac peaks identified in the PHH and HLC datasets. (B) Enrichment profiles of H3K27ac, H3K4me1, and H3K27me3 across the CYP3A4 locus. Profiles are shown for one replicate …
Figure 4 with 2 supplements
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 …
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Figure 4—source data 1
Individual measurements and statistical tests related to Figure 4 and supplements.
- https://cdn.elifesciences.org/articles/71591/elife-71591-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Validation of inducible overexpression (iOX) of combinations of 3TFs and nuclear receptors.
(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) …
Figure 4—figure supplement 2
Forward programming of human induced pluripotent stem cells (hiPSCs) into hepatocytes with 4TFs.
(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 …
Figure 5 with 2 supplements
4TF FoP-Heps are transcriptionally equivalent to hepatocyte-like cells (HLCs).
(A) CYP3A4 activity levels in eFoP-Heps at day 20 (n=6) and day 30 (n=6), iFoP-Heps at day 0 (n=6) and day 30 (n=3), against direct differentiation HLCs (n=6) and primary human hepatocytes (PHHs) …
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Figure 5—source data 1
Individual measurements and statistical tests related to Figure 5 and corresponding supplements.
- https://cdn.elifesciences.org/articles/71591/elife-71591-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Characterisation of 4TF FoP-Heps.
(A) mRNA levels of phase II (UGT1A1) biotransformation enzymes, lipid (APOA1, FASN, PPARα) and bile acid metabolism (NR1H4), and (B) 4TFs (HNF1A, FOXA3, HNF6 RORγ) in human embryonic stem cell …
Figure 5—figure supplement 2
Characterisation of the transcriptome of 4TF eFoP-Heps.
Dotplot showing the top 15 hits on gene ontology enrichment analysis on genes differentially expressed between eFoP and foetal liver (A, B), adult primary human hepatocytes (PHHs) (C, D), human …
Figure 6
RORc promotes functionality of 4TF FoP-Heps.
(A) Immunofluorescence staining for LDL in FoP-Heps at day 20 of forward programming. Scale bars, 200 µm. Nuclei were counterstained with DAPI (blue). (B) Comparison of mRNA levels of SERPINA1, ALB, …
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Figure 6—source data 1
Individual measurements and statistical tests related to Figure 6.
- https://cdn.elifesciences.org/articles/71591/elife-71591-fig6-data1-v1.xlsx
Author response image 1
CYP3A4 Activity in long-term cultures.
hiPSCs expressing the 4TF combination were induced to differentiate for up to 40 days, with CYP3A4 activity measure at days 20, 30 and 40 (left panel). HLCs where differentiated by directed …
Author response image 2
Human Albumin secretion in mice sera.
eFoP (hESC-derived) or iFoP (hIPSC-derived) cells differentiated up to 20 days were dissociated into small clusters, and 1 million cells mixed with growth-factor reduced Matrigel and injected (per …
Author response image 3
PCA plot.
PCA of undifferentiated hiPSCs, HLCs derived from hESC (hESC_HLCs) and hiPSC (hiPSC_HLCs), freshly harvested PHHs (fPHHs) or plated PHHs (pPHHs), where generated with the top 1000 (left panel), 5000 …
Author response image 4
Expression of nuclear receptors in PHHs.
mRNA levels of AR and ESR1 (ERa) genes in primary human hepatocytes from 4 donors including 1 female (F) and 3 males (M1, M2, M3). Data were normalised to the average of 2 housekeeping genes (PBGD …
Author response image 5
Cellular localisation of AR.
Immunofluorescence staining of HNF1A an AR in hESCs targeted with 3TFs+AR after 24h of iOX with dox. Scale bar, 100µm.
Author response image 6
Dox removal during forward programming.
hiPSCs overexpressing the 4TF combination were induced to differentiate for 20 days. Dox was removed from 10 and from day 15. iFoP-Heps where assessed for CYP3A4 activity levels (left panel). …
Author response image 7
CYP3A4 Activity in long term cultures.
hiPSCs expressing the 4TF combination were induced to differentiate for up to 40 days, with CYP3A4 activity measured at days 20, 30 and 40.
