Highly efficient differentiation of functional hepatocytes from human induced pluripotent stem cells

Abstract

Human induced pluripotent stem cells (hiPSCs) hold great potential for use in regenerative medicine, novel drug development, and disease progression/developmental studies. Here, we report highly efficient differentiation of hiPSCs toward a relatively homogeneous population of functional hepatocytes. hiPSC-derived hepatocytes (hiHs) not only showed a high expression of hepatocyte-specific proteins and liver-specific functions, but they also developed a functional biotransformation system including phase I and II metabolizing enzymes and phase III transporters. Nuclear receptors, which are critical for regulating the expression of metabolizing enzymes, were also expressed in hiHs. hiHs also responded to different compounds/inducers of cytochrome P450 as mature hepatocytes do. To follow up on this observation, we analyzed the drug metabolizing capacity of hiHs in real time using a novel ultra performance liquid chromatography-tandem mass spectrometry. We found that, like freshly isolated primary human hepatocytes, the seven major metabolic pathways of the drug bufuralol were found in hiHs. In addition, transplanted hiHs engrafted, integrated, and proliferated in livers of an immune-deficient mouse model, and secreted human albumin, indicating that hiHs also function in vivo. In conclusion, we have generated a method for the efficient generation of hepatocytes from induced pluripotent stem cells in vitro and in vivo, and it appears that the cells function similarly to primary human hepatocytes, including developing a complete metabolic function. These results represent a significant step toward using patient/disease-specific hepatocytes for cell-based therapeutics as well as for pharmacology and toxicology studies.

Keywords: Hepatocyte differentiation; Induced pluripotent stem cells; Liver regeneration; Stem cell transplantation.

Figure 1.

Differentiation of human induced pluripotent stem cells toward hepatocytes. (A–F): The cells were analyzed by flow cytometry for the percentage of positive cells for CXCR4 (A), SOX17 (B), and FOXA2 (C) at day 8 during induction of definitive endoderm; for AFP (D) at day 9 after differentiation; and for ALB (E) and α1-AT (F) at day 18 after differentiation. (G, N): Definitive endodermal cells were stained with the primary antibodies goat anti-SOX 17 (green) (I) and FOXA2 (red) (J) during induction of human induced pluripotent stem (iPS) cells to definitive endoderm; the differentiated cells were stained with the primary antibodies monoclonal antibody against AFP (red) (M) and goat anti-ALB (green) (N) during differentiation of human iPS cells toward hepatocytes. (G, H, K, L): Negative controls of SOX17 (G), FOXA2 (H), AFP (K), and ALB (L) stained with isotype antibodies. All immunohistochemistry analyses were merged with 4′,6′-diamidino-2-phenylindole nucleic acid staining (G–N). (O): Relative expression of ALB, α1-AT, TAT, and HNF4α in hiHs and hPHs determined by quantitative reverse transcription-polymerase chain reaction. (P, Q): Polymerase chain reaction was used to determine expression of the liver-associated genes G-6-P and TAT (P), and liver-associated transcriptional factors and BMP signaling (Q). Scale bars = 100 μm (G–N). Details of primers and antibodies can be found in the supplemental online tables. Abbreviations: α1-AT, α1-antitrypsin; AFP, α-fetoprotein; ALB, albumin; BMP, bone morphogenetic protein; G-6-P, glucose-6-phosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hiH, human induced pluripotent stem cell-derived hepatocytes at day 25; hiH1, human induced pluripotent stem cell-derived hepatocytes at day 10; hPH, human primary hepatocyte; HNF4α, hepatocyte nuclear factor 4α; iPSC, induced pluripotent stem cell; TAT, tyrosine aminotransferase.

Figure 2.

Gene expression in hiHs. (A, B): Expression of phase I (red) and phase II (black) enzymes in hiHs determined by Western blot (A) and reverse transcription-polymerase chain reaction (B). hiH1 and hiH2 are the cells from different batches of differentiation. (D, F, H): Merge of immunostainings for expressions of MRP1 (D), OATP2 (F), and Glut2 (H) with nucleic acid staining by 4′,6′-diamidino-2-phenylindole (DAPI). (C, E, G): Negative controls of MRP1 (C), OAPT2 (E), and Glut2 (G) stained with isotype antibodies, respectively, in immunohistochemistry analysis merged with DAPI nucleic acid staining. Scale bars = 100 μm (C–F). Abbreviations: CYP, cytochrome P450; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Glut2, glucose transporter 2; GST, glutathione S-transferase; hEH, human embryonic stem cell-derived hepatocyte; hiH, human induced pluripotent stem cell-derived hepatocyte; hPH, human primary hepatocyte; iPSC, induced pluripotent stem cell; MRP1, multidrug-resistant protein 1; OATP2, organic anion transporting polypeptide 2; UGT, UDP-glucuronosyl-S-transferase.

Figure 3.

