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Small Molecule Drugs Promote Repopulation of Transplanted Hepatocytes by Stimulating Cell Dedifferentiation

  • Author Footnotes
    # These authors contributed equally to this work.
    Mengmeng Jiang
    Footnotes
    # These authors contributed equally to this work.
    Affiliations
    School of Life Science and Technology, Shanghai Tech University, Shanghai 201210, China

    CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

    University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
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  • Author Footnotes
    # These authors contributed equally to this work.
    Ren Guo
    Footnotes
    # These authors contributed equally to this work.
    Affiliations
    CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
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  • Yan Ai
    Affiliations
    School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, China
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  • Gang Wang
    Affiliations
    Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, China
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  • Peilan Tang
    Affiliations
    School of Pharmaceutical Science, Nanchang University, Nanchang 330006, PR China
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  • Xiaohui Jia
    Affiliations
    CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

    University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
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  • Bingqing He
    Affiliations
    School of Life Science and Technology, Shanghai Tech University, Shanghai 201210, China

    CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

    University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China
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  • Qianting Yuan
    Affiliations
    CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
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  • Xin Xie
    Correspondence
    Corresponding author., 189 Guo Shou Jing Road, Shanghai 201203, China. Tel: (86) 186-0211-0377; Fax: 0086-21-50800721. .
    Affiliations
    School of Life Science and Technology, Shanghai Tech University, Shanghai 201210, China

    CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

    University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China

    School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, China

    State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
    Search for articles by this author
  • Author Footnotes
    # These authors contributed equally to this work.
Open AccessPublished:January 13, 2023DOI:https://doi.org/10.1016/j.jhepr.2023.100670

      Highlights:

      • The transplanted hepatocytes dedifferentiate into hepatic progenitor cells (HPCs) before repopulation.
      • Y-27632 (Y) and CHIR99021 (C) convert mouse hepatocytes into HPCs & support long-term culture (>30 passages) in vitro.
      • YC stimulate the proliferation of transplanted hepatocytes in Fah-/- liver by promoting the conversion into HPCs.
      • Two clinically used drugs target the same pathways as YC, also promote hepatocytes proliferation in vitro and in vivo.

      Abstract

      Background & Aims

      Normal liver has strong regenerative capacity. However, for patients with end-stage liver disease, the regenerative capacity is damaged, and liver transplantation is extremely limited by the availability of donor organs. Thus, hepatocyte transplantation emerged as a possible solution. However, an important obstacle to hepatocyte therapy is the low level of engraftment and proliferation of transplanted hepatocytes, which do not survive long enough to exert therapeutic effects. Exploring the mechanisms of hepatocyte proliferation in vivo and finding a way to promote the growth of transplanted hepatocytes are very important.

      Methods

      Hepatocyte transplantation was performed in Fah-/- mice to explore the mechanisms of hepatocyte proliferation in vivo. Guided by the in vivo regeneration mechanisms, compounds were identified to promote hepatocyte proliferation in vitro. The in vivo effects of these compounds on transplanted hepatocytes were then evaluated.

      Results

      The transplanted mature hepatocytes were found to dedifferentiate into hepatic progenitor cells (HPCs), which proliferate and then convert back to mature state at the completion of liver repopulation. The combination of two small molecules Y-27632 (Y, Rock inhibitor) and CHIR99021 (C, Wnt agonist) could convert mouse primary hepatocytes into HPCs, which could be passaged for more than 30 passages in vitro. Moreover, YC could stimulate the proliferation of transplanted hepatocytes in Fah-/- liver by promoting the conversion into HPCs. Netarsudil (N) and LY2090314 (L), two clinically used drugs which target the same pathways as YC, could also promote hepatocyte proliferation in vitro and in vivo, by facilitating HPC conversion.

      Conclusions

      Our work suggests drugs promoting hepatocyte dedifferentiation may facilitate the growth of transplanted hepatocytes in vivo and may facilitate the application of hepatocyte therapy.

      Lay summary

      Hepatocyte transplantation may provide treatment option for patients with end-stage liver disease. However, one important obstacle to hepatocyte therapy is the low level of engraftment and proliferation of the transplanted hepatocytes. Here we demonstrate that the transplanted hepatocytes dedifferentiate into HPCs, which proliferate and re-differentiate after repopulation. Small molecule compounds which promote hepatocyte proliferation in vitro by facilitating dedifferentiation, could promote the growth of transplanted hepatocytes in vivo and may facilitate the application of hepatocyte therapy.

      Graphical abstract

      Keywords

      ABBREVIATIONS:

      Fah (Fumarylacetoacetate Hydrolase), AFP (Alpha fetoprotein), Alb (Albumin), td (tdTomato), Sox9 (SRY-Related HMG Box Gene 9), Cdc20 (Cell Division Cycle 20), Dlk1 (Delta Like Non-Canonical Notch Ligand 1), Cdk1 (Cyclin Dependent Kinase 1), Cd133 (Prominin-1), Fn14 (Fibroblast growth factor-inducible 14), Cxcr4 (C-X-C chemokine receptor 4), Gata4 (GATA Binding Protein 4), Ck19 (Cytokeratin19), EPCAM (Epithelial Cell Adhesion Molecule), HNF4α (Hepatocyte Nuclear Factor 4 Alpha), PAS (Periodic Acid-Schiff), EGF (Epidermal Growth Factor), HGF (Hepatocyte Growth Factor), DEX (Dexamethasone), ITS (Insulin-Transferrin-Selenium), OSM (Oncostatin M), NTBC (2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclo-hexanedione), ALT (Alanine transaminase), AST (Aspartate aminotransferase), ALP (Alkaline Phosphatase), TBIL (Total bilirubin), Iso (Isolated), HCM (Hepatocyte culture medium), HMM (Hepatic maturation medium), (i)HPCs ((induced) Hepatic progenitor cells), iMHs (Induced mature hepatocytes), Y (Y27632), C (CHIR99021), A (A-83-01), N (Netarsudil), L (LY2090314)

      Chemicals promote hepatocyte growth in vivoConflict of interest

      The authors declare no competing interests.

