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Direct reprogramming of somatic cells into induced hepatocytes: Cracking the Enigma code

  • Matthias Rombaut
    Correspondence
    Corresponding authors. Address: Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium; Tel.: +32 (0)2 477 45 17.
    Affiliations
    Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium
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  • Joost Boeckmans
    Affiliations
    Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium
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  • Robim M. Rodrigues
    Affiliations
    Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium
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  • Leo A. van Grunsven
    Affiliations
    Liver Cell Biology Research Group, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium
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  • Tamara Vanhaecke
    Affiliations
    Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium
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  • Joery De Kock
    Correspondence
    Corresponding authors. Address: Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium; Tel.: +32 (0)2 477 45 17.
    Affiliations
    Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium
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Open AccessPublished:May 11, 2021DOI:https://doi.org/10.1016/j.jhep.2021.04.048

      Summary

      There is an unmet need for functional primary human hepatocytes to support the pharmaceutical and (bio)medical demand. The unique discovery, a decade ago, that somatic cells can be drawn out of their apparent biological lockdown to reacquire a pluripotent state has revealed a completely new avenue of possibilities for generating surrogate human hepatocytes. Since then, the number of papers reporting the direct conversion of somatic cells into induced hepatocytes (iHeps) has burgeoned. A hepatic cell fate can be established via the ectopic expression of native liver-enriched transcription factors in somatic cells, thereby bypassing the need for an intermediate (pluripotent) stem cell state. That said, understanding and eventually controlling the processes that give rise to functional iHeps remains challenging. In this review, we provide an overview of the state-of-the-art reprogramming cocktails and techniques, as well as their corresponding conversion efficiencies. Special attention is paid to the role of liver-enriched transcription factors as hepatogenic reprogramming tools and small molecules as facilitators of hepatic transdifferentiation. To conclude, we formulate recommendations to optimise, standardise and enrich the in vitro production of iHeps to reach clinical standards, and propose minimal criteria for their characterisation.

      Keywords

      Introduction

      Globally, end-stage liver disease is an important cause of death for which orthotopic liver transplantation is often the only curative treatment.
      • Iansante V.
      • Chandrashekran A.
      • Dhawan A.
      Cell-based liver therapies: past, present and future.
      The demand for donor livers therefore far exceeds the current supply. Alternatively, primary human hepatocytes (PHHs) can be transplanted, but difficulties in achieving effective ex vivo expansion while maintaining functionality, present a major obstacle to their large-scale application.
      • Zeilinger K.
      • Freyer N.
      • Damm G.
      • Seehofer D.
      • Knöspel F.
      Cell sources for in vitro human liver cell culture models.
      To overcome this hurdle, increasing efforts have been directed towards generating surrogate hepatocytes from other cell sources. The introduction of human induced pluripotent stem (hiPSC) technology by Takahashi & Yamanaka revolutionised the field of regenerative and personalised medicine, challenging the traditional view that a cell’s identity is sealed after undergoing development. Since this discovery, it is possible to reprogramme a patient’s somatic cells to a pluripotent stem cell-state with unlimited proliferative capacity.
      • Takahashi K.
      • Tanabe K.
      • Ohnuki M.
      • Narita M.
      • Ichisaka T.
      • Tomoda K.
      • et al.
      Induction of pluripotent stem cells from adult human fibroblasts by defined factors.
      Human hepatocyte-like cells have been derived from hiPSCs (hiPSC-HLCs) by mimicking liver development in vitro (extensively reviewed by Heslop and Duncan
      • Heslop J.A.
      • Duncan S.A.
      The use of human pluripotent stem cells for modeling liver development and disease.
      ). These hiPSC-HLCs are widely employed for in vitro drug testing and hepatotoxicity screening,
      • Davidson M.D.
      • Ware B.R.
      • Khetani S.R.
      Stem cell-derived liver cells for drug testing and disease modeling.
      ,
      • Sirenko O.
      • Cromwell E.F.
      Determination of hepatotoxicity in iPSC-derived hepatocytes by multiplexed high content assays.
      and could be used for liver cell therapy in the near future.
      • Iansante V.
      • Chandrashekran A.
      • Dhawan A.
      Cell-based liver therapies: past, present and future.
      In the spirit of changing the cell fate of somatic cells, in 2011, two research groups demonstrated that it is possible to draw mouse fibroblasts out of their biological lockdown and directly convert them to induced hepatocytes (iHeps). They achieved this by ectopic expression of defined liver-enriched transcription factors (LETFs), namely GATA4, hepatocyte nuclear factor (HNF) 1A, and forkhead box (FOX) A3,
      • Huang P.
      • He Z.
      • Ji S.
      • Sun H.
      • Xiang D.
      • Liu C.
      • et al.
      Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors.
      or HNF4A plus FOXA1, FOXA2 or FOXA3.
      • Sekiya S.
      • Suzuki A.
      Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors.
      It was only a few years later that hepatic transdifferentiation was established on a human level, albeit with a different hepatic reprogramming cocktail.
      • Kogiso T.
      • Nagahara H.
      • Otsuka M.
      • Shiratori K.
      • Dowdy S.F.
      Transdifferentiation of human fibroblasts into hepatocyte-like cells by defined transcriptional factors.
      By interacting with the transcriptional machinery, LETFs can gradually activate the silenced liver-specific gene expression programme, while silencing the somatic gene regulatory network. Furthermore, LETFs can interact with other transcription factors (TFs) and multiple co-activators or co-repressors, allowing for the formation of a platform for recruitment of a transcriptional complex, ultimately governing the tissue-restricted expression of liver-specific genes at specific stages of liver development and homeostasis (excellently reviewed by Lau et al.,
      • Lau H.H.
      • Ng N.H.J.
      • Loo L.S.W.
      • Jasmen J.B.
      • Teo A.K.K.
      The molecular functions of hepatocyte nuclear factors – in and beyond the liver.
      Schrem et al.
      • Schrem H.
      • Klempnauer J.
      • Borlak J.
      Liver-enriched transcription factors in liver function and development. Part I: the hepatocyte nuclear factor network and liver-specific gene expression.
      and Lemaigre
      • Lemaigre F.P.
      Mechanisms of liver development: concepts for understanding liver disorders and design of novel therapies.
      ), and orchestrating direct hepatic reprogramming.
      Human hepatocyte-based therapies provide a valid alternative to orthotopic liver transplantation for many life-threatening liver diseases, but are hampered by primary cell shortage, ineffective ex vivo expansion and immunogenicity of allogeneic donor cells.
      A decade after the introduction of direct hepatic reprogramming, we critically discuss the latest direct hepatic reprogramming cocktails and the minimal combination of LETFs required to convert somatic cells towards a hepatic cell fate. To conclude, we discuss recommendations to further optimise, standardise and enrich the in vitro production of human surrogate hepatocytes by direct hepatic reprogramming and propose minimal criteria for their characterisation.

      Protagonists of the direct hepatic reprogramming cocktail

       LETFs as key drivers for direct hepatic conversion

      During hepatic transdifferentiation the somatic identity is downregulated while the endogenous interconnected hepatic cross-regulatory network is gradually activated. The mechanisms underlying direct hepatic reprogramming were elucidated by Lim and colleagues.
      • Lim K.T.
      • Lee S.C.
      • Gao Y.
      • Kim K.P.
      • Song G.
      • An S.Y.
      • et al.
      Small molecules facilitate single factor-mediated hepatic reprogramming.
      They showed a sequential transition initiated by extinguishing the somatic fibroblast gene expression programme, followed by an activation of the mesenchymal-to-epithelial (MET) programme and the hepatic gene expression profile, respectively.
      • Lim K.T.
      • Lee S.C.
      • Gao Y.
      • Kim K.P.
      • Song G.
      • An S.Y.
      • et al.
      Small molecules facilitate single factor-mediated hepatic reprogramming.
      In Table 1, we provide an overview of the direct hepatic reprogramming cocktails, their corresponding conversion efficiency and kinetics, and a characterisation of the iHeps they generate. Studies are primarily divided by cell source of origin (mouse and human), and are reported in chronological order.
      Pioneer factors establish competence for lineage conversion by making epigenetic modifications that are required to start the liver programme. Therefore, they are pinpointed as indispensable for direct hepatic reprogramming cocktails.
      Table 1An overview of the state-of-the-art direct hepatic reprogramming cocktails and reprogramming techniques to generate iHeps, and their corresponding characterisation.
      Reprogramming cocktailCell typeGene transfer methodConversion efficiencyConversion kineticsIn vitro characterisationIn vivo characterisationOther tested TF(s) and combinationsRef.
      Genes involved in direct hepatic reprogrammingIn vitro functionalityMouse modelIn vivo functionality
      Mouse
      GATA4, HNF1A, FOXA3Mouse embryonic & adult fibroblastsLentiviralAlb+ (+/-23%)21 daysTJP1, CDH1, ALB, TTR, TF, AFP, CK19, HNF4A and CK18Glycogen storage, LDL uptake, CYP activity, ICG uptake (20%), albumin secretion, drug metabolism (phenacetin, testosterone and diclofenac)Fah-/-/Rag2-/- mouse5/12 survived 8 weeks, showed increased body weight and 5-80% Fah+ iHep engraftmentScreening by withdrawal (FOXA2, FOXA3, HNF1A, HNF4A, HNF6, GATA4 FOXA1, and HLF)
      • Huang P.
      • He Z.
      • Ji S.
      • Sun H.
      • Xiang D.
      • Liu C.
      • et al.
      Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors.
      HNF4A, FOXA1Mouse embryonic & adult fibroblastsRetroviralMorphological conversion to iHeps (0,3%), thereof +/-85% E-cad+ and Alb+n.s.MRP2, MRP4, ZO-1, ALB, AFP, COMT1, NAT2, MAOA, MAOB, TPMT, GS, GSTA4Glycogen storage (80%), LDL uptake, albumin secretion, urea production, triglycerides synthesis, CYP activity, ICG uptake, drug metabolismFah-/- mouse40% survived longer than 10 weeks, decrease in ALT, ALP and bilirubinScreening by withdrawal of 12 transcription factors
      • Sekiya S.
      • Suzuki A.
      Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors.
      HNF4A, FOXA2
      HNF4A, FOXA3
      HNF1B, FOXA3Mouse embryonic & adult fibroblastsLentiviral, followed by directed hepatic differentiationE-cad+ (0,4%)15 daysALB, TTR, HNF4A, CK8, CK18, CK19, AFP, SOX9, EPCAM, DLK1, PAN-CK, LGR5n.a.Fah-/- mouse;

      DDC-induced mouse
      4/11 survived 8 weeks, 11,6% repopulation, Alb expression, decrease in ALT, AST and bilirubin20 candidate factors
      • Yu B.
      • He Z.Y.
      • You P.
      • Han Q.W.
      • Xiang D.
      • Chen F.
      • et al.
      Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors.
      56,4%12 daysALB, HNF4A, SERPINA1, GJB1, CYP3A11, CYP7A1, G6PGlycogen storage, G6P activity, albumin secretionn.a.n.a.
      GATA4, HNF1A, FOXA3Mouse embryonic & adult fibroblastsoriP/EBNA1-based episomal systemE-cad+ (0,12%)30 daysAFP, ALB HNF1A, FOXA3, GATA4, CEBPA, HNF4A, TTR, CK8, CK18, CLDN2, CDH, APOA1, CYP39A1, ZO-1, CROT, and AKR1C13Glycogen storage (>70%), ICG uptake (>50%), LDL uptake, albumin secretion, urea production, upregulation

