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Mouse liver repopulation with hepatocytes generated from human fibroblasts

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Abstract

Human induced pluripotent stem cells (iPSCs) have the capability of revolutionizing research and therapy of liver diseases by providing a source of hepatocytes for autologous cell therapy and disease modelling. However, despite progress in advancing the differentiation of iPSCs into hepatocytes (iPSC-Heps) in vitro1,2,3, cells that replicate the ability of human primary adult hepatocytes (aHeps) to proliferate extensively in vivo have not been reported. This deficiency has hampered efforts to recreate human liver diseases in mice, and has cast doubt on the potential of iPSC-Heps for liver cell therapy. The reason is that extensive post-transplant expansion is needed to establish and sustain a therapeutically effective liver cell mass in patients, a lesson learned from clinical trials of aHep transplantation4. Here, as a solution to this problem, we report the generation of human fibroblast-derived hepatocytes that can repopulate mouse livers. Unlike current protocols for deriving hepatocytes from human fibroblasts, ours did not generate iPSCs but cut short reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells and subsequently hepatocytes (iMPC-Heps) could be efficiently differentiated. For this purpose we identified small molecules that aided endoderm and hepatocyte differentiation without compromising proliferation. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to those of aHeps. Unfractionated iMPC-Heps did not form tumours, most probably because they never entered a pluripotent state. Our results establish the feasibility of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy.

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Figure 1: Protocol for stepwise iMPC-Hep generation.
Figure 2: Characterization of iMPC-EPCs.
Figure 3: Characterization of iMPC-Heps.
Figure 4: Post-transplant proliferation and maturation of iMPC-Heps.

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Gene Expression Omnibus

Data deposits

Results of the microarray analysis have been deposited in the Gene Expression Omnibus database under accession no. GSE52309.

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Acknowledgements

We thank the Gladstone Institutes’ Bioinformatics Core for data analysis, A. Grimm for discussion, and P. Derish for manuscript editing. H.W. is supported by funding from the California Institute for Regenerative Medicine (CIRM; RN2-00950) and the National Institutes of Health (NIH; P30 DK26743). S.D. is supported by funding from CIRM, NIH and the Gladstone Institutes. S.Z. is supported by CIRM research training grant TG2-01160. M.R. is a research fellow in the Biomedical Exchange Program funded by the German Academic Exchange Service. J.H. is an Ethicon-Society of University Surgeons Fellow. A.N.M. is supported by CIRM research training grant TG2-01153.

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Contributions

S.Z., M.R. and J.H. are joint first authors; H.W. and S.D. are joint senior authors. S.Z., M.R., J.H., H.W. and S.D. designed the experiments. S.Z. and A.N.M. performed the reprogramming and directed differentiation experiments. M.R. and J.H. performed the transplantation experiments. M.R., J.H. and A.N.M. analysed the transplantation experiments and performed additional in vitro analyses. A.R.W. and L.Z.B. performed the liquid chromatography–tandem mass spectrometry analyses. S.Z., M.R., J.H., H.W. and S.D. wrote the manuscript. H.W. and S.D. edited the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Holger Willenbring or Sheng Ding.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Reprogramming of human fibroblasts into endoderm progenitor cells without activation of pluripotency markers.

a, qRT–PCR shows expression levels of the endoderm-specific genes SOX17 and FOXA2 during the reprogramming process (combination of initiation and reprogramming steps of the protocol) relative to starting cells at day 0. Results are means and s.e.m. for biological replicates (n = 3). b, Immunostainings show co-expression of SOX17 and FOXA2 in colonies at day 28. Scale bars, 100 μm. c, Immunostainings show absence of SOX17 and FOXA2 and the pluripotency-specific markers OCT4 and NANOG in parental fibroblasts. Scale bars, 100 μm. d, Small molecules increase the number of colonies positive in FOXA2 immunostaining at day 28. Medium containing activin A was additionally supplemented with the indicated small molecules. Results are means and s.e.m. for biological replicates (n = 3). e, qRT–PCR shows absence of endogenous (endo) OCT4 and NANOG gene expression during reprogramming to endoderm. Gene expression levels are shown relative to ESCs. Results are means and s.e.m. for biological replicates (n = 3). f, Flow cytometry shows absence of cells expressing the pluripotency marker TRA-1-60 at the end of the reprogramming process. Cells at day 0 and ESCs were used as controls. At least 10,000 events were collected. g, Flow cytometry for TRA-1-60 and NANOG of 10,000 cells from a culture of human fibroblasts transduced with retroviruses expressing OCT4, SOX2 and KLF4 and grown under iPSC reprogramming conditions for 30 days shows that both markers are effective in delineating rare cells reprogrammed to pluripotency. Because the number of NANOG-positive cells is higher than the number of TRA-1-60-positive cells, and virtually all TRA-1-60-positive cells are NANOG positive, NANOG seems to be a more sensitive marker in this process. All results were replicated in at least three independent experiments.