      Financial support

      This work was supported by grants from the Chinese Academy of Sciences (XDA16021308, XDA16010202), National Key R&D Program of China (No. 2022YFA1104701), the National Natural Science Foundation of China (82121005, 32000504, 81730099), the China Postdoctoral Science Foundation (2018M642117).

      Authors’ contributions

      J.M. and R.G. conducted most of the experiments, analyzed the results, and wrote the paper; Y.A. G.W. P.T. X.J. B. H. and Q.Y. provided technical assistance with animal studies; X.X. conceived the idea for the project, supervise the study, analyzed the results and wrote the paper. All authors reviewed the results and approved the final version of the manuscript.

      Data availability statement

      All data files are available upon request.

      Introduction

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      . To study the mechanism of hepatocyte proliferation and finding ways to promote hepatocyte growth after transplantation may help to remove some of these obstacles.
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      . However, these studies didn’t explore whether hepatocytes dedifferentiate into HPCs before proliferation. Other studies have found that in chronic liver injury, hepatocytes can dedifferentiate into HPC state to restore the liver mass. In diet-induced chronic liver injury, hepatocytes have been shown to convert to cholangiocyte-like cells (Sox9+EpCAM+ cells) and supply new hepatocytes to repair damaged tissues
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      . Another study utilized hepatocyte-chimeric mice showed that bipotential HPCs were derived from chronically injured mature hepatocytes and could revert back to hepatocytes
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      Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes.
      . Present evidences suggest that HPCs may emerge from hepatocytes via dedifferentiation upon liver injury and contribute to liver regeneration. However, whether such process also exist in the repopulation after hepatocytes transplantation remain unclear.
      Mature hepatocytes were difficult to culture or grow in vitro, while cells with HPC characteristics, such as Lgr5+ hepatocytes from mice liver
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      or Epcam+ hepatocytes from human liver
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      Long-term culture of genome-stable bipotent stem cells from adult human liver.
      could be expanded in vitro. Recently, several studies have shown that small molecule chemicals or cytokines could convert mature hepatocytes into HPCs which can proliferate in vitro. In 2017, the combination of small molecules A-83-01 (inhibitor of TGF-β signaling), Y27632 (inhibitor of ROCK kinase) and CHIR99021 (agonist of WNT signaling) has been used to grow mice hepatocytes in vitro by inducing hepatocyte dedifferentiation
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      . Immediately after, another combination of A-83-01, Y27632, CHIR99021, S1P and LPA have been found to induce mice hepatocytes to HPCs conversion and growth in vitro
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      . One year later, a combination of Wnt3a, A-83-01 and Y27632 has been found to support human hepatocytes proliferation via dedifferentiation to HPCs
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      . Then, a study reported that A-83-01 and CHIR99021 were enough support the dedifferentiation and proliferation of human primary hepatocytes
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      . It seems that inducing hepatocytes dedifferentiation into HPCs is a common mechanism to promote their growth in vitro. We wonder whether the conditions used to facilitate hepatocyte to HPC conversion in vitro can stimulate hepatocyte growth in vivo after transplantation.
      Here, we report that HPC-dependent regeneration occurs in Fah-/- mice receiving hepatocyte transplantation. We also identify small molecules which promote hepatocyte dedifferentiation in vitro can facilitate the growth of transplanted hepatocytes in vivo.

      Results

      Hepatocytes are reprogrammed to a hepatic progenitor state during repopulation in vivo