      CYPs after treatment with inducer (CYP1A1, CYP1A2, CYP2A5, CYP2D22 and CYP3A13)
      FRG mouseSurvival beyond 45 daysHNF4A, FOXA1; HNF4A, FOXA3
      • Kim J.
      • Kim K.P.
      • Lim K.T.
      • Lee S.C.
      • Yoon J.
      • Song G.
      • et al.
      Generation of integration-free induced hepatocyte-like cells from mouse fibroblasts.
      OCT4, SOX2, KLF4, MYC, HNF4A, CEBPA, NR1I2Mouse embryonic fibroblastsLentiviralThy1-/Alb+ (+/- 17%); Thy1-/CYP7A1+ (+/- 17%)25 daysALB, DLK1, KRT18, TAT and CYP7A1glycogen storage, LDL uptake, ICG uptake, albumin secretion, urea productionCCl4-injured mouseEngraftableScreening (GATA4, GATA6, FOXA1, FOXA2, FOXA3, HNF1A, HNF1B, HHEX, TBX3, PROX1, HNF4A, OC1, OC2, NR1I2, UPF1, CREB1, USF1, RXRA, CEBPA, CEBPB)
      • Pournasr B.
      • Asghari-Vostikolaee M.H.
      • Baharvand H.
      Transcription factor-mediated reprograming of fibroblasts to hepatocyte-like cells.
      MYC, KLF4, HNF4A, FOXA1Mouse embryonic fibroblastsRetroviraln.s.15 daysFOXA2, HNF4A,HNF1A, C-MET, TTR, ALB, CK18, CYP1A2, CDH, ZO-1, CYP1A1Urea production, albumin secretion, glycogen storage, LDL uptake, ICG uptake, accumulation lipidsCCl4-injured mouse; FRG mouseinfiltration of reactive HSCs and macrophages increased, fibrin clearance increased, AST and ALT decreased; Prolonged survivalKLF4, HNF4A, FOXA1; MYC, HNF4A, FOXA1; HNF4A, FOXA1; HNF1A; HNF4A
      • Lim K.T.
      • Lee S.C.
      • Gao Y.
      • Kim K.P.
      • Song G.
      • An S.Y.
      • et al.
      Small molecules facilitate single factor-mediated hepatic reprogramming.
      MYC, KLF4, HNF4A, FOXA3Mouse embryonic fibroblastsRetroviraln.s.15 daysFOXA2, HNF4A,HNF1A, C-MET, TTR, ALB, CK18, CYP1A2, CDH, ZO-1, CYP1A1, CYP3A44Urea production, albumin secretion, glycogen storage, LDL uptake, ICG uptake, accumulation lipidsCCl4-injured mouse; FRG mouseinfiltration of reactive HSCs and macrophages increased, fibrin clearance increased, AST and ALT decreased; Prolonged survivalHNF4A, FOXA3; HNF4A, FOXA3 + 1TF (CEBPA, DBP, FOXA2, GATA4, OC1)
      • Lim K.T.
      • Lee S.C.
      • Gao Y.
      • Kim K.P.
      • Song G.
      • An S.Y.
      • et al.
      Small molecules facilitate single factor-mediated hepatic reprogramming.
      ,
      • Park S.
      • In Hwang S.
      • Kim J.
      • Hwang S.
      • Kang S.
      • Yang S.
      • et al.
      The therapeutic potential of induced hepatocyte-like cells generated by direct reprogramming on hepatic fibrosis.
      HNF1A, A-83-01, CHIR99021, BMP-4Mouse embryonic fibroblastsRetroviralE-cad+ (26,2%); Alb+ (8,2%); Aat+ (5,4%)35 daysALB, SERPINA1, MRP2, MRP3, MAOB, MGST1, SULT1A1, CK18, ZO1Albumin secretion, CYP activity, LDL uptake, glycogen storage, ICG uptake, accumulation lipidsFRG mouseProlonged survivalHNF4A, A-83-01, CHIR99021; HNF1A, A-83-01, CHIR99021
      • Lim K.T.
      • Lee S.C.
      • Gao Y.
      • Kim K.P.
      • Song G.
      • An S.Y.
      • et al.
      Small molecules facilitate single factor-mediated hepatic reprogramming.
      ,
      • Park S.
      • In Hwang S.
      • Kim J.
      • Hwang S.
      • Kang S.
      • Yang S.
      • et al.
      The therapeutic potential of induced hepatocyte-like cells generated by direct reprogramming on hepatic fibrosis.
      HNF4A, FOXA3, KDM2BMouse embryonic fibroblastsLentiviraln.s.11 daysTTR, TAT, ASGR, G6P, GATA4, CDH1, ALB, CYP2B9, CYP3A4, HNF4A, LGR5, DLK1, EPCAMGlycogen storage, ICG uptake, albumin secretion, LDL uptake, CYP3A4 activityCCl4-injured mousesurvival rate: 100% iHeps vs. 80% fibroblasts, engraftable iHeps, albumin expressionHNF4A, FOXA3
      • Zakikhan K.
      • Pournasr B.
      • Nassiri-Asl M.
      • Baharvand H.
      Enhanced direct conversion of fibroblasts into hepatocyte-like cells by Kdm2b.
      FOXA3, GATA4, HNF1A, HNF4AIn vitro: myofibroblasts derived from primary mouse hepatic stellate cells

      In vivo: Mouse myofibroblasts
      In vitro: lentiviral (polycistronic system)

      In vivo: adenoviral (polycistronic system)
      In vitro: Alb+ (+/-12%)

      In vivo: < 4%
      In vitro: 21 days

      In vivo: 30 days
      ALB, SERPINA1, APOA1, CK18, FOXA1, FOXA2, GJB1, ACTBGlycogen storage, LDL uptake, albumin secretion, CYP1A2 and CYP3A activityCCl4-injured mouse<4% reprogramming efficiency, albumin secretion, urea production, glycogen storage, ICG & LDL uptake, accumulation lipids, CYP activity (CYP3A, 1A1, 2C9, and 1A2), upregulation CYP1A1, UGT1A1, ABCC2, and OATP after inductionScreening by withdrawal (FOXA1, FOXA2, FOXA3, GATA4, HNF1A, HNF4A and CEBPA)
      • Song G.
      • Pacher M.
      • Balakrishnan A.
      • Yuan Q.
      • Tsay H.C.
      • Yang D.
      • et al.
      Direct reprogramming of hepatic myofibroblasts into hepatocytes in vivo attenuates liver fibrosis.
      FOXA1, FOXA2, FOXA3, GATA4, HNF1A, HNF4AMouse myofibroblastsIn vivo: adeno-associated viral0.87% of all hepatocytes in the livern.s.n.a.n.a.CCl4-injured mousenormal albumin secretion, CYP activity and urea productionn.a.
      • Rezvani M.
      • Español-Suñer R.
      • Malato Y.
      • Dumont L.
      • Grimm A.A.
      • Kienle E.
      • et al.
      In vivo hepatic reprogramming of myofibroblasts with AAV vectors as a therapeutic strategy for liver fibrosis.
      HNF4A, FOXA3Mouse mesenchymal stem cellsPiggyBac transposon (polycistronic system)E-cad+ (25,1%)12 daysCDH, ALB, CK18, G6P, AFP, CK19, ACTB, CYP3A11, CYP2E1, CYP1A2, TAT, TTR, SERPINA1Urea production, accumulation lipids, glycogen storage, ICG uptake, albumin secretionn.a.n.a.n.a.
      • Katayama H.
      • Yasuchika K.
      • Miyauchi Y.
      • Kojima H.
      • Yamaoka R.
      • Kawai T.
      • et al.
      Generation of non-viral, transgene-free hepatocyte like cells with piggyBac transposon.
      FOXA1 + CRVPTDMouse embryonic fibroblasts & adult fibroblastsRetroviralE-cad+ (+/-55%); Alb+ (+/-36%)18 daysAFP, ALB, CK8, TTR, CDH, VTN, CLDN3, HNF4A, TF, CK18, CK19, EPCAMGlycogen storage, LDL uptake, CYP activity (CYP1A1, CYP1A2, CYP2B10, CYP2C29, CYP2C38, CYP2D22, CYP2E1, CYP3A11 and CYP3A13), drug metabolisation (phenacetin, tolbutamide, testosterone, diclofenac)Fah-/- mouse4/12 survived 8 weeks & livers were as normal as these of the surviving hepatocyte-transplanted mice, ALB & FAH expression, ALT, AST, ALP and total bilirubin markedly reducedn.a.
      • Guo R.
      • Tang W.
      • Yuan Q.
      • Hui L.
      • Wang X.
      • Xie X.
      Chemical cocktails enable hepatic reprogramming of mouse fibroblasts with a single transcription factor.
      FOXA2 + CRVPTD
      FOXA3 + CRVPTD
      FOXA3, HNF4AMouse embryonic fibroblastsLipofectamine 2000 (mRNA)n.s.12 daysALB, CDH, AFP, HNF4A, CK18, ASGR1, NR1I2 and CYP1A2Glycogen storage (>70%), ICG uptake, albumin secretion, xenobiotic metabolic