Expression of nuclear receptors in human induced pluripotent stem cell-derived hepatocytes (hiHs). (B, D, F, H): Merge of immunostainings for expressions of the nuclear receptors CAR, AhR, CPR, and PXR with nucleic acid by 4′,6′-diamidino-2-phenylindole (DAPI) in hiHs determined by immunohistochemistry. (A, C, E, G): Negative controls of CAR, AhR, CPR, and PXR stained with the corresponding isotype antibodies in immunohistochemistry analysis merged with nucleic acid staining by DAPI. Scale bars = 100 μm (A–H). Abbreviations: AhR, aryl hydrocarbon receptor; CAR, constitute androstane receptor; CPR, cytochrome P450 reductase; PXR, pregnane X receptor.

Figure 4.

Liver function assays of hiHs. (A): hiHs were stained at 37°C for 60 minutes with indocyanine green (ICG) at day 18 after differentiation, and then the cellular uptake of ICG was examined by microscopy after the cells were washed with phosphate-buffered saline (PBS). (B, C): After PBS was replaced by the culture medium and the cells were incubated at 37°C again, the cellular excretion of ICG was examined by microscopy at 1 hour (B) and 3.5 hours (C) after the removal of ICG. (D): hiHs were analyzed by periodic acid-Schiff's staining for glycogen storage at day 20 after differentiation. Scale bars = 100 μm (A–D). (E): The supernatants were collected during the differentiation of human induced pluripotent stem cells, and albumin secretion by hiHs in supernatants was determined by enzyme-linked immunosorbent assay. The total secreted albumin was normalized to the cell numbers at days 26–27. (F): The supernatants were collected at 72 hours after hiHs were treated with the inducers Rifa and Phe at day 22 after differentiation. The increase of CYP3A4 activity was assessed by measurement of luciferase activity with the P450-Glo CYP3A4 assay kit. Abbreviations: ALB, albumin; hiH, human induced pluripotent stem cell-derived hepatocyte; Phe, phenobarbital; Rifa, rifampicin.

Figure 5.

Metabolites (peaks) identified with MRM transition code and liquid chromatography (LC) RT and MS/MS fragment patterns. (A): Peaks a and b: Metabolites with MRM transition of m/z 278.2/204.2 and RTs of 1.672 and 1.979 minutes (from hiHs) and 1.575 and 1.990 minutes (from hPHs) were identified from oxidations/+16.0. Peaks c and d: Metabolites with MRM transition of m/z 276.2/202.2 and RTs of 2.885 and 2.423 minutes (from hiHs) and 2.929 and 2.438 minutes (from hPHs) were identified from ketone formation and/or methylation/+14.0. Peak e: Metabolites with MRM transition of m/z 260.2/186.2 and RTs of 2.134 minutes (from hiHs) and 2.149 minutes (from hPHs) were identified from dehydrogenation/−2.0. Peak f: Metabolites with MRM transition of m/z 438.2/262.2 and RTs of 1.000 minute (from hiHs) and 1.006 minutes (from in hPHs) were identified from glucuronidation/+289.0. Peak g: Metabolites with MRM transition of m/z 424.2/188.2 and RTs of 1.996 minutes (from in hiHs) and 2.010 minutes (from hPHs) were identified from conjugation of glucose/+162.0. Peak h: Metabolites with MRM transition of m/z 551.2/188.2 and RTs of 1.993 minutes (from hiHs) and 1.990 minutes (from hPHs) were identified from conjugation of glutathione/+289.0. (B): The major LC/MS/MS fragments of metabolites from each pathway are listed with those of parent bufuralol. Details of mass spectrometry methods, metabolite identification, and validation can be found in the supplemental online data. Abbreviations: hiH, human induced pluripotent stem cell-derived hepatocyte; hPH, human primary hepatocyte; LC/MS/MS, liquid chromatography tandem mass spectrometry; MRM, multiple reaction monitoring; RT, retention time.

Figure 6.

Time course effects of metabolites after treatment with BF and their metabolic pathways in hiHs and hPHs. (A): Supernatants from hiHs and hPHs treated with BF were collected at 24 and 48 hours after treatment, and the amount of metabolite products was determined as analyte peak area and normalized to cell number used. Metabolites identified by MRM transition codes and liquid chromatography RTs (shown in Fig. 5) from each pathway showed time course effects in both hiHs and hPHs. (B): Summary of in vitro metabolic pathways of bufuralol in hiHs and hPHs. The metabolic pathways of drug bufuralol in hiHs were shown to be the same as those in hPHs. Abbreviations: BF, bufuralol; hiH, human induced pluripotent stem cell-derived hepatocyte; hPH, human primary hepatocyte; RT, retention time.

Figure 7.

Analysis of human albumin expression by human induced pluripotent stem cell-derived hepatocytes (hiHs) in mouse livers after transplantation. (A–D): Liver sections from transplanted mice were immunostained with primary goat anti-human albumin antibody in livers of NOD/SCID mice sacrificed at 4 weeks after transplantation with hiHs. Large numbers of hiHs engrafted around central veins (A), integrated in the parenchyma of mouse livers (B), and proliferated along at least five adjacent central veins (CV1–CV5) in the livers of mice treated with retrorsine 2 weeks prior to transplantation (C–F). All immunostainings of liver sections were merged with nucleic acid staining by 4′,6-diamidino-2-phenylindole. Scale bars = 100 μm (A–F). Abbreviation: CV, central vein.