      To trace the hepatocytes after transplantation in vivo, tdTomato+-hepatocytes (td-Hepa) were isolated from mice obtained by crossing R26RtdTomato mice with the Albumin-Cre mice
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      (Figs. S1A and B). Fah-/- mice require 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione (NTBC) supply for survival
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      Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I (vol 12, pg 266, 1996).
      . This is an ideal model to study the proliferation and function of transplanted hepatocytes in vivo
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      • Wakefield L.
      • Wilson E.M.
      • et al.
      Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes.
      . Therefore, after NTBC withdrawal, td-Hepa were transplanted into Fah-/- mice via intra-splenic injection through a left-flank incision under tribromoethanol anesthesia (Fig. 1A). The body weight of these animals kept dropping in the first three weeks, then gradually recover (Fig. 1B). Liver samples of Fah-/- mice receiving td-Hepa were collected at different time points (day 4, 7, 14, 30, 60, 90 and 120). Comparing to the livers of Fah-/- mice before NTBC withdrawal, the cell cycle genes (CyclinB1, Cdc20 and Cdk1) and HPC genes (Afp, Sox9, Dlk1, Cd133 and Fn14) in the livers of Fah-/- mice receiving td-Hepa were upregulated from day 4, reached a peak around day 30 and then decreased to normal (Fig. 1C). The td-Hepa showed clonal expansion at D30 and almost fully occupied the liver at D120 (Fig. 1D and Fig. S1C). It is not surprising that only the td-Hepa were positive for Fah staining (Figs. S1C and D). Interestingly, only the repopulating (D30) td-Hepa in Fah-/- mice were positively for AFP staining but not the repopulated (D120) td-Hepa (Fig. 1D). These results suggest that the transplanted hepatocytes dedifferentiate to HPC stage to proliferate and then redifferentiate after repopulation.
      Figure thumbnail gr1
      Fig. 1Hepatocytes were reprogrammed to a progenitor state after transplantation (A) A schematic view of Fah-/- mice transplanted with td-Hepa. (B) Body weight change of Fah-/- mice transplanted with td-Hepa for 120 days (n=3). (C) Quantitative RT-PCR analysis of cell cycle genes (CyclinB1, Cdc20 and Cdk1) and hepatic progenitor genes (Afp, Sox9, Dlk1, Cd133 and Fn14) in liver samples of Fah-/- mice transplanted with td-Hepa for 120 days (n=3). (D) Immunofluorescence staining of AFP in frozen liver sections of Fah-/- mice before transplantation (Fah-/- (D0)), or 30 and 120 days after td-Hepa transplantation (Fah-/--td-Hepa (D30) and (D120). The liver of Alb-td mice was used as control. (E) Statistical analysis of AFP staining in (D). All data are means ± SEM. ***p < 0.001 (Student’s t test).

      Reprogram of hepatocytes into expandable HPCs by Y27632 and CHIR99021

      Previous study has shown that a cocktail of small molecules A-83-01 (inhibitor of TGF-β signaling), Y27632 (inhibitor of ROCK kinase) and CHIR99021 (agonist of Wnt signaling) (AYC) can convert rat and mouse hepatocytes into HPCs with high proliferative capacity
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      • Yamamoto Y.
      • et al.
      Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity.
      . We wonder whether the combination could be simplified to facilitate in vivo application. To optimize the chemicals, we cultured hepatocytes in the classical hepatocyte culture medium (HCM, containing epidermal growth factor (EGF) and hepatocyte growth factor (HGF)) supplemented with AYC or any two of the three chemicals (YC, AY and AC). Consist with the general notion
      • Katsuda T.
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      Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity.
      ,
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      Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors.
      , no proliferation of hepatocytes was observed in vehicle group (DMSO) at day 14 (Fig. 2A and B). As expected, AYC stimulated significant proliferation as the cell number increased about 20 times, which was similar as reported
      • Katsuda T.
      • Kawamata M.
      • Hagiwara K.
      • Takahashi R.U.
      • Yamamoto Y.
      • et al.
      Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity.
      (Fig. 2A and B). Interestingly, YC was found to induce similar speed of hepatocyte growth as AYC, while AY or AC could only promote moderate proliferation (Fig. 2A and B). The proliferating cells in YC and AYC groups at D14 shown typical epithelial morphology with a high nucleus/cytoplasm ratio, which is a typical feature of HPCs
      • Katsuda T.
      • Kawamata M.
      • Hagiwara K.
      • Takahashi R.U.
      • Yamamoto Y.
      • et al.
      Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity.
      (Fig. 2A). Immunofluorescence staining revealed that the proliferating cells in YC group expressed the highest level of AFP and Dlk1, which was also expressed in AYC group, but absent in fresh isolated hepatocytes, or cells cultured for 14 days in other conditions (Fig. 2C and D, Figs. S2A and B). Quantitative RT-PCR analysis also confirmed more cell cycle genes and HPC genes were upregulated in YC-iHPCs (D14) (Figs. S2C and D). Comparing to hepatocytes, the proliferating cells in YC group were also highly positive for cell cycle marker Ki67, but the marker for mature hepatocytes Albumin was significantly reduced (Fig. 2E and F). So, these cells were named YC-induced HPCs (YC-iHPCs). Moreover, YC-iHPCs (D14) could be differentiated into mature hepatocytes with a widely used hepatic maturation medium (HMM)
      • Katsuda T.
      • Kawamata M.
      • Hagiwara K.
      • Takahashi R.U.
      • Yamamoto Y.
      • et al.
      Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity.
      with minor modifications. After culturing YC-iHPCs (D14) in HMM for 7 days, YC-iHPCs transformed into cells with typical mature hepatocyte morphology (Figs. S2E and F), which were named YC-induced mature hepatocytes (YC-iMHs). The YC-iMHs were negative for AFP and Ki67, but the Albumin was significantly increased (Fig. 2E and F). Quantitative RT-PCR analysis also confirmed more HPCs genes and cell cycle genes were upregulated in YC-iHPCs (D14) but greatly reduced in YC-iMHs (D21) (Fig. S2G), while the genes related to mature hepatocyte functions were downregulated in YC-iHPCs (D14) but then upregulated in YC-iMHs (D21) (Fig. S2H). To test whether YC could support long-term culture, YC-iHPCs (D14) were passaged every 5 to 7 days (∼90% confluence) in YC-HCM. YC-iHPCs could be passaged for more than 30 times and the cumulative cell number increased from 1 x 105 to about 1 x 1040 in ∼150 days without any apparent morphological changes (Fig. 2G and Fig. S2I). YC-iHPCs at P10 and P30 expressed high levels of the HPC markers Sox9 and Ck19 and cell cycle markers CyclinD1 and Ki67 (Figs. S2J and K). Importantly, YC-iHPCs at P30 could be differentiated into YC-iMHs which were highly positive for PAS staining and expressed high levels of Albumin and HNF4α (Fig. 2H). The strategy for using YC to promote hepatocyte proliferation in vitro was summarized in Fig. 2I. Taken together, the combination of two chemicals YC are enough to reprogram mature hepatocytes into expandable HPCs in vitro, and these cells could be differentiated into more mature hepatocyte-like cells.
      Figure thumbnail gr2
      Fig. 2Reprogram of hepatocytes into expandable HPCs by Y27632 and CHIR99021 (A, B) Representative morphology (A) and growth curves (B) of primary hepatocytes (D0) cultured in various combinations of A83-01 (A), Y27632 (Y) and CHIR99021 (C) for 14 days (D14) (n=4). ***p < 0.001 (two-way ANOVA). (C) Immunofluorescence staining of AFP in hepatocytes cultured in various combinations of A, Y and C for 14 days. (D) Statistical data of the immunofluorescence staining data in (C) (eight random fields for each group). (E) Immunofluorescence staining of AFP, Ki67 and Albumin in Hepa (D0), YC-iHPCs (D14) and YC-iMHs (D21). (F) Statistical data of AFP, Ki67 and Albumin in (E) (eight random fields for each group). (G) Calculated cumulative cell numbers of YC-iHPCs for 30 passages. (H) Representative images of morphology, PAS staining, and immunofluorescence staining of Albumin and Hnf4α in YC-iMHs (P30). (I) A schematic view for hepatocytes expansion using chemical cocktail YC. Nuclei were stained with Hoechst 33342. Scale bars represent 100 μm. All data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 (Student’s t test unless specified otherwise).