      Activity (>50%)
      Alb-TRECK SCID mouse; Fah1RTyrc/RJAlbumin positive; Fah productionn.a.
      • Yoon S.
      • Kang K.
      • Cho Y.D.
      • Kim Y.
      • Buisson E.M.
      • Yim J.H.
      • et al.
      Nonintegrating direct conversion using mRNA into hepatocyte-like cells.
      FOXA3, HNF1A, GATA4Mouse embryonic fibroblastsLentiviral (polycistronic system)n.s.16 daysALB, CDH1, CYP1A2, CYP3A11, MRP2, AFP, HNF4A, TTR, TF, SOX9, HNF1B, TJP1AST and LDH activity, CYP3A activity, albumin secretionn.a.n.a.n.a.
      • Chen C.
      • Pla-Palacín I.
      • Baptista P.M.
      • Shang P.
      • Oosterhoff L.A.
      • van Wolferen M.E.
      • et al.
      Hepatocyte-like cells generated by direct reprogramming from murine somatic cells can repopulate decellularized livers.
      Human
      FOXA2, HNF4A, CEBPB, MYCHuman neonatal & adult fibroblastsRetroviraln.s.20 daysALB, CYP3A4, SERPINA1,GAPDHAlbumin secretion, glycogen storage, ICG uptaken.a.n.a.All combinations of FOXA2 with MYC, CEBPB or HNF4A
      • Kogiso T.
      • Nagahara H.
      • Otsuka M.
      • Shiratori K.
      • Dowdy S.F.
      Transdifferentiation of human fibroblasts into hepatocyte-like cells by defined transcriptional factors.
      FOXA3, HNF1A, HNF4AHuman foetal, adult fibroblasts & mesenchymal stem cellsLentiviralfoetal: Alb+/Aat+ (+/-20%); adult: Alb+/Aat+ (+/-10%)14 daysALB, TTR, ASGR1, TF, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP3A4, MDR1, MRP2, MRP3, BSEP, NTCP, OATP1, OATP2, OATPB, RXRB, GR, RXRGAlbumin and Aat secretion, glycogen storage, LDL uptake, ICG uptake, accumulation lipids, upregulation CYPs after treatment with inducer (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9), drug metabolisation (phenacetin, coumarin, dextrometorphan)Con A-injured mouse; FRG mouse5/14 completely recovered, normal AST & ALT levels; 5/15 survived 9 weeks, 0,3-4,2% repopulationScreening (FOXA3, GATA4, HNF1B, HNF4A, HHEX, PROX1, CEBPB, KLF4)
      • Huang P.
      • Zhang L.
      • Gao Y.
      • He Z.
      • Yao D.
      • Wu Z.
      • et al.
      Direct reprogramming of human fibroblasts to functional and expandable hepatocytes.
      ,
      • Ni X.
      • Gao Y.
      • Wu Z.
      • Ma L.
      • Chen C.
      • Wang L.
      • et al.
      Functional human induced hepatocytes (hiHeps) with bile acid synthesis and transport capacities: a novel in vitro cholestatic model.
      ,
      • Sun L.
      • Wang Y.
      • Cen J.
      • Ma X.
      • Cui L.
      • Qiu Z.
      • et al.
      Modelling liver cancer initiation with organoids derived from directly reprogrammed human hepatocytes.
      HNF1A, HNF4A, HNF6, ATF5, PROX1, CEBPAHuman embryonic & adult fibroblastsViralAlb+ (90%); Aat+ (+/-100%)25 daysCYP3A4, CYP1A2, CYP2B6, CYP2C9, CYP2C19, CDH, FOXA1, FOXA2, FOXA3, ALB, LRH1, UGT1A1, UGT2B7, UGT2B15, MGST1, NNMT, MRP6Glycogen storage, LDL uptake, ICG uptake, albumin secretion, accumulation lipids, no Afp secretionTet-uPA/Rag2-/- γc-/- mouserepopulate 30%, 313 μg/ml human albumin, expression of CYP3A4, CYP2C9, CYP1A2, CYP2E1, CYP2C19, and CYP2D6- each transcription factor; HNF4A, HNF1A and HNF6
      • Du Y.
      • Wang J.
      • Jia J.
      • Song N.
      • Xiang C.
      • Xu J.
      • et al.
      Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming.
      HNF1A + 2TFs (FOXA1 or FOXA3 or HNF4A)Human embryonic fibroblastsSynthetic modified mRNA (daily cationic lipid transfection)n.s.5 daysALB, AFPAlbumin and Afp secretionn.a.n.a.n.a.
      • Simeonov K.P.
      • Uppal H.
      Direct reprogramming of human fibroblasts to hepatocyte-like cells by synthetic modified mRNAs.
      FOXA2, GATA4, FOXA1, FOXA3, HNF4A, HNF1AHuman embryonic fibroblastsSynthetic modified mRNA (cationic lipid transfection)n.s.5 daysALB, AFP, TLR3, APOA1,

      APOH, FGB, SERPINA1, CXCL9, CXCL10, ODC1, miR-122, miR-145, miR-192, miR-194
      Albumin and Afp secretionn.a.n.a.11 TFs (+CEBPA, GATA6, HHEX, HNF1B, and HNF6A)
      • Simeonov K.P.
      • Uppal H.
      Direct reprogramming of human fibroblasts to hepatocyte-like cells by synthetic modified mRNAs.
      ATF5, PROX1, FOXA2, FOXA3, HNF4AHuman foetal fibroblasts (MRC5)LentiviralAlb+/Aat+ (+/-27%); Asgr1+ (+/-22%)28 daysALB, AFP, CYP3A7, SERPINA1, CYP1A2, CYP2C19, CYP3A4, CYP2C9, CYP2D6, UGT1A1, NTCP, ATF5, PROX1, FOXA2, and FOXA3Albumin secretion, CYP1A2 and CYP3A4 activityn.a.n.a.Screening by withdrawal (ATF5, CEBPA, PROX1, FOXA2, FOXA3, HNF1A, HNF4A, HNF6, and GATA4)
      • Nakamori D.
      • Akamine H.
      • Takayama K.
      • Sakurai F.
      • Mizuguchi H.
      Direct conversion of human fibroblasts into hepatocyte-like cells by ATF5, PROX1, FOXA2, FOXA3, and HNF4A transduction.
      FOXA3 + CRVPTDHuman urine-derived epithelial-like cellsLentiviralAlb+ (+/-34%)24 daysALB, ASGR1, CK18, HNF4A, TTR, TF, ZO-1, GJB1, CYP1A2, CYP3A4, CYP2B6, CYP2D6, CYP2C8, CYP2C9, NTCP, MRP2, AHR, PXR, RXRA and RXRBAlbumin and Aat secretion, glycogen storage, LDL uptake, accumulation lipidsCon A-injured mouse3 of 12 mice survived 7 days, ALT, AST, and total bilirubin gradually decreasedn.a.
      • Tang W.
      • Guo R.
      • Shen S.
      • Zheng Y.
      • Lu Y.
      • Jiang M.
      • et al.
      Chemical cocktails enable hepatic reprogramming of human urine-derived cells with a single transcription factor.
      HNF1A + CRVPTD
      HNF4A + CRVPTD
      FOXA3, HNF1A, HNF4AHuman skin fibroblastsLentiviraln.s.10 daysCAR, FXR, HNF1A, ALB, MRP2, SERPINA1, GGT, UGT1A1, ASGR1, TF, HNF4An.a.n.a.n.a.n.a.
      • Wang L.Y.
      • Liu L.P.
      • Ge J.Y.
      • Yuan Y.Y.
      • Sun L.L.
      • Xu H.
      • et al.
      A multiple-cell microenvironment in a 3-dimensional system enhances direct cellular reprogramming into hepatic organoids.
      HNF1A, FOXA3, HNF4AHuman hepatoma (HCCLM3 & Huh-7)Adenoviraln.s.14 daysCYP1A2, CYP2C19, CYP3A4, ALB, PXR, GR, RXRB, PEPCK, GS, AAT, G6P, TF, ALDOB, MRP2, NTCP, OATPBGlycogen storage, LDL uptake, urea production, albumin secretion, CYP activity (CYP1A2, CYP2C19, CYP3A4), upregulation CYPs after inducer (CYP1A2, CYP2C19, CYP2C8, CYP3A4), drug metabolism (phenacetin, testosterone)Fah-/-/Rag2-/- mouseHuman Fah, Alb and Aat, 4,69% repopulation, better survival rate with iHeps than with PHHn.a.
      • Cheng Z.
      • He Z.
      • Cai Y.
      • Zhang C.
      • Fu G.
      • Li H.
      • et al.
      Conversion of hepatoma cells to hepatocyte-like cells by defined hepatocyte nuclear factors.
      HNF4A, HNF6A, GATA4, FOXA2, HHEX, c-MYCHuman embryonic and neonatal fibroblastsLentiviral, followed by directed hepatic differentiationAlb+ (2,7%); Alb+ (+/-75%)15 days; 40 daysALB, AFP, EPCAM, CK8, CK18, HNF1B, DLK1n.a.Tet-uPA/Rag2-/- γc-/- mouse; NPG mouse+/-50% human Alb+, CYP3A4, CYP2E1, CYP2D6, CYP2C9, CYP2C8, CYP1A2, CYP2C19, NTCP, MRP2; No tumour developmentn.a.
      • Xie B.
      • Sun D.
      • Du Y.
      • Jia J.
      • Sun S.
      • Xu J.
      • et al.
      A two-step lineage reprogramming strategy to generate functionally competent human hepatocytes from fibroblasts.
      Alb+ (>90%)25 daysALB, CDH1, HNF1A, CEBPA, AAT, LXR, FXR, PXR, PPARALDL uptake, albumin secretion, accumulation lipids, glycogen storage, CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP1A2, CYP2B6 and CYP2C8, UGTs, NTCP, steatosis, phospholipidosisn.a.n.a.
      HNF4A, FOXA3, HNF1AHuman neonatal dermal fibroblastsInducible polycistronic lentiviraln.s.10 daysGLS2, HGD, CYP7A1, GPT1, ALB, SERPINA1, ALDH4A1, GPT1Glycogen storage, glutamine/glutamate conversion and secretion, albumin secretionCB17/Icr-Prkdc scid/Crl miceHuman albuminIndividual TFs
      • Ballester M.
      • Bolonio M.
      • Santamaria R.
      • Castell J.V.
      • Ribes-Koninckx C.
      • Bort R.
      Direct conversion of human fibroblast to hepatocytes using a single inducible polycistronic vector.
      HNF1A, GATA4, FOXA3human urinary epithelial cellsLentiviraln.s.20 daysALB, CK8, CK18, TTR, ASGPR1, SERPINA1, CYP2B6, CYP2A9, CYP3A4, CYP2C19, CYP1A2Glycogen storage, accumulation lipids, ICG uptake, upregulation CYPs after inducer (CYP2B6, CYP2C9, CYP3A4, CYP2C19, CYP1A2)n.a.n.a.Screening (FOXA2, FOXA3, GATA4, HNF1A, HNF1B, HNF4A)
      • Wu H.
      • Du C.
      • Yang F.
      • Zheng X.
      • Qiu D.
      • Zhang Q.
      • et al.
      Generation of hepatocyte-like cells from human urinary epithelial cells and the role of autophagy during direct reprogramming.