Extended Data Figure 2 Analysis of FOXA2 and NANOG expression at the colony and single-cell level during human fibroblast-to-iMPC-EPC reprogramming.

a, Diagram showing time points of analysis. b, Quantification of FOXA2-positive (red) and NANOG-positive (blue) colonies forming during the reprogramming process. Results are means ± s.e.m. for biological replicates (n = 3). c, Representative immunostainings show FOXA2-positive colonies emerging at day 16 of the reprogramming process and absence of NANOG-positive colonies or cells at all time points. Scale bars, 100 μm. d, Flow cytometry shows a gradual increase in the number of FOXA2-positive cells beginning at day 16 of the reprogramming process, whereas NANOG-positive cells are absent at all time points. Parental fibroblasts, ESCs and iMPC-EPCs were used as controls. At least 10,000 events were collected. All results were replicated in at least three independent experiments.

Extended Data Figure 3 Reprogramming of human fibroblasts into iMPC-EPCs occurs earlier and is more efficient than reprogramming into iPSCs.

a, Diagram showing duration of treatment with doxycycline and time allowed for reprogramming to occur until analysis. b, Quantification of iMPC-EPC and iPSC colonies forming from human fibroblasts cultured under iMPC-EPC and iPSC reprogramming conditions, respectively, in response to different durations of treatment with doxycycline. iMPC-EPC and iPSC colonies were identified by immunostaining with FOXA2 and NANOG, respectively. Results are means and s.e.m. for biological replicates (n = 3). All results were replicated in at least three independent experiments.

Extended Data Figure 4 Expansion and further characterization of iMPC-EPCs.

a, Medium containing both CHIR and A83 promotes iMPC-EPC colony expansion. Cells treated with carrier dimethylsulphoxide (DMSO) were used as a control. Scale bars, 100 μm. b, Supplementing medium containing both CHIR and A83 with EGF and bFGF further increases the number of iMPC-EPC colonies forming after passaging. Results are means and s.e.m. for biological replicates (n = 3). c, Immunostainings show that expanded (passage 7) iMPC-EPCs remain positive for FOXA2 and negative for NANOG. ESCs were used as a control. Scale bars, 100 μm. d, Immunostainings show HNF4α expression in an iMPC-EPC colony after expansion (passage 4) but not at day 21 of the reprogramming process, indicating that expansion induces HNF4α expression. Scale bars, 100 μm. e, Immunostaining shows that iMPC-EPCs acquire expression of the hepatic differentiation marker AFP after exposure to bFGF and BMP4 for 4 days. f, Immunostaining shows that iMPC-EPCs acquire expression of the pancreatic differentiation marker PDX1 after exposure to retinoic acid, GDC-0449 (Sonic Hedgehog inhibitor) and LDN-193189 (BMP inhibitor) for 4 days. Scale bars, 100 μm. All results were replicated in at least three independent experiments.

Extended Data Figure 5 Directed differentiation of iMPC-EPCs into iMPC-Heps.

a, Immunostainings show that almost all iMPC-EPCs express AFP after sequential exposure to bFGF, BMP4, Dex, HGF and OSM, whereas only a subset of the cells acquire expression of ALB and AAT. Scale bars, 100 μm. b, qRT–PCR at day 18 of the hepatocyte specification step of the protocol shows an additive effect of A83 and C-E in inducing ALB gene expression. Gene expression levels are shown relative to those of iMPC-EPCs treated with DMSO. Results are means and s.e.m. for technical replicates (n = 3). All results were replicated in at least three independent experiments.

Extended Data Figure 6 Analysis of hepatocyte function of iMPC-Heps in vitro.

a, Periodic acid–Schiff (PAS) staining shows that iMPC-Heps contain glycogen. Adding Dil-ac-low-density lipoprotein (LDL) fluorescent substrate to the culture medium shows that iMPC-Heps take up LDL. Incubation with BODIPY 493/503 or staining with Oil-red-O (ORO) shows storage of lipids in iMPC-Heps. Parental fibroblasts were used as a negative control. Scale bars, 100 μm. b, iMPC-Heps (red) produce urea. The concentrations of urea measured in cell culture medium at the indicated time points are shown relative to the concentrations of urea measured in fresh medium. Parental fibroblasts (blue) were used as a negative control. Results are means ± s.e.m. for biological replicates (n = 3). c, qRT–PCR shows higher expression of several hepatocyte-specific genes including ALB and SERPINA1, and lower expression of AFP, a marker of immature hepatocytes, in iMPC-Heps than in iPSC-Heps generated using current standard protocols. Gene expression of many CYP450 enzymes is also higher in iMPC-Heps than in iPSC-Heps, indicating that iMPC-Heps have a more mature hepatocyte phenotype than iPSC-Heps. Gene expression levels in iPSC-Heps were set to 1. Results are means and s.e.m. for technical replicates (n = 3). d, iMPC-Heps secrete more ALB and have higher CYP3A family, CYP3A4 and CYP2C19 activities than iPSC-Heps generated with the iMPC-EPC/Hep generation protocol, referred to as iPSC-Heps (NP). Results are means and s.e.m. for biological replicates (n = 3); Student’s t-test, asterisk, P < 0.05; two asterisks, P < 0.01. All results were replicated in at least three independent experiments.