      Combination of Y27632 and CHIR99021 promotes hepatocyte proliferation in vivo

      Next, we sought to evaluate whether YC could promote hepatocyte proliferation in vivo. According to the concentration of Y27632 (10 μM) and CHIR99021 (3 μM) used in vitro, Y27632 (10 mg/kg) and CHIR99021 (14 mg/kg) were tested in Fah-/- mice by oral administration (Fig. 3A). After NTBC withdrawal, all Fah-/- mice without transplantation and giving vehicle (Sham-Vehicle) or YC (Sham-YC) showed continuous body weight loss (Fig. 3C) and died in about 25 days (Fig 3B). In contrast, Fah-/- mice receiving td-Hepa and then treated with vehicle (td-Hepa-Vehicle) showed a significantly less body weight loss (Fig. 3C), and 16 out of 22 Fah-/- mice survived for more than 1 month (Fig. 3B). Fah-/- mice receiving td-Hepa and YC-treatment (td-Hepa-YC) showed even less body weight loss (Fig. 3C), and 24 out of 25 Fah-/- mice survived for more than 1 month, the survival rate was significantly improved comparing to td-Hepa-Vehicle group (Fig. 3B). The surviving animals were sacrificed at Day 28 or D56 for analysis (Fig. 3A and B). The highly increased serum levels of AST, ALT, ALP and total bilirubin (TBIL) due to NTBC withdrawal were significantly reduced in td-Hepa-Vehicle group (Fig. 3D), and were further reduced in td-Hepa-YC group (Fig. 3D) at D28. The repopulated td-Hepa in YC-treated mice were significantly more abundant than in the vehicle group (Fig. 3E and 3F) at D28. The repopulated td-Hepa at D28 were almost 100% positive for Fah staining in both groups (Fig 3G and H) at D28. However, the td-Hepa in YC-treated animals expressed higher level of the HPCs markers AFP, Dlk1 and Ck19 than that in vehicle-treated mice (Fig. 3G and H) at D28. Quantitative RT-PCR confirmed that the expression of cell cycle genes (CyclinB1, Cdc20 and Cdk1) and HPCs genes (Afp, Cd133 and Dlk1) were gradually increased in livers after transplantation (Fig. 3I), and YC-treatment further upregulated these genes (Fig. 3I). The repopulated td-Hepa were isolated by FACS at D28 after transplantation, more than 90% of the cells were tdTomato positively (Fig. 3J and 3K). Quantitative RT-PCR confirmed that the cell cycle genes (Cdc20, CyclinB1) and HPCs genes (Cxcr4, Sox9, Gata4, Dlk1, Afp, Ck19, Cd24, Cd34, Cd44 and Cd133) were significantly upregulated in YC-treated group (Fig. 3L).
      Figure thumbnail gr3
      Fig. 3Y27632 and CHIR99021 promote proliferation of transplanted hepatocytes in vivo (A) A schematic view of YC treatment in Fah-/- mice transplanted with td-Hepa. (B, C) Survival curves (B) and body weight change (C) of Fah-/- mice receiving vehicle (Sham-Vehicle, starting n=12) or YC (Sham-YC, starting n=8), or Fah-/- mice transplanted with td-Hepa and then treated with vehicle (td-Hepa-Vehicle, starting n=22) or YC (td-Hepa-YC, starting n=25) for 56 days after NTBC withdrawal. Most of the surviving mice were sacrificed at Day 28 for analysis. Three of the surviving mice in vehicle group continued to receive vehicle till Day 56. Three of the mice in YC group continued to receive YC till Day56, and another three in YC group started to receive vehicle from D28-D56. Statistical differences between two groups were analyzed from D0-D21. *p < 0.05, **p< 0.01, ***p< 0.001 versus td-Hepa-Vehicle group (Log-rank test for B and two-way ANOVA test for C). (D) Serum levels of ALT, AST, ALP and TBIL in the Fah-/- mice after transplantation, animal numbers were the same as the survived mice in (C), Fah-/- mice before NTBC withdrawal (Fah-/- (D0)) were used as controls. (E) Representative tdTomato images of the whole liver (left) and frozen sections of liver (right) of td-Hepa-Vehicle and td-Hepa-YC mice (D28), scale bar represents 1 mm. (F) Quantitative analysis of tdTomato positive areas in (E) (td-Hepa-Vehicle group, n=8, td-Hepa-YC group, n=13). (G) Immunofluorescence staining of Fah and progenitor markers (AFP, Dlk1 and Ck19) in frozen liver sections of td-Hepa-Vehicle and td-Hepa-YC mice (D28). (H) Statistical data of the intensity of Fah, AFP, Dlk1 and Ck19 staining in (G) (n=3). (I) Quantitative RT-PCR analysis of cell cycle genes (CyclinB1, Cdc20, Cdk1) and hepatic progenitor genes (Afp, Cd133 and Dlk1) in livers of td-Hepa transplanted Fah-/- mice treated with vehicle or YC at day 7, 14 and 28 (n=3). Fah-/- before NTBC withdrawal (D0) was used as control. (J) Representative morphology and fluorescence images of td-Hepa isolated (by FACS) from td-Hepa-Vehicle and td-Hepa-YC mice (D28). (K) Statistical data of the positive of tdTomato+ hepatocytes in (J) (n=3). (L) Quantitative RT-PCR analysis of cell cycle genes (Cdc20 and CyclinB1) and hepatic progenitor genes (Cxcr4, Sox9, Gata4, Dlk1, Afp, Ck19, Cd24, Cd34, Cd44, and Cd133) in td-Hepa isolated from td-Hepa-Vehicle and td-Hepa-YC mice (D28) (n=3). Primary td-Hepatocytes were used as controls. Nuclei were stained with Hoechst 33342. Scale bars represent 100 μm. All data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 (Student’s t test unless specified otherwise).
      To further confirm that YC stimulated growth of transplanted hepatocytes via dedifferentiation, the global gene expression profiles in the livers were compared. Gene set enrichment analysis (GSEA) showed there was a clear enrichment of genes related to cell cycle regulation, stem cell proliferation and somatic stem cell population maintenance in td-Hepa-YC group comparing to td-Hepa-Vehicle group at D28 (Fig. S3A). Transcriptomic comparison was also carried out among Fah-/- (D0, before NTBC removal), td-Hepa-Vehicle and td-Hepa-YC groups at D28 (Fig. S3B). Genes related to cell cycle regulation, stem cell proliferation and somatic stem cell population maintenance were upregulated in livers receiving td-Hepa transplantation, and even higher in YC-treated group (Fig. S3B).