       Synergy between HNF1A, HNF4A and the FOXA family

      As can be seen in Table 1, HNF1A and/or HNF4A are usually present in direct hepatic reprogramming cocktails, mainly in combination with a member of the FOXA family. HNF4A initiates the hepatic differentiation cascade during liver development and it co-regulates the transcription of 1,575 hepatic genes throughout adulthood.
      • Duncan S.A.
      Transcriptional regulation of liver development.
      ,
      • Odom D.T.
      • Zizlsperger N.
      • Gordon D.B.
      • Bell G.W.
      • Rinaldi N.J.
      • Murray H.L.
      • et al.
      Control of pancreas and liver gene expression by HNF transcription factors.
      The role of HNF1A in liver development is not entirely clear, yet it can be classified as a rather mature LETF that binds to at least 222 hepatocyte-specific genes in mature hepatocytes, all of which are involved in crucial hepatic pathways.
      • Odom D.T.
      • Zizlsperger N.
      • Gordon D.B.
      • Bell G.W.
      • Rinaldi N.J.
      • Murray H.L.
      • et al.
      Control of pancreas and liver gene expression by HNF transcription factors.
      A highly synergistic mechanism is conceivable between HNF1A and HNF4A as they can both bind to the same sets of hepatic genes. This suggests that mature hepatic gene expression depends on multi-input motifs regulated by multiple LETFs.
      • Odom D.T.
      • Zizlsperger N.
      • Gordon D.B.
      • Bell G.W.
      • Rinaldi N.J.
      • Murray H.L.
      • et al.
      Control of pancreas and liver gene expression by HNF transcription factors.
      In non-hepatic somatic cells, most genes involved in hepatic metabolism are typically embedded in heterochromatin and therefore not accessible for LETFs to engage in gene transcription.
      • Allshire R.C.
      • Madhani H.D.
      Ten principles of heterochromatin formation and function.
      Pioneer factors (e.g. GATA & FOXA family) can recognise and bind to target DNA sequences in heterochromatin, enabling a remodelling of the adjoining epigenetic landscape that provides access to LETFs, chromatin modifiers and chromatin remodellers.
      • Iwafuchi-Doi M.
      • Zaret K.S.
      Pioneer transcription factors in cell repogramming.
      As such, they establish competence for lineage conversion and are seen as crucial in direct hepatic reprogramming cocktails.
      • Ribeiro M.M.
      • Okawa S.
      • del Sol A.
      TransSynW: a single-cell RNA-sequencing based web application to guide cell conversion experiments.
      Overexpression of epigenetic modifiers that promote an open chromatin configuration, such as histone demethylases (KDM2B
      • Zakikhan K.
      • Pournasr B.
      • Nassiri-Asl M.
      • Baharvand H.
      Enhanced direct conversion of fibroblasts into hepatocyte-like cells by Kdm2b.
      ), also facilitate hepatic transdifferentiation. The first two reports of direct hepatic reprogramming on mouse fibroblasts showed that single expression of HNF1A
      • Huang P.
      • He Z.
      • Ji S.
      • Sun H.
      • Xiang D.
      • Liu C.
      • et al.
      Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors.
      or HNF4A
      • Sekiya S.
      • Suzuki A.
      Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors.
      in combination with a member of the FOXA family was sufficient to generate iHeps that can mature to functional hepatocytes in vivo.
      • Sekiya S.
      • Suzuki A.
      Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors.
      Strikingly, Huang et al. found that upon screening of different LETFs involved in liver development and hepatic differentiation, the removal of HNF4A in their direct reprogramming cocktail promoted the formation of epithelial colonies, the first key step in direct hepatic reprogramming. Further removal of a single LETF out of their 3TF reprogramming cocktail (GATA4, HNF1A, FOXA3) reduced the possibility of generating epithelial colonies. They also found that the replacement of FOXA3 by FOXA2 resulted in less induction of hepatic gene expression and epithelial colony formation.
      • Huang P.
      • He Z.
      • Ji S.
      • Sun H.
      • Xiang D.
      • Liu C.
      • et al.
      Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors.
      On a side note, Horisawa and colleagues showed that all FOXA family members bind to regions distal to the transcription start site. Yet, only FOXA3 transferred to proximal regions and was able to bind RNA polymerase II, ultimately inducing the lineage conversion.
      • Horisawa K.
      • Udono M.
      • Ueno K.
      • Ohkawa Y.
      • Nagasaki M.
      • Sekiya S.
      • et al.
      The dynamics of transcriptional activation by hepatic reprogramming factors.
      Sekiya and Suzuki showed that the removal of HNF4A from the viral pool resulted in a reduction of albumin and alfa-fetoprotein expression, whereas the expression level of E-cadherin was hardly affected by the removal of any of the LETFs. Consequently, expression of HNF4A with different LETFs was investigated and they showed that combined expression of HNF4A and any member of the FOXA family resulted in expression of hepatic markers.
      • Sekiya S.
      • Suzuki A.
      Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors.
      Lim and colleagues compared epithelial formation in response to HNF1A or HNF4A overexpression in mouse fibroblasts and showed that HNF1A is better at inducing MET (14.5% vs. 1.4% E-cadherin+ colonies).
      • Lim K.T.
      • Lee S.C.
      • Gao Y.
      • Kim K.P.
      • Song G.
      • An S.Y.
      • et al.
      Small molecules facilitate single factor-mediated hepatic reprogramming.
      That HNF1A plays a more influential role than HNF4A during direct hepatic reprogramming in mouse fibroblasts was also confirmed by Rezvani et al.
      • Rezvani M.
      • Español-Suñer R.
      • Malato Y.
      • Dumont L.
      • Grimm A.A.
      • Kienle E.
      • et al.
      In vivo hepatic reprogramming of myofibroblasts with AAV vectors as a therapeutic strategy for liver fibrosis.
      As can be seen in Table 1, direct hepatic reprogramming cocktails for hepatic transdifferentiation of human fibroblasts are more complex and generally require the combined expression of HNF1A and HNF4A with other LETFs. This is supported by the finding that HNF1A and HNF4A both bind to the same sets of human hepatic genes and both play a central role in the complex interconnected hepatic cross-regulatory circuitry.
      • Odom D.T.
      • Zizlsperger N.
      • Gordon D.B.
      • Bell G.W.
      • Rinaldi N.J.
      • Murray H.L.
      • et al.
      Control of pancreas and liver gene expression by HNF transcription factors.
      ,
      • Odom D.T.
      • Dowell R.D.
      • Jacobsen E.S.
      • Nekludova L.
      • Rolfe P.A.
      • Danford T.W.
      • et al.
      Core transcriptional regulatory circuitry in human hepatocytes.
      Hwang et al. obtained a metastable state by only overexpressing HNF1A, suggesting that it is not possible to achieve a stable reprogrammed hepatic identity contingent on ectopic expression of a single LETF in human fibroblasts.
      • Hwang S.I.
      • Kwak T.H.
      • Kang J.H.
      • Kim J.
      • Lee H.
      • Kim K.P.
      • et al.
      Metastable reprogramming state of single transcription factor-derived induced hepatocyte-like cells.
      This shows that the defined minimal LETF combinations in mice are notably different from the extensive direct hepatic reprogramming cocktails needed to direct a hepatic cell fate conversion in human somatic cells and that the extrapolation of mouse to man is rather unreliable, as the DNA binding sites and the interconnected hepatic cross-regulatory circuitry differ in human and mouse liver cells.
      • Odom D.T.
      • Dowell R.D.
      • Jacobsen E.S.
      • Gordon W.
      • Danford T.W.
      • MacIsaac K.D.
      • et al.
      Tissue-specific transcriptional regulation has diverged significantly between human and mouse.
      ,
      • Simeonov K.P.
      • Uppal H.
      Direct reprogramming of human fibroblasts to hepatocyte-like cells by synthetic modified mRNAs.
      Huang and colleagues showed that in their 3TF hepatic reprogramming cocktail (HNF1A, FOXA3, HNF4A) the concomitant expression of HNF1A is mandatory to induce the hepatic reprogramming of human fibroblasts.
      • Huang P.
      • Zhang L.
      • Gao Y.
      • He Z.
      • Yao D.
      • Wu Z.
      • et al.
      Direct reprogramming of human fibroblasts to functional and expandable hepatocytes.
      In addition, Simeonov et al. demonstrated that HNF1A is a mandatory LETF to induce a direct hepatic conversion in human fibroblasts.
      • Simeonov K.P.
      • Uppal H.
      Direct reprogramming of human fibroblasts to hepatocyte-like cells by synthetic modified mRNAs.
      Contrarily, Nakamori and colleagues showed that withdrawing HNF1A from their direct hepatic reprogramming cocktail did not change hepatic marker expression, suggesting that HNF1A might not play an important role in human hepatic cell fate conversion. Furthermore, they confirmed that HNF4A plays a crucial role in their direct hepatic reprogramming cocktail as hepatic markers drastically decreased upon withdrawal of HNF4A.
      • Nakamori D.
      • Akamine H.
      • Takayama K.
      • Sakurai F.
      • Mizuguchi H.
      Direct conversion of human fibroblasts into hepatocyte-like cells by ATF5, PROX1, FOXA2, FOXA3, and HNF4A transduction.
      These discrepancies clearly highlight that there is no consensus regarding whether HNF1A or HNF4A plays the more important role in direct hepatic conversion. Yet, it is clear that mouse fibroblasts exhibit a higher propensity for lineage conversion than human fibroblasts, which are seemingly in a stronger biological lockdown.

       Pluripotency-promoting factors improve and accelerate iHep generation

      Concomitant expression of pluripotency-promoting TFs (“OSKM”)
      • Kogiso T.
      • Nagahara H.
      • Otsuka M.
      • Shiratori K.
      • Dowdy S.F.
      Transdifferentiation of human fibroblasts into hepatocyte-like cells by defined transcriptional factors.
      ,
      • Pournasr B.
      • Asghari-Vostikolaee M.H.
      • Baharvand H.
      Transcription factor-mediated reprograming of fibroblasts to hepatocyte-like cells.
      ,
      • Serrano F.
      • García-Bravo M.
      • Blazquez M.
      • Torres J.
      • Castell J.V.
      • Segovia J.C.
      • et al.
      Silencing of hepatic fate-conversion factors induce tumorigenesis in reprogrammed hepatic progenitor-like cells.
      makes the somatic cells convert towards a temporary stem cell-like state that allows them to proliferate before reaching hepatic maturity. Furthermore, it has been shown that these TFs accelerate the sequential cell fate transition.
      • Lim K.T.
      • Lee S.C.
      • Gao Y.
      • Kim K.P.
      • Song G.
      • An S.Y.
      • et al.
      Small molecules facilitate single factor-mediated hepatic reprogramming.
      Even though the presence of MET activators Kruppel like factor 4 (KLF4) and c-MYC accelerates the conversion kinetics and, consequently, enhances the efficiency of iHep generation,
      • Lim K.T.
      • Lee S.C.
      • Gao Y.
      • Kim K.P.
      • Song G.
      • An S.Y.
      • et al.
      Small molecules facilitate single factor-mediated hepatic reprogramming.
      these 2 activators must be excluded from direct hepatic conversion cocktails because of their oncogenic properties.
      • Serrano F.
      • García-Bravo M.
      • Blazquez M.
      • Torres J.
      • Castell J.V.
      • Segovia J.C.
      • et al.
      Silencing of hepatic fate-conversion factors induce tumorigenesis in reprogrammed hepatic progenitor-like cells.
      ,
      • Nakagawa M.
      • Koyanagi M.
      • Tanabe K.
      • Takahashi K.
      • Ichisaka T.
      • Aoi T.
      • et al.
      Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts.
      Overexpression of SV40 large T antigen can bypass the inability of iHeps to proliferate.
      • Huang P.
      • Zhang L.
      • Gao Y.
      • He Z.
      • Yao D.
      • Wu Z.
      • et al.
      Direct reprogramming of human fibroblasts to functional and expandable hepatocytes.
      ,
      • Guo R.
      • Tang W.
      • Yuan Q.
      • Hui L.
      • Wang X.
      • Xie X.
      Chemical cocktails enable hepatic reprogramming of mouse fibroblasts with a single transcription factor.
      • Wang L.Y.
      • Liu L.P.
      • Ge J.Y.
      • Yuan Y.Y.
      • Sun L.L.
      • Xu H.
      • et al.
      A multiple-cell microenvironment in a 3-dimensional system enhances direct cellular reprogramming into hepatic organoids.
      • Shi X.-L.
      • Gao Y.
      • Yan Y.
      • Ma H.
      • Sun L.
      • Huang P.
      • et al.
      Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes.
      • Tang W.
      • Guo R.
      • Shen S.
      • Zheng Y.
      • Lu Y.
      • Jiang M.
      • et al.
      Chemical cocktails enable hepatic reprogramming of human urine-derived cells with a single transcription factor.
      Yet, this forced proliferation is at the expense of hepatic functionality. The knockdown of p53 with siRNA has been shown to be a valid alternative for growing iHeps in vitro
      • Du Y.
      • Wang J.
      • Jia J.
      • Song N.
      • Xiang C.
      • Xu J.
      • et al.
      Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming.
      ,
      • Xie B.
      • Sun D.
      • Du Y.
      • Jia J.
      • Sun S.
      • Xu J.
      • et al.
      A two-step lineage reprogramming strategy to generate functionally competent human hepatocytes from fibroblasts.
      and has been shown to increase pluripotent reprogramming efficiencies.
      • Okita K.
      • Yamakawa T.
      • Matsumura Y.
      • Sato Y.
      • Amano N.
      • Watanabe A.
      • et al.
      An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells.
      However, it is also well-known that diminishing the function of p53 may cause the induction of genomic mutations, as p53 is prominently involved in DNA repair following damage.
      • Williams A.B.
      • Schumacher B.
      p53 in the DNA-damage-repair process.
      ,
      • Marión R.M.
      • Strati K.
      • Li H.
      • Murga M.
      • Blanco R.
      • Ortega S.
      • et al.
      A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity.
      To be clinically relevant, cells must be prevented from acquiring the ability to proliferate uncontrollably, as seen in iPSC reprogramming.
      • Hwang S.I.
      • Kwak T.H.
      • Kang J.H.
      • Kim J.
      • Lee H.
      • Kim K.P.
      • et al.
      Metastable reprogramming state of single transcription factor-derived induced hepatocyte-like cells.
      Yet, these iHeps can still be used in preclinical hepatotoxicity screenings and extracorporeal bio-artificial liver devices for acute liver failure.
      • Shi X.-L.
      • Gao Y.
      • Yan Y.
      • Ma H.
      • Sun L.
      • Huang P.
      • et al.
      Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes.