Extended Data Figure 7 Quantification and isolation of repopulating nodules formed by transplanted iMPC-Heps.

a, Immunostainings show a small and a large nodule of iMPC-Heps detected with a human-specific ALB antibody at 3 and 9 months after transplantation. Scale bars, 100 μm. b, Multiple large nodules of iMPC-Heps identified by FAH immunostaining at 9 months after transplantation. Scale bar = 100 μm. c, Size distribution of nodules of iMPC-Heps 9 months after transplantation based on ALB and FAH immunostaining. d, Example of an iMPC-Hep nodule identified by ALB immunostaining for isolation by laser-capture microscopy (LCM). Blood vessels (numbers) were used as additional markers of the location of a nodule in an adjacent, unfixed cryosection. e, Confirmation of successful isolation of an iMPC-Hep nodule by ALB immunostaining after LCM. The middle image shows a cryosection fixed and immunostained for ALB after LCM to confirm specific isolation of a nodule. The left and right images show ALB immunostainings of cryosections flanking the cryosection used for LCM. Scale bars, 100 μm. All results were replicated in at least three independent experiments.

Extended Data Figure 8 Assessment of in vivo maturation of iMPC-Heps by global gene expression profiling.

a, Venn diagram showing the number of genes significantly (P < 0.05) differentially expressed between iMPC-Heps and aHeps in vivo. Of 17,367 reliably detected genes, 132 are differentially expressed; 78 are expressed higher in iMPC-Heps, and 54 are expressed higher in aHeps. The complete results of the global gene expression profiling—including the genes that are differentially expressed between aHeps and iMPC-Heps in vivo—are shown in Supplementary Table 2. be, Further analysis of results from global gene expression profiling using gene sets of the hepatocyte function-related Gene Ontology (GO) terms REACTOME CYTOCHROME P450 ARRANGED BY SUBSTRATE TYPE (b), BILE ACID METABOLIC PROCESS (c), GLUCOSE METABOLIC PROCESS (d) and RESPONSE TO XENOBIOTIC STIMULUS (e). GO terms and annotated genes were obtained from Molecular Signatures Database (MSigDB) v.4.0. Heatmaps were generated individually for each GO term; a representative colour legend is shown. All results are from one microarray analysis.

Extended Data Figure 9 Assessment of in vivo maturation of iMPC-Heps by immunostaining.

a, Co-immunostaining for ALB and AFP shows lack of expression of the immature hepatocyte-specific marker AFP in iMPC-Hep and aHep nodules. Human fetal liver was used as a positive control. Scale bars, 100 μm. b, c, Co-immunostainings for ALB and CYP3A4 (b) or CYP2D6 (c) show expression of these mature hepatocyte-specific markers in iMPC-Heps. The CYP450 antibodies detect the mouse homologues of CYP3A4 and CYP2D6, which—as in humans—seem to be expressed in hepatocytes but not in non-parenchymal liver cells. Scale bars, 100 μm. All results were replicated in at least two independent experiments.

Extended Data Figure 10 Therapeutic efficacy and safety of iMPC-Heps.

a, Kaplan–Meier survival curve shows that 106 transplanted iMPC-Heps, iPSC/ESC-Heps or aHeps are not effective in rescuing mice from death from acute liver failure. Log-rank test P = 0.4426 between iMPC-Heps and iPSC/ESC-Heps, P = 0.4031 between iMPC-Heps and aHeps. b, Kaplan–Meier survival curve shows similar efficacy of 106 transplanted aHeps and iMPC-Heps, but not iPSC/ESC-Heps, in preventing death in mice with chronic liver failure. Log-rank test P < 0.01 between iMPC-Heps and iPSC/ESC-Heps, P = 0.9501 between iMPC-Heps and aHeps. The number of mice in each group is shown in parentheses. c, Haematoxylin/eosin staining shows a dysplastic nodule in the liver of an FRG mouse transplanted with iMPC-Heps. Scale bar, 100 μm. d, Co-immunostaining with human-specific β2-microglobulin (B2M) and ALB antibodies shows that the cells within a dysplastic nodule (dashed line) are negative for both markers and are therefore of mouse origin. Scale bars, 100 μm. Nodules of iMPC-Heps or aHeps are shown as controls. All results were replicated in at least two independent experiments.

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Supplementary Tables

This file contains Supplementary Tables 1 and 3. (PDF 140 kb)

Supplementary Table 2

This file contains the global gene expression profile. (XLS 13124 kb)

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Zhu, S., Rezvani, M., Harbell, J. et al. Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature 508, 93–97 (2014). https://doi.org/10.1038/nature13020

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