      Combination of Y27632 and CHIR99021 does not affect hepatocyte engraftment and maturation

      At D56, the surviving transplanted mice had been treated with vehicle or YC for 56 days, or treated with YC for 28 days and then treated with vehicle for another 28 days (Fig. 3A). The livers from all three groups at Day 56 were as normal as the Alb-td-mice (Fig. S4A), td-Hepa repopulated more than 95% of the liver in these animals (Fig. S4A). The highly increased serum levels of AST, ALT, ALP and total bilirubin after NTBC withdrawal were almost returned to normal after transplantation (Fig. S4B). The repopulated td-Hepa in Fah-/- mice were almost 100% positive for Fah, Albumin and Cyp1a2 staining (Figs. S5A–C). The repopulated td-Hepa expressed pericentral marker Cyp2e1 only in the pericentral region (Fig. S5D), in consistent with previous studies
      • Halpern K.B.
      • Shenhav R.
      • Matcovitch-Natan O.
      • Toth B.
      • Lemze D.
      • et al.
      Single-cell spatial reconstruction reveals global division of labour in the mammalian liver.
      . And at D56, the transplanted td-Hepa in Fah-/- mice were negative for HPC markers AFP and Dlk1 staining (Figs. S5E and F).
      Successful hepatocyte transplantation requires efficient engraftment (typically within 3 days after transplantation) and then repopulation
      • Wang C.
      • Zhang L.
      • Sun Z.
      • Yuan X.
      • Wu B.
      • et al.
      Dedifferentiation-associated inflammatory factors of long-term expanded human hepatocytes exacerbate their elimination by macrophages during liver engraftment.
      . A previous study
      • Wang C.
      • Zhang L.
      • Sun Z.
      • Yuan X.
      • Wu B.
      • et al.
      Dedifferentiation-associated inflammatory factors of long-term expanded human hepatocytes exacerbate their elimination by macrophages during liver engraftment.
      has demonstrated that blocking macrophage-mediated elimination of transplanted hepatocytes facilitate engraftment and later-on repopulation. We also accessed whether YC treatment affect hepatocyte engraftment. Fah-/- mice receiving td-Hepa were treated with YC or Vehicle for 3 days and the engraftment of td-Hepa did not show a significant difference between YC and vehicle groups (Figs. S6A and S6B). Taken together, these results indicate that YC treatment does not affect the engraftment stage and the maturation after repopulation, but only affect the proliferation stage.