       Hepatic maturation factors enhance functionality of obtained iHeps

      To further improve the direct conversion towards more mature iHeps, it is opportune to include hepatic maturation factors in the direct hepatic reprogramming cocktail. Hepatic maturation factors are defined as LETFs that are differentially expressed between foetal liver cells and freshly isolated PHHs.
      • Du Y.
      • Wang J.
      • Jia J.
      • Song N.
      • Xiang C.
      • Xu J.
      • et al.
      Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming.
      Du et al. showed that activating transcription factor (ATF) 5, prospero homeobox transcription factor (PROX) 1 and CCAAT/enhancer binding protein (CEBP) A are maturation factors that are crucial for the metabolic maturation of hepatic pathways in iHeps.
      • Du Y.
      • Wang J.
      • Jia J.
      • Song N.
      • Xiang C.
      • Xu J.
      • et al.
      Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming.
      CEBPB
      • Kogiso T.
      • Nagahara H.
      • Otsuka M.
      • Shiratori K.
      • Dowdy S.F.
      Transdifferentiation of human fibroblasts into hepatocyte-like cells by defined transcriptional factors.
      and nuclear receptor subfamily 1 group I member 2 (NR1I2)
      • Pournasr B.
      • Asghari-Vostikolaee M.H.
      • Baharvand H.
      Transcription factor-mediated reprograming of fibroblasts to hepatocyte-like cells.
      are also potent hepatic maturation factors. NR1I2 regulates a plethora of genes involved in phase I, phase II and phase III detoxification reactions. As the metabolism of xenobiotics largely defines a functional hepatocyte, the incorporation of hepatic maturation factors in direct hepatic reprogramming cocktails is of great interest.
      Inclusion of maturation factors in the direct hepatic reprogramming cocktail enhances the hepatic functionality of iHeps. These factors are defined as LETFs that differ between foetal and adult liver cells.

       Towards expandable hepatoblasts with hepatic progenitor factors

      Yu and colleagues were the first to incorporate a LETF of early hepatogenesis, namely HNF1B, in their reprogramming cocktail. Since HNF1B is responsible for normal hepatic bud formation and gut regionalisation during early hepatic development,
      • Lokmane L.
      • Haumaitre C.
      • Garcia-Villalba P.
      • Anselme I.
      • Schneider-Maunoury S.
      • Cereghini S.
      Crucial role of vHNF1 in vertebrate hepatic specification.
      it will induce and maintain expression of early hepatic TFs and immature hepatic markers.
      • Yu B.
      • He Z.Y.
      • You P.
      • Han Q.W.
      • Xiang D.
      • Chen F.
      • et al.
      Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors.
      As such, they managed to reprogramme fibroblasts into bipotential hepatic stem cells with the ability to proliferate. Another study based a direct hepatic reprogramming cocktail on the concept of tissue generation in lower animals.
      • Tanaka E.M.
      • Reddien P.W.
      The cellular basis for animal regeneration.
      This gene expression plasticity inspired Xie et al. to generate expandable induced hepatic progenitor cells that could then be coaxed into abundant competent human induced hepatocytes (hiHeps) by hepatic differentiation. They included HHEX in the direct hepatic reprogramming cocktail, which is highly expressed in human foetal liver cells, to simulate the earlier stages of hepatogenesis. Global gene expression analysis showed that the human induced hepatic progenitor cells they obtained share a similar gene expression pattern with human foetal liver cells. Furthermore, genes enriched in human hepatic progenitors are greatly upregulated in the human-induced hepatic progenitors. After propagation of up to 30 passages, maturation was obtained by culturing the human-induced hepatic progenitors in a hepatocyte medium that has shown good results for the cultivation of PHHs. As such, the obtained hiHeps showed upregulation of crucial and mature hepatic markers, reaching similar levels as in freshly isolated PHHs and adult liver tissue.
      • Xie B.
      • Sun D.
      • Du Y.
      • Jia J.
      • Sun S.
      • Xu J.
      • et al.
      A two-step lineage reprogramming strategy to generate functionally competent human hepatocytes from fibroblasts.

       Small molecules to facilitate direct hepatic conversion

      Small molecules used to improve hiPSC-HLC generation could be applied to increase the efficiency of direct hepatic conversion and possibly, in the future, to generate chemically iHeps without the need for gene transfer. Hou and colleagues showed that iPSCs can be generated from mouse somatic cells by using a combination of only 7 small molecules: CRFVPTD (CHIR99021, RepSox, Forskolin, Valproic acid, Parnate, TTNPB, DZNep).
      • Hou P.
      • Li Y.
      • Zhang X.
      • Liu C.
      • Guan J.
      • Li H.
      • et al.
      Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds.
      This breakthrough triggered Guo et al. to translate this process to direct hepatic reprogramming. They report the direct conversion of mouse embryonic fibroblasts into iHeps by a chemical cocktail (CRVPTD) in combination with a single hepatocyte-specific pioneer factor (FOXA1, FOXA2 or FOXA3).
      • Guo R.
      • Tang W.
      • Yuan Q.
      • Hui L.
      • Wang X.
      • Xie X.
      Chemical cocktails enable hepatic reprogramming of mouse fibroblasts with a single transcription factor.
      The same research group managed to repeat the same process on a human cell level by overexpressing FOXA3, HNF1A or HNF4A.
      • Tang W.
      • Guo R.
      • Shen S.
      • Zheng Y.
      • Lu Y.
      • Jiang M.
      • et al.
      Chemical cocktails enable hepatic reprogramming of human urine-derived cells with a single transcription factor.
      The addition of CHIR99021 potentiates self-renewal of pluripotent stem cells and rapid proliferation of somatic cells by inhibition of glycogen synthase kinase (GSK) 3, which mediates activation of the Wnt/β-catenin pathway. Yet, it has also been shown that the small molecule facilitates somatic reprogramming, and thus the transition of mesenchyme to epithelia.
      • Lim K.T.
      • Lee S.C.
      • Gao Y.
      • Kim K.P.
      • Song G.
      • An S.Y.
      • et al.
      Small molecules facilitate single factor-mediated hepatic reprogramming.
      ,
      • Li W.
      • Ding S.
      Small molecules that modulate embryonic stem cell fate and somatic cell reprogramming.
      Parnate (lysine-specific demethylase 1 inhibitor),
      • Li W.
      • Ding S.
      Small molecules that modulate embryonic stem cell fate and somatic cell reprogramming.
      DZNep (histone methylation inhibitor)
      • Miranda T.B.
      • Cortez C.C.
      • Yoo C.B.
      • Liang G.
      • Abe M.
      • Kelly T.K.
      • et al.
      DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation.
      and valproic acid (histone deacetylase inhibitor)
      • Li W.
      • Ding S.
      Small molecules that modulate embryonic stem cell fate and somatic cell reprogramming.
      are epigenetic modifiers that modulate the chromatin structure by creating an open, transcriptionally active euchromatin configuration at gene coding and regulatory regions. As such, they facilitate gene transcription towards the hepatic lineage. RepSox is an activin receptor-like kinase (ALK) inhibitor that – by inhibiting the canonical activin/nodal/transforming growth factor (TGF)-β pathway – inhibits TGF-β-induced epithelial-to-mesenchymal transition (EMT) and enhances MET.
      • Tojo M.
      • Hamashima Y.
      • Hanyu A.
      • Kajimoto T.
      • Saitoh M.
      • Miyazono K.
      • et al.
      The ALK-5 inhibitor A-83-01 inhibits Smad signaling and epithelial-to-mesenchymal transition by transforming growth factor-β.
      By activating the retinoic acid receptor with the small molecule TTNPB, hepatic nuclear receptor-mediated pathways are modulated, as such promoting hepatic lineage conversion.
      • Ang L.T.
      • Tan A.K.Y.
      • Autio M.I.
      • Goh S.H.
      • Choo S.H.
      • Lee K.L.
      • et al.
      A roadmap for human liver differentiation from pluripotent stem cells.
      Lim et al. showed that 2 small molecules (A-83-01 & CHIR99021) could replace the pluripotent reprogramming TFs KLF4 and c-MYC in the direct hepatic reprogramming cocktail.
      • Lim K.T.
      • Lee S.C.
      • Gao Y.
      • Kim K.P.
      • Song G.
      • An S.Y.
      • et al.
      Small molecules facilitate single factor-mediated hepatic reprogramming.
      Wu et al. also enriched their direct hepatic reprogramming cocktail with A-83-01 and CHIR99021
      • Wu H.
      • Du C.
      • Yang F.
      • Zheng X.
      • Qiu D.
      • Zhang Q.
      • et al.
      Generation of hepatocyte-like cells from human urinary epithelial cells and the role of autophagy during direct reprogramming.
      and Zakikhan et al. with A-83-01 alone.
      • Zakikhan K.
      • Pournasr B.
      • Nassiri-Asl M.
      • Baharvand H.
      Enhanced direct conversion of fibroblasts into hepatocyte-like cells by Kdm2b.
      As A-83-01 is an ALK 4/5/7 inhibitor, it induces MET and promotes direct hepatic conversion.
      • Tojo M.
      • Hamashima Y.
      • Hanyu A.
      • Kajimoto T.
      • Saitoh M.
      • Miyazono K.
      • et al.
      The ALK-5 inhibitor A-83-01 inhibits Smad signaling and epithelial-to-mesenchymal transition by transforming growth factor-β.
      From a tissue regeneration point of view, Xie et al. aspired to generate proliferating human induced hepatic progenitors that become functionally competent hiHeps after an additional maturation step. In a first hepatic conversion step, they used CHIR99021, lysophosphatidic acid, SB431542, and sphingosine-1-phosphate to generate induced hepatic progenitors; in the final maturation step the medium is supplemented with forskolin and SB431542.
      • Xie B.
      • Sun D.
      • Du Y.
      • Jia J.
      • Sun S.
      • Xu J.
      • et al.
      A two-step lineage reprogramming strategy to generate functionally competent human hepatocytes from fibroblasts.
      Lysophosphatidic acid and sphingosine-1-phosphate are known inducers of proliferation and play a key role in hepatic tissue regeneration.
      • Khomich O.
      • Ivanov A.V.
      • Bartosch B.
      Metabolic hallmarks of hepatic stellate cells in liver fibrosis.
      Similar to A-83-01, SB431542 suppresses the canonical activin/nodal/TGF-β pathway, which enhances MET, and is both beneficial for hepatic progenitor generation and for hepatocyte maturation.
      • Sgodda M.
      • Mobus S.
      • Hoepfner J.
      • Sharma A.D.
      • Schambach A.
      • Greber B.
      • et al.
      Improved hepatic differentiation strategies for human induced pluripotent stem cells.
      To potentiate the directed hepatic differentiation process, a cAMP-dependent protein kinase A (PKA) activator, like the small molecule forskolin, can be added to the culture medium.
      Importantly, Boon et al. showed that high levels of extracellular amino acids induce a metabolic-competent state in hiPSCs-HLCs and HepG2 cells, even reaching similar levels to those in PHHs.
      • Boon R.
      • Kumar M.
      • Tricot T.
      • Elia I.
      • Ordovas L.
      • Jacobs F.
      • et al.
      Amino acid levels determine metabolism and CYP450 function of hepatocytes and hepatoma cell lines.
      Supplementing the rich direct hepatic reprogramming medium with a supraphysiological concentration of amino acids might thus push hepatic transdifferentiation towards an unprecedented level of hepatic maturity (Fig. 1).
      Expandable induced hepatic progenitor cells can be obtained by including hepatic progenitor factors in the direct hepatic reprogramming cocktail. These factors are highly expressed in human foetal liver cells and mimic the earlier stages of hepatogenesis.
      Figure thumbnail gr1
      Fig. 1Fundamental issue of directly converting somatic cells to iHeps.
      Conversion methods that result in a good conversion efficiency are difficult to translate to the clinic and transdifferentiation methods that are compatible with clinical applications result in a low conversion efficiency. iHeps, induced hepatocytes.