Tables
Table 1
Sequences of primers used for cloning.
| Gene | Primer | Sequence 5'–3' (GSG linker sequence underlined) |
|---|---|---|
| HNF1A | Start_F | CAC TTT TGT CTT ATA CTT ACT AGT GCC ACC ATG GTT TCT AAA CTG AGC CAG CTG CAG |
| HNF1A | P2A_R | TTC CAC GTC TCC TGC TTG CTT TAA CAG AGA GAA GTT CGT GGC TCC GGA GCC CTG GGA GGA AGA GGC CAT CTG G |
| HNF4A | E2A_F | TAT GCT CTC TTG AAA TTG GCT GGA GAT GTT GAG AGC AAC CCT GGA CCT GTC AGC GTG AAC GCG CCC CT |
| HNF4A | Stop_R | AGA GGA TCC CCG GGT ACC GAG CTC GAA TTC CTA GAT AAC TTC CTG CTT GGT GAT GGT CG |
| HNF4A | P2A_F | TCT CTG TTA AAG CAA GCA GGA GAC GTG GAA GAA AAC CCC GGT CCT GTC AGC GTG AAC GCG CCC CT |
| HNF4A | T2A_R | CTC CTC CAC GTC ACC GCA TGT TAG AAG ACT TCC TCT GCC CTC TCC GGA GCC GAT AAC TTC CTG CTT GGT GAT GGT CG |
| HNF4A | Start_F | CAC TTT TGT CTT ATA CTT ACT AGT GCC ACC ATG GTC AGC GTG AAC GCG CCC |
| HNF4A | P2A_R | TTC CAC GTC TCC TGC TTG CTT TAA CAG AGA GAA GTT CGT GGC TCC GGA GCC GAT AAC TTC CTG CTT GGT GAT GGT CG |
| FOXA3 | T2A_F | AGT CTT CTA ACA TGC GGT GAC GTG GAG GAG AAT CCC GGC CCT CTG GGC TCA GTG AAG ATG GAG GC |
| FOXA3 | E2A_R | CTC AAC ATC TCC AGC CAA TTT CAA GAG AGC ATA ATT AGT ACA CTG TCC GGA GCC GGA TGC ATT AAG CAA AGA GCG GGA ATA G |
| FOXA3 | P2A_F | TCT CTG TTA AAG CAA GCA GGA GAC GTG GAA GAA AAC CCC GGT CCT CTG GGC TCA GTG AAG ATG GAG GC |
| FOXA3 | T2A_R | CTC CTC CAC GTC ACC GCA TGT TAG AAG ACT TCC TCT GCC CTC TCC GGA GCC GGA TGC ATT AAG CAA AGA GCG GGA ATA G |
| FOXA3 | T2A_F | AGT CTT CTA ACA TGC GGT GAC GTG GAG GAG AAT CCC GGC CCT CTG GGC TCA GTG AAG ATG GAG GC |
| FOXA3 | Stop_R | AGA GGA TCC CCG GGT ACC GAG CTC GAA TTC CTA GGA TGC ATT AAG CAA AGA GCG GGA ATA G |
| HNF6 | P2A_F | TCT CTG TTA AAG CAA GCA GGA GAC GTG GAA GAA AAC CCC GGT CCT AAC GCG CAG CTG ACC ATG GAA GC |
| HNF6 | T2A_R | CTC CTC CAC GTC ACC GCA TGT TAG AAG ACT TCC TCT GCC CTC TCC GGA GCC TGC TTT GGT ACA AGT GCT TGA TGA AGA AGA T |
| HNF6 | T2A_F | AGT CTT CTA ACA TGC GGT GAC GTG GAG GAG AAT CCC GGC CCT AAC GCG CAG CTG ACC ATG GAA GC |
| HNF6 | Stop_R | AGA GGA TCC CCG GGT ACC GAG CTC GAA TTC CTA TGC TTT GGT ACA AGT GCT TGA TGA AGA AGA T |
| RORy | E2A_F | TAT GCT CTC TTG AAA TTG GCT GGA GAT GTT GAG AGC AAC CCT GGA CCT GAC AGG GCC CCA CAG AGA CAG |
| RORy | Stop_R | AGA GGA TCC CCG GGT ACC GAG CTC GAA TTC CTA CTT GGA CAG CCC CAC AGG TGA C |
| ESR1 | E2A_F | TAT GCT CTC TTG AAA TTG GCT GGA GAT GTT GAG AGC AAC CCT GGA CCT ACC ATG ACC CTC CAC ACC AAA GCA |
| ESR1 | Stop_R | AGA GGA TCC CCG GGT ACC GAG CTC GAA TTC CTA GAC CGT GGC AGG GAA ACC CTC |
| AR | E2A_F | TAT GCT CTC TTG AAA TTG GCT GGA GAT GTT GAG AGC AAC CCT GGA CCT GAA GTG CAG TTA GGG CTG GGA AG |
| AR | Stop_R | AGA GGA TCC CCG GGT ACC GAG CTC GAA TTC CTA CTG GGT GTG GAA ATA GAT GGG CTT G |
Table 2
Sequences of primers used for qPCR.