      Netarsudil and LY2090314 promote hepatocyte proliferation in vitro and repopulation in vivo

      Y27632 and CHIR99021 haven’t been tested in clinical studies. For possible future clinical application, we’d like to find a combination with available drugs. Netarsudil (N) is also a ROCK inhibitor which is used to treat open-angle glaucoma in clinic
      • Batra M.
      • Gupta S.
      • Nair A.B.
      • Dhanawat M.
      • Sandal S.
      • et al.
      Netarsudil: A new ophthalmic drug in the treatment of chronic primary open angle glaucoma and ocular hypertension.
      , and LY2090314 (L) is a WNT agonist which has been tested in phase 2 clinical trials to treat Leukemia
      • Rizzieri D.A.
      • Cooley S.
      • Odenike O.
      • Moonan L.
      • Chow K.H.
      • et al.
      An open-label phase 2 study of glycogen synthase kinase-3 inhibitor LY2090314 in patients with acute leukemia.
      . So, we tested the combination of different concentration of N (0 to 0.1 μM) and L (0 to 3 μM) in hepatocytes culture (Fig. S7A), and the combination of N at 0.1 μM and L at 1 μM yielded best result (Figs. S7B and C), so this NL combination was used for further studies. The primary hepatocytes cultured in NL grow rapidly (Fig. 4A and 4B). The proliferating cells at D14 showed similar morphology as YC-iHPCs and expressed high levels of cell cycle genes (Cdc20, CyclinB1, Cdk1, Pcna and Bcl2) and HPCs genes (Afp, Ck19, Epcam, Gata4, Cd34, Cd44, Cd133), so these cells were named as NL-iHPCs (Fig. 4C). After culturing in HMM for 7 days, the NL-iHPCs could be differentiated into mature hepatocytes (NL-iMHs, Fig. 4D). Immunofluorescence staining revealed that NL-iMHs were highly positive for HNF4α and Albumin at levels comparable to primary hepatocytes (Fig. 4E and 4F). Corresponding doses of N (0.3 mg/kg) and L (3 mg/kg) were then tested in Fah-/- mice by oral administration in the way same as YC (Fig. 3A), but all sacrificed at D28. Similar to YC-treatment, Fah-/- mice receiving td-Hepa and then treated with NL (td-Hepa-NL) showed significantly less body weight loss and no animal death comparing to td-Hepa-Vehicle group, which already showed significant therapeutic effect comparing to Sham-Vehicle group (Fig. 4G). The AST, ALT, ALP and TBIL were further reduced by the treatment of NL in mice receiving td-Hepa transplantation (Fig. 4H), and the repopulated td-Hepa in NL-treated mice were significantly more abundant than in vehicle group (Fig. 4I and 4J). Immunofluorescence staining revealed that the repopulated td-Hepa at D28 were almost 100% positive for Fah staining, and the levels were similar in both NL- and vehicle-treated groups (Fig. 4K and 4L), but the AFP level was significantly higher in repopulated td-Hepa in the NL-treated animals (Fig. 4K and 4L). Taken together, these results indicate that the drug combination NL could enhance the repopulation of hepatocytes in vivo by promoting the reprogramming of hepatocytes into HPCs.
      Figure thumbnail gr4
      Fig. 4Netarsudil and LY2090314 promote hepatocyte expansion in vitro and repopulation in vivo (A, B) Representative images (A) and growth curves (B) of primary hepatocytes cultured with Netarsudil (N, 0.1 μM) and LY2090314 (L, 1 μM) for 0-14 days. (n=3), ***p<0.001 (two-way ANOVA). (C) Quantitative RT-PCR analysis of cell cycle genes (Cdc20, CyclinB1, Cdk1, Pcna, and Bcl2) and hepatic progenitor genes (Afp, Ck19, Epcam, Gata4, Cd34, Cd44 and Cd133) in NL-iHPCs (D14) (n=3). Primary td-Hepatocytes were used as controls (D) Representative phase contrast images of NL-iMHs (D21). (E, F) Immunofluorescence staining (E) and statistical data (F) of HNF4a and Albumin in Hepa (D0) and NL-iMHs (D21) (eight random fields for each group). (G) Body weight change (top) and survival curves (down) of Fah-/- mice without transplantation (n=6), or receiving td-Hepa transplantation and then treated with vehicle (td-Hepa-Vehicle, n=21) or NL (td-Hepa-NL, n=20) for 28 days after NTBC withdrawal. Statistical differences between two groups were analyzed from D0-D21 for body weight change, *p < 0.05 versus td-Hepa-vehicle group (two-way ANOVA for body weight change, Log-rank test for survival curves). (H) Serum levels of ALT, AST, ALP and TBIL in the Fah-/- mice without transplantation (Sham-Vehicle, n=4), or Fah-/- mice transplanted with td-Hepa and then treated with vehicle (td-Hepa-Vehicle, n=14) or NL (td-Hepa-NL, n=20). Fah-/- mice before NTBC withdrawal (Fah-/- (D0), n=4) were used as controls. (I) Representative tdTomato images of the whole liver (left) and frozen sections of liver (right) of td-Hepa transplanted Fah-/- mice receiving vehicle or NL (D28), scale bar represents 1 mm. (J) Quantitative analysis of tdTomato positive areas in (I), (td-Hepa-Vehicle group, n=5, td-Hepa-NL group, n=8). (K) Immunofluorescence staining of Fah and AFP in frozen liver sections of td-Hepa transplanted Fah-/- mice receiving vehicle or NL (D28). (L) Statistical data of the intensity of Fah and AFP staining in (K). Nuclei were stained with Hoechst 33342. Scale bars represent 100 μm. All data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 (Student’s t test unless specified otherwise).