      Minimal criteria for iHep characterisation and reporting

      Phenotypic features of iHeps can be defined in vitro based on 4 levels: i) morphology, ii) gene expression, iii) protein expression and iv) hepatic functionality and should always be compared to PHHs as the golden standard. In addition, iHep functionality can be evaluated in an in vivo setting, when therapeutic evaluation is envisaged. Currently, it is extremely difficult to unambiguously compare iHep study outcomes, because no reporting standards have been defined. It is clear that in many studies the in vitro characterisation of iHeps is limited to gene and protein expression (Table 1). Often, the expression of hepatic markers is only compared to the original cell source (e.g. fibroblasts) and not PHHs. Therefore, we strive for a consensus that scientific papers about iHeps should at least report on a predefined set of hepatic markers and compare their expression to PHHs. Importantly, Nakamori et al. showed that hiHeps still retain expression of fibroblast markers, while hepatic markers are strongly upregulated.
      • Nakamori D.
      • Akamine H.
      • Takayama K.
      • Sakurai F.
      • Mizuguchi H.
      Direct conversion of human fibroblasts into hepatocyte-like cells by ATF5, PROX1, FOXA2, FOXA3, and HNF4A transduction.
      It is therefore of equal relevance to report on the downregulation of markers of the somatic cell source.
      • Huang P.
      • He Z.
      • Ji S.
      • Sun H.
      • Xiang D.
      • Liu C.
      • et al.
      Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors.
      ,
      • Lim K.T.
      • Lee S.C.
      • Gao Y.
      • Kim K.P.
      • Song G.
      • An S.Y.
      • et al.
      Small molecules facilitate single factor-mediated hepatic reprogramming.
      ,
      • Nakamori D.
      • Akamine H.
      • Takayama K.
      • Sakurai F.
      • Mizuguchi H.
      Direct conversion of human fibroblasts into hepatocyte-like cells by ATF5, PROX1, FOXA2, FOXA3, and HNF4A transduction.
      ,
      • Pournasr B.
      • Asghari-Vostikolaee M.H.
      • Baharvand H.
      Transcription factor-mediated reprograming of fibroblasts to hepatocyte-like cells.
      ,
      • Du Y.
      • Wang J.
      • Jia J.
      • Song N.
      • Xiang C.
      • Xu J.
      • et al.
      Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming.
      ,
      • Xie B.
      • Sun D.
      • Du Y.
      • Jia J.
      • Sun S.
      • Xu J.
      • et al.
      A two-step lineage reprogramming strategy to generate functionally competent human hepatocytes from fibroblasts.
      ,
      • Chen C.
      • Pla-Palacín I.
      • Baptista P.M.
      • Shang P.
      • Oosterhoff L.A.
      • van Wolferen M.E.
      • et al.
      Hepatocyte-like cells generated by direct reprogramming from murine somatic cells can repopulate decellularized livers.
      • Park S.
      • In Hwang S.
      • Kim J.
      • Hwang S.
      • Kang S.
      • Yang S.
      • et al.
      The therapeutic potential of induced hepatocyte-like cells generated by direct reprogramming on hepatic fibrosis.
      • Kim J.
      • Kim K.P.
      • Lim K.T.
      • Lee S.C.
      • Yoon J.
      • Song G.
      • et al.
      Generation of integration-free induced hepatocyte-like cells from mouse fibroblasts.
      Better-characterised iHeps will help us uncover important shortcomings that can be acted upon to generate fully functional and metabolically competent iHeps in the future. Analysing the transcriptome and/or proteome of single cells can aid with this, as it enables the reliable identification of different constituent cell types. This will give us a better understanding of the direct hepatic reprogramming efficiency and the presence of non-hepatic or inefficiently reprogrammed cell types. Transcriptomic and proteomic data, generated during the phenotypic characterisation of iHeps, should be deposited in publicly accessible repositories, to significantly improve reporting transparency and to allow for the phenotypic comparison of different iHeps, PHHs and other hepatocyte-like cells. Ultimately, the hepatic functionality of iHeps should be reported and compared to PHHs. Defining the phase I and II biotransformation capacity and other functional hepatic traits (e.g. glycogen storage, LDL uptake, albumin secretion, urea production) should be standard criteria for evaluating iHeps. We propose that all studies should report absolute hepatic functionality data and not only relative fold changes to the non-hepatic cell of origin (e.g. fibroblast). This would allow us to unambiguously compare the functionality of the generated iHeps to other hepatocyte-like cells and PHHs. In case a therapeutic evaluation is envisaged, the functionality of the obtained iHeps should be tested in a relevant in vivo animal model, with – at least – a report on their ability to engraft and attenuate liver damage parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin when compared to PHHs. Altogether, we propose a set of minimal criteria to characterise iHeps for both laboratory-based scientific investigations and for (pre-)clinical studies (Box 1). The aim of this proposition is solely to provide unambiguous characterisation criteria of iHeps at 5 different levels, based on the currently available knowledge.
      To date, unambiguous comparison among study outcomes is difficult, because the characterisation of iHeps has not been standardised. A set of minimal criteria to report on iHeps for both laboratory-based scientific investigations and for (pre-)clinical studies adds transparency to this research field and will help move human iHeps from bench-to-bedside.
      Minimal criteria to report on iHeps.

      Recommendations for generation of iHeps in view of potential clinical applications