| Gene | Forward | Reverse |
|---|---|---|
| AFP | TGCGGCCTCTTCCAGAAACT | TAATGTCAGCCGCTCCCTCG |
| ALB | CCTTTGGCACAATGAAGTGGGTAACC | CAGCAGTCAGCCATTTCACCATAG |
| APOA1 | AGACAGCGGCAGAGACTATG | CCAGTTGTCAAGGAGCTTTAGG |
| CYP2A6 | CAGCACTTCCTGAATGAG | AGGTGACTGGGAGGACTTGAGGC |
| CYP2C8 | CATTACTGACTTCCGTGCTACAT | CTCCTGCACAAATTCGTTTTCC |
| CYP2C9 | GCCGGCATGGAGCTGTTTTTAT | GCCAGGCCATCTGCTCTTCTT |
| CYP3A4 | TGTGCCTGAGAACACCAGAG | GTGGTGGAAATAGTCCCGTG |
| FASN | GCAAGCTGAAGGACCTGTCT | AATCTGGGTTGATGCCTCCG |
| FOXA3 | TGGGCTCAGTGAAGATGGAG | GGGGATAGGGAGAGCTTAGAG |
| G6PC | GTGTCCGTGATCGCAGACC | GACGAGGTTGAGCCAGTCTC |
| HHEX | GCCCTTTTACATCGAGGACA | AGGGCGAACATTGAGAGCTA |
| HNF1A | TGGCCATGGACACGTACAG | GCTGCTTGAGGGTACTTCTG |
| HNF4A | CATGGCCAAGATTGACAACCT | TTCCCATATGTTCCTGCATCAG |
| HNF6 | GTGTTGCCTCTATCCTTCCCAT | CGCTCCGCTTAGCAGCAT |
| NANOG | CATGAGTGTGGATCCAGCTTG | CCTGAATAAGCAGATCCATGG |
| NR1H4 | ACTGAACTCACCCCAGATCAA | TGGTTGCCATTTCCGTCAAA |
| PBGD | GGAGCCATGTCTGGTAACGG | CCACGCGAATCACTCTCATCT |
| PCK1 | ACACAGTGCCCATCCCCAAA | GGTGCGACCTTTCATGCACC |
| POU5F1 | AGTGAGAGGCAACCTGGAGA | ACACTCGGACCACATCCTTC |
| PPARa | CCCTCCTCGGTGACTTATCC | CGGTCGCACTTGTCATACAC |
| PPARy | GAGCCTGCATCTCCACCTTAT | AGAAACCCTTGCATCCTTCACA |
| RORy | CTACGGCAGCCCCAGTTT | GCTGGCATGTCTCCCTGTA |
| RPLP0 | GGCGTCCTCGTGGAAGTGAC | GCCTTGCGCATCATGGTGTT |
| SERPINA1 | CCACCGCCATCTTCTTCCTGCCTGA | GAGCTTCAGGGGTGCCTCCTCTG |
| SOX17 | CGCACGGAATTTGAACAGTA | GGA TCAGGGACCTGTCACAC |
| TBX3 | TGGAGCCCGAAGAAGAGGTG | TTCGCCTTCCCGACTTGGTA |
| UGT1A1 | TGATCCCAGTGGATGGCAGC | CAACGAGGCGTCAGGTGCTA |
| UGT1A6 | GGAGCCCTGTGATTTGGAGAGT | GACCCCGGTCACTGAGAACC |
Table 3
Lisf of primary antibodies.
| Protein | Supplier | Catalog number | Host | Concentration |
|---|---|---|---|---|
| Albumin | Bethyl Laboratories | A80-229A | goat | 1:100 |
| Alpha-1 Antitrypsin | Dako | A0012 | rabbit | 1:100 |
| Alpha-Fetoprotein | Dako | A0008 | rabbit | 1:100 |
| HNF4A | Abcam | ab92378 | rabbit | 1:100 |
| HNF1A | Santa cruz | sc-135939 | mouse | 1:50 |
| HNF6 | Santa cruz | sc-13050 | rabbit | 1:100 |
| FOXA3 | Santa cruz | sc-166703 | mouse | 1:50 |
| RORc | Abcam | ab221359 | rabbit | 1:100 |
| ERɑ | Abcam | ab32063 | rabbit | 1:100 |
| AR | Abcam | ab108341 | rabbit | 1:100 |