      Discussion

      Here we demonstrate that the transplanted hepatocytes undergo dedifferentiation to HPCs and then convert back to mature state after repopulation. Small molecules that can induce hepatocyte to HPC conversion in vitro can be used to stimulate the same process in vivo after hepatocyte transplantation and facilitate the growth and repopulation. It is also possible that same strategy can be used to stimulate in situ liver regeneration in various types of liver injury if hepatocyte dedifferentiation is the major route of regeneration.
      However, whether the regeneration of liver after various types of injury requires the hepatocyte-HPC-hepatocyte conversion remains to be debated. In partial hepatectomy (PHx), a hepatocyte fate-tracing study has showed that about 98% newly formed hepatocytes were derived from preexisting hepatocytes, and the remaining small fraction might be derived from preexisting HPCs
      • Malato Y.
      • Naqvi S.
      • Schurmann N.
      • Ng R.
      • Wang B.
      • et al.
      Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration.
      . A recent study using single-cell RNA-seq and ATAC-Seq showed that after PHx, some hepatocytes acquired chromatin landscapes and transcriptomes similar to fetal hepatocytes, suggesting dedifferentiation to HPCs
      • Chen T.Y.
      • Oh S.
      • Gregory S.
      • Shen X.L.
      • Diehl A.M.
      Single-cell omics analysis reveals functional diversification of hepatocytes during liver regeneration.
      . Liver regeneration in CCl4-induced acute or chronic injury have been shown to attribute to the proliferation of mature hepatocytes
      • Espanol-Suner R.
      • Carpentier R.
      • Van Hul N.
      • Legry V.
      • Achouri Y.
      • et al.
      Liver progenitor cells yield functional hepatocytes in response to chronic liver injury in mice.
      . In another toxin-induced liver injury study, genetic labeling suggested that hepatocytes were the source for hepatocyte renewal and regeneration
      • Yanger K.
      • Knigin D.
      • Zong Y.W.
      • Maggs L.
      • Gu G.Q.
      • et al.
      Adult Hepatocytes Are Generated by Self-Duplication Rather than Stem Cell Differentiation.
      , although whether these hepatocytes pass through a HPC stage remain unclear. Another study with DDC diet-induced liver injury indicated that mature hepatocytes may convert into cholangiocytes-like cells, which serve as hepatic progenitors with the ability of clonal proliferation and then differentiate into functional hepatocytes
      • Tanimizu N.
      • Nishikawa Y.
      • Ichinohe N.
      • Akiyama H.
      • Mitaka T.
      Sry HMG box protein 9-positive (Sox9+) epithelial cell adhesion molecule-negative (EpCAM-) biphenotypic cells derived from hepatocytes are involved in mouse liver regeneration.
      . Another study also reported that bipotential adult liver progenitors are derived from chronically injured mature hepatocytes
      • Tarlow B.D.
      • Pelz C.
      • Naugler W.E.
      • Wakefield L.
      • Wilson E.M.
      • et al.
      Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes.
      . It seems hepatocyte to HPC conversion is a rather common phenomenon after liver injury and may facilitate regeneration. In our transplantation model, it was very clear that HPC markers gradually increased and peaked at day 30, and at that time point, most of the transplanted cells expressed AFP, indicating the conversion of hepatocytes to HPCs, accompanying cell growth. So, promoting hepatocyte dedifferentiation may also facilitate the regeneration of liver from other injuries.
      A number of pathways have been proposed to drive hepatocyte to HPC dedifferentiation in vivo, including Notch, WNT and YAP signaling pathways. One study using mice with a liver-specific deletion of RBP-Jκ (an essential component of the canonical Notch pathway) has demonstrated that Notch signaling is required for hepatocyte reprogramming under DDC diet-induced liver injury
      • Yanger K.
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      • Shapira S.N.
      • Maddipati R.
      • et al.
      Robust cellular reprogramming occurs spontaneously during liver regeneration.
      . Hes1, a target gene of RBP-Jκ, is also important for the conversion of hepatocytes into primitive ductular cells in DDC-treated chronically injured liver
      • Sekiya S.
      • Suzuki A.
      Hepatocytes, Rather than Cholangiocytes, Can Be the Major Source of Primitive Ductules in the Chronically Injured Mouse Liver.
      . β-catenin conditional knockout has been shown to reduce the number of HPCs in DDC diet-induced liver injury, demonstrating that the WNT/β-catenin pathway plays a key role in the proliferation of HPCs
      • Apte U.
      • Thompson M.D.
      • Cui S.
      • Liu B.
      • Cieply B.
      • et al.
      Wnt/beta-catenin signaling mediates oval cell response in rodents.
      . Similarly, β-catenin overexpression results in higher number of proliferating HPCs in DDC diet–induced liver injury
      • Thompson M.D.
      • Awuah P.
      • Singh S.
      • Monga S.P.
      Disparate cellular basis of improved liver repair in beta-catenin-overexpressing mice after long-term exposure to 3,5-diethoxycarbonyl-1,4-dihydrocollidine.
      . YAP-driven transcriptional program has also been reported to be crucial for the process of liver regeneration after DDC injury and specifically for the reprogramming of hepatocytes towards a progenitor, biliary-like fate
      • Pepe-Mooney B.J.
      • Dill M.T.
      • Alemany A.
      • Ordovas-Montanes J.
      • Matsushita Y.
      • et al.
      Single-Cell Analysis of the Liver Epithelium Reveals Dynamic Heterogeneity and an Essential Role for YAP in Homeostasis and Regeneration.
      . Overexpression of active YAP in hepatocytes may drive reprogramming via Notch2 transcriptional regulation, suggesting that YAP-Notch is indeed a crucial axis for this process
      • Yimlamai D.
      • Christodoulou C.
      • Galli G.G.
      • Yanger K.
      • Pepe-Mooney B.
      • et al.
      Hippo Pathway Activity Influences Liver Cell Fate.
      .
      Many of the compounds/supplements used to induce in vitro hepatocyte to HPC conversion also target these pathways. In our study, CHIR99021 and LY2090314 are inhibitors of the GSK-3β, which may lead to WNT pathway activation. Activation of WNT/β-catenin signaling has also been reported to promote hepatocyte proliferation and liver regeneration in PHx, by upregulating cell-cycle regulators
      • Russell J.O.
      • Monga S.P.
      Wnt/beta-Catenin Signaling in Liver Development, Homeostasis, and Pathobiology.
      . Y27632 and Netarsudil are Rho-associated kinase (ROCK) inhibitors. ROCK regulates cellular growth, adhesion, migration, metabolism, and apoptosis through control of the actin cytoskeletal assembly and cell contraction
      • Kurosawa H.
      Application of Rho-associated protein kinase (ROCK) inhibitor to human pluripotent stem cells.
      . There are limited reports on the direct roles of ROCK pathway in inducing hepatocyte to HPC dedifferentiation. However, ROCK inhibitors have now been recognized as useful tools to promote the survival of multiple types of stem cells
      • Centonze G.
      • Centonze S.
      • Ponzone L.
      • Calautti E.
      ROCK 'n TOR: An Outlook on Keratinocyte Stem Cell Expansion in Regenerative Medicine via Protein Kinase Inhibition.
      .
      Up till now, we have observed the Fah-/- mice receiving td-Hepa for 170 days (the first 56 days receiving YC treatment) and found the livers of these mice to be normal. But we believe the safety of such treatment should to be assessed in large-scale and long-term experiments in future. In conclusion, drugs/conditions promoting hepatocytes dedifferentiation may promote the growth of transplanted hepatocytes in vivo and may facilitate the application of hepatocyte therapy.