       Optimising viral-induced direct hepatic reprogramming

      Most of the current direct hepatic reprogramming protocols overexpress several LETFs, yet they are expressed by individual viral vectors carrying a single transgene. Hence, a heterogenous cell population is obtained with inconsistent transductions of LETFs, subsequently resulting in variable hepatic conversion efficiencies. By combining several transgenes into 1 construct, a multicistronic vector system is obtained (Fig. 1). The traditional way to co-express multiple genes in a single mRNA is by including an internal ribosome entry site (IRES) sequence or a 2A oligopeptide sequence in between open reading frames. Yet, over the years, the 2A co-expression system has shown to be more reliable for stoichiometric expression,
      • Liu Z.
      • Chen O.
      • Wall J.B.J.
      • Zheng M.
      • Zhou Y.
      • Wang L.
      • et al.
      Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector.
      since expression of the transgene before the IRES is generally significantly higher than that of the second transgene.
      • Mizuguchi H.
      • Xu Z.
      • Ishii-Watabe A.
      • Uchida E.
      • Hayakawa T.
      IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector.
      Furthermore, 2A sequences only comprise 60–80 bp, which enables a multicistronic sequence of several transgenes, whereas the number of transgenes is limited when using IRES (588 bp).
      • Chen C.
      • Pla-Palacín I.
      • Baptista P.M.
      • Shang P.
      • Oosterhoff L.A.
      • van Wolferen M.E.
      • et al.
      Hepatocyte-like cells generated by direct reprogramming from murine somatic cells can repopulate decellularized livers.
      By constructing an all-in-one polycistronic hepatic reprogramming cassette,
      • Chen C.
      • Pla-Palacín I.
      • Baptista P.M.
      • Shang P.
      • Oosterhoff L.A.
      • van Wolferen M.E.
      • et al.
      Hepatocyte-like cells generated by direct reprogramming from murine somatic cells can repopulate decellularized livers.
      ,
      • Song G.
      • Pacher M.
      • Balakrishnan A.
      • Yuan Q.
      • Tsay H.C.
      • Yang D.
      • et al.
      Direct reprogramming of hepatic myofibroblasts into hepatocytes in vivo attenuates liver fibrosis.
      • Katayama H.
      • Yasuchika K.
      • Miyauchi Y.
      • Kojima H.
      • Yamaoka R.
      • Kawai T.
      • et al.
      Generation of non-viral, transgene-free hepatocyte like cells with piggyBac transposon.
      • Ballester M.
      • Bolonio M.
      • Santamaria R.
      • Castell J.V.
      • Ribes-Koninckx C.
      • Bort R.
      Direct conversion of human fibroblast to hepatocytes using a single inducible polycistronic vector.
      cells reprogramme synchronously and a homogenous cell population is obtained as viral transduction results in co-expression of the direct hepatic reprogramming cocktail. Also, implementing a gene-inducible system
      • Hwang S.I.
      • Kwak T.H.
      • Kang J.H.
      • Kim J.
      • Lee H.
      • Kim K.P.
      • et al.
      Metastable reprogramming state of single transcription factor-derived induced hepatocyte-like cells.
      ,
      • Ballester M.
      • Bolonio M.
      • Santamaria R.
      • Castell J.V.
      • Ribes-Koninckx C.
      • Bort R.
      Direct conversion of human fibroblast to hepatocytes using a single inducible polycistronic vector.
      allows for proliferation prior to initiating direct conversion, making it possible to generate a cell line and subsequently facilitating high-throughput screening and generation of good manufacturing practice-grade iHeps. Besides this, these systems make it possible to temporarily and quantitatively activate or suppress transgenes, as well as exhibiting a higher efficiency and less side effects than the traditional overexpression of transgenes.
      • Kallunki T.
      • Barisic M.
      • Jäättelä M.
      • Liu B.
      How to choose the right inducible gene expression system for mammalian studies?.
      To minimise the risks linked to viral gene transfer, so-called “safe harbour” loci may be used in the future in order to direct cell fate conversions (Fig. 1). These safe harbour sites can be targeted using genome editing technology, resulting in stable transgene expression with minimal adverse effects on global or local gene expression.
      • DeKelver R.C.
      • Choi V.M.
      • Moehle E.A.
      • Paschon D.E.
      • Hockemeyer D.
      • Meijsing S.H.
      • et al.
      Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome.
      Yet, several articles warn of variable or inhibited transgene expression and therefore safe harbour loci should be carefully assessed.
      • Ordovás L.
      • Boon R.
      • Pistoni M.
      • Chen Y.
      • Wolfs E.
      • Guo W.
      • et al.
      Efficient recombinase-mediated cassette exchange in hPSCs to study the hepatocyte lineage reveals AAVS1 locus-mediated transgene inhibition.
      ,
      • Bhagwan J.R.
      • Collins E.
      • Mosqueira D.
      • Bakar M.
      • Johnson B.B.
      • Thompson A.
      • et al.
      Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour.
      Another way to circumvent the problem of transgene-integrated cytotoxicity is by using non-integrating viral vectors. Adenoviral (AV)
      • Song G.
      • Pacher M.
      • Balakrishnan A.
      • Yuan Q.
      • Tsay H.C.
      • Yang D.
      • et al.
      Direct reprogramming of hepatic myofibroblasts into hepatocytes in vivo attenuates liver fibrosis.
      ,
      • Cheng Z.
      • He Z.
      • Cai Y.
      • Zhang C.
      • Fu G.
      • Li H.
      • et al.
      Conversion of hepatoma cells to hepatocyte-like cells by defined hepatocyte nuclear factors.
      and adeno-associated viral (AAV)-mediated
      • Rezvani M.
      • Español-Suñer R.
      • Malato Y.
      • Dumont L.
      • Grimm A.A.
      • Kienle E.
      • et al.
      In vivo hepatic reprogramming of myofibroblasts with AAV vectors as a therapeutic strategy for liver fibrosis.
      direct hepatic reprogramming have already been tested as in vivo gene therapy approaches for hepatic malignancies. Song et al. established an in vivo AV-mediated gene therapy targeting myofibroblasts, resulting in a hepatic conversion efficiency of less than 4%. As such, they managed to attenuate the development of liver fibrosis, yet only in mild cases, presumably due to the amount of myofibroblasts present in the damaged liver.
      • Song G.
      • Pacher M.
      • Balakrishnan A.
      • Yuan Q.
      • Tsay H.C.
      • Yang D.
      • et al.
      Direct reprogramming of hepatic myofibroblasts into hepatocytes in vivo attenuates liver fibrosis.
      Using the same delivery method, Cheng and colleagues converted hepatoma cell lines into iHeps that did not give rise to hepatomas after transplantation. In addition, xenografted hepatomas efficiently shrank after intratumoral injection of the hepatic reprogramming cocktail, showing a successful hepatic reprogramming of hepatoma cells in vivo.
      • Cheng Z.
      • He Z.
      • Cai Y.
      • Zhang C.
      • Fu G.
      • Li H.
      • et al.
      Conversion of hepatoma cells to hepatocyte-like cells by defined hepatocyte nuclear factors.
      Since A(A)V vectors are already broadly being used in the clinic for a plethora of gene therapies, there is potential for clinical translation.
      • Rezvani M.
      • Español-Suñer R.
      • Malato Y.
      • Dumont L.
      • Grimm A.A.
      • Kienle E.
      • et al.
      In vivo hepatic reprogramming of myofibroblasts with AAV vectors as a therapeutic strategy for liver fibrosis.
      Yet, AV-mediated pluripotent reprogramming generally has a lower conversion efficiency (0.0002%),
      • Malik N.
      • Rao M.S.
      A review of the methods for human iPSC derivation.
      repeated transductions are required because of transgene dilution during cell proliferation
      • Cieślar-Pobuda A.
      • Knoflach V.
      • Ringh M.V.
      • Stark J.
      • Likus W.
      • Siemianowicz K.
      • et al.
      Transdifferentiation and reprogramming: overview of the processes, their similarities and differences.
      and seldom (0.001-1%) do these non-integrating viral vectors randomly integrate into the host cell’s genome.
      • Mitani K.
      • Kubo S.
      Adenovirus as an integrating vector.
      However, non-integrating Sendai viral vectors tend to be present for longer in the host cell, making pluripotent reprogramming efficiency significantly higher (1%).
      • Malik N.
      • Rao M.S.
      A review of the methods for human iPSC derivation.
      Although they are more expensive to produce and they require increased biosafety measures, Sendai viral vectors are seen as the next step for non-integrating viral-mediated direct hepatic reprogramming in order to generate footprint-free, clinical-grade iHeps.

       Integration of computational tools to improve direct hepatic reprogramming

      The inefficient and time-consuming trial and error approach that is often used to identify the optimal minimal TF combination to induce transdifferentiation can be avoided by using computational tools (excellently reviewed by Bian and Cahan
      • Bian Q.
      • Cahan P.
      Computational tools for stem cell biology.
      ). Generally, these in silico tools use lineage conversion prediction algorithms to identify the optimal combination of key regulators that activate the cellular programme of interest with maximal gene network coverage, while avoiding redundancy. This partly led to the discovery of iPSCs since the research group of Dr. Shinya Yamanaka used the FANTOM database to select candidate TFs for pluripotent reprogramming. The FANTOM consortium also formed the basis for Mogrify, a direct cellular reprogramming platform to develop ex vivo cell and in vivo reprogramming therapies.
      • Rackham O.J.L.
      • Firas J.
      • Fang H.
      • Oates M.E.
      • Holmes M.L.
      • Knaupp A.S.
      • et al.
      A predictive computational framework for direct reprogramming between human cell types.
      “eegc” is an R-tool that allows for a systemic evaluation of the cellular transition stage (insufficient, inactive, over and reverse) of the direct conversion process. In addition, the tool suggests potential TFs to be included in the direct reprogramming cocktail based on lack of activation of the gene regulatory network of the cell type of interest.
      • Zhou X.
      • Meng G.
      • Nardini C.
      • Mei H.
      Systemic evaluation of cellular reprogramming processes exploiting a novel R-tool: eegc.
      “TransSynW” is a web application which predicts cell conversions based on TFs and furthermore, identifies marker genes for assessing the lineage conversion’s effectiveness. The tool prioritises pioneer factors above tissue-specific TFs in different reprogramming cocktails, as they have a chromatin opening role. For hepatic transdifferentiation, it categorises HNF4A, FOXA3, and FOXA2 as non-specific pioneer factors and NR1I2, ZFP750, ZFHX4, HNF1A, and ZBTB48 as specific TFs, possibly uncovering new LETFs to study and potentially improve the direct hepatic conversion efficiency.
      • Ribeiro M.M.
      • Okawa S.
      • del Sol A.
      TransSynW: a single-cell RNA-sequencing based web application to guide cell conversion experiments.
      Recently, the integrative gene regulatory network (IRENE) model was published. Besides a combination of TFs governing the target’s cell type, it also considers the stochastic upregulation of specific cofactors, the remodelling of the epigenetic landscape during cell fate transition and a PiggyBac transposon gene delivery system. Since it systematically integrates a plethora of interaction and expression data to reconstruct the target’s core gene regulatory network and their cofactors, it offers the most accurate and efficient method to simulate a lineage conversion.
      • Jung S.
      • Appleton E.
      • Ali M.
      • Church G.M.
      • del Sol A.
      A computer-guided design tool to increase the efficiency of cellular conversions.

       Generation of footprint-free and clinical-grade iHeps

      Generally, the use of viral gene transfer hampers the clinical applicability of hiHeps because of the risk of integration-associated genotoxicity, (proto-)oncogene activation or deactivation of tumour suppressor genes. Furthermore, epigenetic alterations that take place during direct reprogramming may contribute to genomic instability and cause carcinogenesis. An opportune non-viral gene transfer method is mRNA transfection (Fig. 1). Simeonov et al. & Yoon et al. modified mRNA of LETFs to maximise the half-life. Via cationic lipid transfection of these synthetic modified mRNAs they demonstrated that it is possible to directly convert human fibroblasts into hiHeps.
      • Simeonov K.P.
      • Uppal H.
      Direct reprogramming of human fibroblasts to hepatocyte-like cells by synthetic modified mRNAs.
      ,
      • Yoon S.
      • Kang K.
      • Cho Y.D.
      • Kim Y.
      • Buisson E.M.
      • Yim J.H.
      • et al.
      Nonintegrating direct conversion using mRNA into hepatocyte-like cells.
      Yet, this is a very labour-intensive method as transfection has to be repeated for several consecutive days. The miRNA-302/367 cluster plays a prominent role in the regulation of both pluripotency and differentiation of human embryonic stem cells and as such acts as a powerful pluripotent reprogramming factor.
      • Anokye-Danso F.
      • Trivedi C.M.
      • Juhr D.
      • Gupta M.
      • Cui Z.
      • Tian Y.
      • et al.
      Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency.
      ,
      • Rosa A.
      • Brivanlou A.H.
      A regulatory circuitry comprised of miR-302 and the transcription factors OCT4 and NR2F2 regulates human embryonic stem cell differentiation.
      Concomitant transfection of this miRNA cluster has been shown to increase pluripotent reprogramming 10- to 15-fold compared to the Yamanaka factors alone.
      • Subramanyam D.
      • Lamouille S.
      • Judson R.L.
      • Liu J.Y.
      • Bucay N.
      • Derynck R.
      • et al.
      Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells.
      As miRNAs act as transcriptional regulators of several hundreds of genes, a strong synergistic effect is obtained. Therefore, the knowledge of miRNAs in liver health and development
      • Hsu S hao
      • Ghoshal K.
      MicroRNAs in liver health and disease.
      should be used to guide direct hepatic reprogramming. Aside from mRNA transfection, there are also other interesting non-viral transfer methods already being tested for direct hepatic reprogramming, like a PiggyBac transposon system
      • Katayama H.
      • Yasuchika K.
      • Miyauchi Y.
      • Kojima H.
      • Yamaoka R.
      • Kawai T.
      • et al.
      Generation of non-viral, transgene-free hepatocyte like cells with piggyBac transposon.
      and a oriP/EBNA1-based episomal system.
      • Kim J.
      • Kim K.P.
      • Lim K.T.
      • Lee S.C.
      • Yoon J.
      • Song G.
      • et al.
      Generation of integration-free induced hepatocyte-like cells from mouse fibroblasts.
      Both systems have great potential as they can generate footprint-free and clinical-grade iHeps, yet the low conversion efficiency of these non-integrating reprogramming methods remains a technological obstacle.
      • Malik N.
      • Rao M.S.
      A review of the methods for human iPSC derivation.
      This is possibly due to the fact that in order to direct a cell fate conversion, TFs need to be overexpressed at supraphysiological levels to overcome epigenetic barriers and the lack of cofactors.
      • Vierbuchen T.
      • Wernig M.
      Molecular roadblocks for cellular reprogramming.
      Endogenous targets can be activated by a dead clustered regularly interspaced short palindromic repeats activation (dCRISPRa) system, where the endonuclease activity of Cas9 is mutated and instead linked to transcriptional activators. These transcriptional activators have an activation domain which acts as a platform for the recruitment of a transcriptional complex.
      • Qi L.S.
      • Larson M.H.
      • Gilbert L.A.
      • Doudna J.A.
      • Weissman J.S.
      • Arkin A.P.
      • et al.
      Repurposing CRISPR as an RNA-Guided platform for sequence-specific control of gene expression.
      The dCRISPRa/Cas9 system has already been used successfully to generate iPSCs,
      • Liu P.
      • Chen M.
      • Liu Y.
      • Qi L.S.
      • Ding S.
      CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency.
      ,
      • Weltner J.
      • Balboa D.
      • Katayama S.
      • Bespalov M.
      • Krjutškov K.
      • Jouhilahti E.M.
      • et al.
      Human pluripotent reprogramming with CRISPR activators.
      neuronal cells,
      • Black J.B.
      • Adler A.F.
      • Wang H.G.
      • D’Ippolito A.M.
      • Hutchinson H.A.
      • Reddy T.E.
      • et al.
      Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9-Based transcriptional activators directly converts fibroblasts to neuronal cells.
      muscular tissue,
      • Chakraborty S.
      • Ji H.
      • Kabadi A.M.
      • Gersbach C.A.
      • Christoforou N.
      • Leong K.W.
      A CRISPR/Cas9-based system for reprogramming cell lineage specification.
      ,
      • Liu X.S.
      • Wu H.
      • Ji X.
      • Stelzer Y.
      • Wu X.
      • Czauderna S.
      • et al.
      Editing DNA methylation in the mammalian genome.
      and most recently cardiac progenitor cells.
      • Wang J.
      • Jiang X.
      • Zhao L.
      • Zuo S.
      • Chen X.
      • Zhang L.
      • et al.
      Lineage reprogramming of fibroblasts into induced cardiac progenitor cells by CRISPR/Cas9-based transcriptional activators.

       Towards personalised medicine and “off-the-shelf” iHep cell therapy

      Differences in hepatocyte development and DNA binding sites make it impossible to translate findings in mouse iHeps to a human setting.
      • Odom D.T.
      • Dowell R.D.
      • Jacobsen E.S.
      • Gordon W.
      • Danford T.W.
      • MacIsaac K.D.
      • et al.
      Tissue-specific transcriptional regulation has diverged significantly between human and mouse.
      Furthermore, most of the generated iHeps originate from foetal or embryonic fibroblasts. This tissue is still in development, subsequently meaning that the chromatin is still much more open and thus more accessible for TFs. This holds great potential for in vitro screening purposes but is of low therapeutic value for clinical applications. When the same experiment is repeated on adult fibroblasts, it is found that the obtained iHeps are less functional and the conversion efficiency is also much lower.
      • Lim K.T.
      • Lee S.C.
      • Gao Y.
      • Kim K.P.
      • Song G.
      • An S.Y.
      • et al.
      Small molecules facilitate single factor-mediated hepatic reprogramming.
      ,
      • Kim J.
      • Kim K.P.
      • Lim K.T.
      • Lee S.C.
      • Yoon J.
      • Song G.
      • et al.
      Generation of integration-free induced hepatocyte-like cells from mouse fibroblasts.
      To evade the risk of rejection and the need for a lifelong treatment with immunosuppressants, autologous cell therapy is the ultimate goal. Since fibroblast harvesting is an invasive method, other readily available and ubiquitous human adult cells, such as urine-derived epithelial-like cells
      • Tang W.
      • Guo R.
      • Shen S.
      • Zheng Y.
      • Lu Y.
      • Jiang M.
      • et al.
      Chemical cocktails enable hepatic reprogramming of human urine-derived cells with a single transcription factor.
      ,
      • Wu H.
      • Du C.
      • Yang F.
      • Zheng X.
      • Qiu D.
      • Zhang Q.
      • et al.
      Generation of hepatocyte-like cells from human urinary epithelial cells and the role of autophagy during direct reprogramming.
      and peripheral blood mononuclear cells, should be subjected to direct hepatic reprogramming. To date, 2 clinical trials with autologous iPSC-derived cells have been successfully completed, both reporting stable symptoms, no side effects and no need for immunosuppressants. The first clinical trial was executed in 2014 where autologous iPSC-derived retinal pigment epithelial cells were generated in vitro and transplanted into a patient with neovascular age-related macular degeneration.
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      • et al.
      Autologous induced stem-cell–derived retinal cells for macular degeneration.
      In the second clinical trial in 2017, autologous iPSC-derived dopamine progenitor cells were successfully transplanted into a patient suffering from idiopathic Parkinson's disease.
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      Personalized iPSC-derived dopamine progenitor cells for Parkinson’s disease.
      It is only a matter of time until the first clinical trial with autologous iPSC-HLCs or iHeps is performed to treat end-stage liver disease in humans. However, personalised cell therapy is expensive and very time-consuming.
      Last, but not least, human leukocyte antigen (HLA) homozygous human somatic cells can permit allogenic cell therapy and enable a faster and more cost-effective “off-the-shelf” iHep cell therapy. When donor and patient share the same HLA haplotype it is anticipated that the patient’s immune system will not perceive the transplanted cells as pathogenic and therefore will not induce a dangerous immune response which could lead to complications that are even more severe than the disease itself. Ideally, a biobank is created with HLA homozygous cells for each HLA type, ideally from healthy donors with blood group O, to minimise the risk of allograft rejection.
      • Taylor C.J.
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      • Chaudhry A.N.
      • Bradley J.A.
      • Bolton E.M.
      Generating an iPSC bank for HLA-matched tissue transplantation based on known donor and recipient hla types.
      Unfortunately, within the human population, the HLA genotype is highly variable and HLA homozygous donors are rare. To overcome the concern of HLA mismatching, immunocompatible cells can be generated by targeted disruption of HLA genes via genome editing technology.
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      Development of immunocompatible pluripotent stem cells via CRISPR-based human leukocyte antigen engineering.
      ,
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      Targeted disruption of HLA genes via CRISPR-cas9 generates iPSCs with enhanced immune compatibility.
      This paradigm has been tested by several research groups that compared HLA-matched and HLA-mismatched grafts in non-human primates. Their results show higher graft survival rates and less immune-cell infiltration, confirming the rationale of HLA-matching grafts. Yet, a substantial immune response is still induced in response to HLA-matched grafts when using a single (or no) immunosuppressant, showing that HLA-matching alone is not sufficient to ensure successful long-term engraftment.
      • Kawamura T.
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      Cardiomyocytes derived from MHC-homozygous induced pluripotent stem cells exhibit reduced allogeneic immunogenicity in MHC-matched non-human primates.
      • Aron Badin R.
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      MHC matching fails to prevent long-term rejection of iPSC-derived neurons in non-human primates.
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      MHC matching improves engraftment of iPSC-derived neurons in non-human primates.
      Direct hepatic reprogramming of human somatic cells is a technology that has the potential to provide autologous and HLA haplotype-matched surrogate hepatocytes. Furthermore, it can be used to directly alter cell fate of malignant cells in vivo in liver diseases.

      Conclusion

      After the introduction of pluripotent reprogramming, directly changing cell fate gained broad interest. Although direct hepatic reprogramming is still in its infancy, great progress has been made in the last decade. By overexpressing LETFs in somatic cells, it is possible to directly convert them into iHeps, greatly improving the time-efficiency to generate autologous hepatocytes for clinical application. Each iHep is the result of a unique reprogramming event, giving rise to a heterogeneous cell population. To achieve clinical standards, the direct hepatic reprogramming event needs to be standardised (Fig. 2). A controlled and stoichiometric expression of LETFs can be obtained by using inducible polycistronic constructs and by targeting “safe harbour loci” or episomal-mediated direct hepatic reprogramming, crucial safety issues linked to viral-mediated gene transfer can be circumvented. Ultimately, if the actual knowledge of pluripotent reprogramming is translated to iHep generation, footprint-free and clinical-grade iHeps can be obtained. Knowledge of hiPSC-HLC generation could be used to guide current efforts at hepatic transdifferentiation. Small molecules have been used more frequently in recent years and their added value cannot be ignored. Furthermore, the addition of supraphysiological concentrations of amino acids also holds great promise to improve the hepatic maturation of iHeps.
      Figure thumbnail gr2
      Fig. 2Strategy to generate iHeps for a clinical application and recommendations to report on iHeps.
      To directly reprogramme a somatic cell (e.g. fibroblast) into a surrogate hepatocyte (iHep), at least 1 pioneer factor is required in combination with 1 or 2 core LETFs. In case expandability is required, a progenitor TF needs to be added to the reprogramming cocktail. In case fully matured iHeps are required, at least 1 maturation TF needs to be included. To provide an unambiguous characterisation, the resulting iHeps need to be phenotypically defined at different levels according to a proposed set of minimal criteria covering key properties of hepatocytes. iHeps, induced hepatocytes; LETFs, liver-enriched transcription factors; TF, transcription factor.
      The optimal combination of LETFs remains uncertain, as crucial molecular mechanisms driving the direct hepatic reprogramming process are yet to be elucidated. Pioneer factors play a prominent role in direct hepatic reprogramming cocktails as they modulate epigenetic alterations, allow LETFs to bind to their DNA target sites and as such enable transdifferentiation (Fig. 2). Both HNF1A and HNF4A are known to drive hepatic transdifferentiation, yet there are contradicting conclusions regarding their contribution to the direct hepatic reprogramming cocktail, making it impossible to reach a consensus with our current knowledge. To obtain iHeps with a superior hepatic functionality, a maturation factor can also be included (Fig. 2). Since billions of hiHeps are needed to be clinically relevant, one can also aim to generate expandable progenitor hiHeps (via addition of a hepatic progenitor factor) that can later reach maturity to become functional competent hiHeps (Fig. 2). Lastly, a promising feature of direct hepatic reprogramming with therapeutic implications is the ability to directly alter the cell fate of malignant cells in vivo in liver diseases.
      Since the discovery that somatic cells can be pulled out of their biological lockdown, a subset of the direct hepatic reprogramming code has successfully been deciphered. Crucial hepatic transdifferentiation steps have been uncovered, paving the road towards a full cracking of the code and ultimately moving hiHeps from bench-to-bedside. The proposed minimal criteria for the characterisation of iHeps could play a key role in accomplishing this goal.

       Abbreviations

      AAV, adeno-associated viral; AV, adenoviral; ALK, activin receptor-like kinase; CEBP, CCAAT/enhancer binding protein; dCRISPRa, dead clustered regularly interspaced short palindromic repeats activation; EMT, epithelial-to-mesenchymal transition; FOX, forkhead box; hiHep, human-induced hepatocyte; hiPSC, human-induced pluripotent stem cell; hiPSC-HLC, human hepatocyte-like cell derived from iPSC; HLA, human leukocyte antigen; HNF, hepatocyte nuclear factor; iHep, induced hepatocyte; iPSC, induced pluripotent stem cell; IRES, internal ribosome entry site; LETF, liver-enriched transcription factors; MET, mesenchymal-to-epithelial transition; NR1I2, nuclear receptor subfamily 1 group I member 2; PHHs, primary human hepatocytes; TF, transcription factor.

      Financial support

      This research was funded by the Research Foundation – Flanders (FWO) grant numbers 1S73019N (MR), 1S10518N (JB), G042719N (LvG) & FWO-SBO-S001121 (LvG), Wetenschappelijk Fonds Willy Gepts (WFWG) from the UZ Brussel (JDK & LvG), and the Research Chair Mireille Aerens for Alternatives to Animal Testing .

      Authors' contributions

      MR: Conceptualisation, visualisation, writing – original draft and reviewing and editing; JB: Writing – original draft; RMR: Writing – original draft; LvG: Writing – original draft and reviewing and editing; TV: Supervision, writing – original draft; JDK: Conceptualisation, supervision, writing – original draft and reviewing and editing.

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following is the supplementary data to this article:

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