      Materials And Methods

      Mice

      All mice were housed under controlled humidity and temperature conditions and under 12-hour light/dark cycles. The care and the use of animals were complied with international guidelines and were approved by the Animal Ethics Committee of Shanghai Institute of Materia Media.

      Isolation of mice primary hepatocyte

      Primary hepatocytes were isolated by the classic two-step collagenase perfusion technique from mice (C57BL/6J) at the age of 8-10 weeks. R26RtdTomato mice (Jackson Laboratory) were crossed with Albumin-Cre mice and the offspring Albumin-Cre:R26RtdTomato (Alb-td) mice were used for the isolation of tdTomato+-hepatocytes. The liver was perfused through the inferior vena with 25 mL Perfusion buffer and then 25 mL Enzyme buffer. The hepatocytes were then released into the M199 medium (GIBCO) using sterile surgical scissors. Cell suspension was filtered through a 70 μm cell strainer (Corning). After then the hepatocytes were purified with 50% of percoll (Sigma) gradient at low-speed centrifugation (1,500 rpm, 15 min) then the pellets were dissociated into single cell suspension. Viability of isolated hepatocytes were about 90% as determined by Trypan blue staining. Details please see the Supplementary Materials and Methods.

      Hepatocyte transplantation and samples collection

      Fah-/- mice were fed with7.5 mg/L NTBC in drinking water, Fah-/- mice at the age of 8-12 weeks were used for transplantation. For transplantation, hepatocytes (2.5×106) were suspend in 200 uL 0.9% sodium chloride solution and transplanted into Fah-/- mice via intrasplenic injection through a left-flank incision under 1.25% tribromoethanol anesthesia. After the operation, NTBC was withdrawn from the drinking water. After transplantation, Fah-/- mice were treated with vehicle (0.5% Sodium carboxymethyl cellulose in PBS), YC (10 mg/kg Y27632 and 14 mg/kg CHIR99021, p.o.q.d) or NL (0.3 mg/kg Netarsudil and 3 mg/kg LY2090314, p.o.q.d). The blood and liver samples were collected at indicate time points. Total bilirubin, ALT, ALP, AST in serum were measured (Bioassay system kit).

      Statistical Analysis

      Values are reported as the means ± SEM. p-values were calculated with Student’s t-test, two-way ANOVA test or Log-rank test as indicated in figure legends, p < 0.05 was considered statistically significant. All graphs were plotted with GraphPad Prism software. The Immunofluorescence images were analyzed using ImageJ software.

      Appendix A. Supplementary data

      The following is/are the supplementary data to this article:

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