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Induced pluripotent stem cells: A new era for hepatology

  • Samira Asgari
    Affiliations
    Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

    Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran
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  • Behshad Pournasr
    Affiliations
    Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
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  • Ghasem Hosseini Salekdeh
    Affiliations
    Department of Molecular Systems Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

    Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran
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  • Arefeh Ghodsizadeh
    Affiliations
    Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

    Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran
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  • Michael Ott
    Affiliations
    Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, and TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany
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  • Hossein Baharvand
    Correspondence
    Corresponding author. Address: Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, P.O. Box 19395-4644, Tehran, Iran. Tel.: +98 21 22306485; fax: +98 21 22310406.
    Affiliations
    Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

    Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran
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Open AccessPublished:June 29, 2010DOI:https://doi.org/10.1016/j.jhep.2010.05.009
      Stem cell transplantation has been proposed as an attractive alternative approach to restore liver mass and function. Recent progress has been reported on the generation of induced pluripotent stem (iPS) cells from somatic cells. Human-iPS cells can be differentiated towards the hepatic lineage which presents possibilities for improving research on diseases, drug development, tissue engineering, the development of bio-artificial livers, and a foundation for producing autologous cell therapies that would avoid immune rejection and enable correction of gene defects prior to cell transplantation. This focused review will discuss how human iPS cell advances are likely to have an impact on hepatology.

      Keywords

      Introduction

      Allogeneic liver transplantation is the only effective treatment available to patients with liver failure [
      • Miro J.M.
      • Laguno M.
      • Moreno A.
      • Rimola A.
      Management of end stage liver disease (ESLD): what is the current role of orthotopic liver transplantation (OLT)?.
      ]. A serious shortage of liver donors in combination with the high risk of organ rejection mandates alternative therapeutic approaches. Moreover, operative damage and, in some cases, recurrence of pre-transplant diseases can be considered as additional obstacles [
      • Lorenzini S.
      • Isidori A.
      • Catani L.
      • Gramenzi A.
      • Talarico S.
      • Bonifazi F.
      • et al.
      Stem cell mobilization and collection in patients with liver cirrhosis.
      ]. The discovery of human embryonic stem (ES) cells [
      • Thomson J.A.
      • Itskovitz-Eldor J.
      • Shapiro S.S.
      • Waknitz M.A.
      • Swiergiel J.J.
      • Marshall V.S.
      • et al.
      Embryonic stem cell lines derived from human blastocysts.
      ] has raised the hopes for curing diseases that have poor prognoses. However, after more than a decade of research, several challenges related to ES cell safety, efficacy, and bioethics have not been sufficiently answered. For example, in 2009 the US Food and Drug Administration (FDA) approved a clinical trial of human ES cell-derived oligodendrocyte progenitors in spinal cord injury patients, but it was subsequently placed on hold pending further data regarding safety issues [].
      In a groundbreaking 2006 report, Yamanaka and co-workers surprised the scientific community when they demonstrated that both mouse embryonic fibroblasts and tail tip fibroblasts could be reprogrammed into a pluripotent state similar to that observed in ES cells [
      • Takahashi K.
      • Yamanaka S.
      Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.
      ]. This was achieved by the retroviral transduction of Oct4, Sox2, Klf4, and c-Myc genes. These reprogrammed cells were named induced pluripotent stem (iPS) cells. Subsequently Yamanaka’s and Thomson’s laboratories successfully reprogrammed human somatic cells into iPS cells [
      • 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.
      ,
      • Yu J.
      • Vodyanik M.A.
      • Smuga-Otto K.
      • Antosiewicz-Bourget J.
      • Frane J.L.
      • Tian S.
      • et al.
      Induced pluripotent stem cell lines derived from human somatic cells.
      ]. Human-iPS cells have the hallmarks of ES cell attributes including: morphology, unlimited self-renewal, expression of key pluripotency genes, and a normal karyotype. Additionally, in human-iPS cells proof of functional differentiation into specialized cell lineages of all three embryonic germ layers [
      • Karumbayaram S.
      • Novitch B.G.
      • Patterson M.
      • Umbach J.A.
      • Richter L.
      • Lindgren A.
      • et al.
      Directed differentiation of human-induced pluripotent stem cells generates active motor neurons.
      ,
      • Si-Tayeb K.
      • Noto F.K.
      • Nagaoka M.
      • Li J.
      • Battle M.A.
      • Duris C.
      • et al.
      Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells.
      ,
      • Song Z.
      • Cai J.
      • Liu Y.
      • Zhao D.
      • Yong J.
      • Duo S.
      • et al.
      Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells.
      ,
      • Sullivan G.J.
      • Hay D.C.
      • Park I.H.
      • Fletcher J.
      • Hannoun Z.
      • Payne C.M.
      • et al.
      Generation of functional human hepatic endoderm from human induced pluripotent stem cells.
      ,
      • Zhang D.
      • Jiang W.
      • Liu M.
      • Sui X.
      • Yin X.
      • Chen S.
      • et al.
      Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells.
      ,
      • Zhang J.
      • Wilson G.F.
      • Soerens A.G.
      • Koonce C.H.
      • Yu J.
      • Palecek S.P.
      • et al.
      Functional cardiomyocytes derived from human induced pluripotent stem cells.
      ] have been demonstrated. This wide differentiation potential provides fascinating possibilities and tools for the study of human development and genetic diseases, in addition to their use for drug discovery and regenerative medicine (reviewed in Ref. [
      • Nishikawa S.
      • Goldstein R.A.
      • Nierras C.R.
      The promise of human induced pluripotent stem cells for research and therapy.
      ]) (Fig. 1).
      Figure thumbnail gr1
      Fig. 1Progress towards safer iPS cells and application of differentiated hepatocytes. Methods have evolved from conventional viral integration to virus-free strategies by transgene removal or the avoidance of viral integration. iPS cells have first been generated using integrating retroviral and lentiviral vectors to deliver reprogramming factors such as: OCT4, SOX2, KLF4, and c-MYC (A). Alternatively, iPS cells can be produced by the integration of reprogramming factors using the LoxP site containing plasmids, lentiviruses, or piggyBac transposon mediated gene transfer system followed by removal of transgene sequences from the host genome using Cre or transposase enzymes (B). Another strategy to generate iPS cells without viral integration used non-integrating viruses or plasmids to introduce reprogramming factors (C). Mouse and human iPS cell lines were also derived using vector or adenoviruses that transiently expressed Oct4, Sox2, Klf4, and c-Myc (C). It was also shown that virus-free mouse iPS cells could be generated using repeated plasmid transfections and by nucleofection of a single plasmid construct expressing Oct4, Sox2, Klf4, and c-Myc as a single polycistronic unit (C). Human-iPS cells were also recently generated completely free of vector and transgene sequences by transfection of a non-integrating episomal vector. Another recent strategy to create iPSCs without viral integration has been reported by protein transduction of the four reprogramming proteins fused with a cell-penetrating peptide (CPP) (D). Somatic or iPS cells can be used to treat disease by correction of the underlying genetic defect. The wild-type gene can be used to replace the defective gene by homologous recombination of somatic cells or iPS cells. The differentiated hepatic cell lineages provide fascinating possibilities and tools for the study of disease models, tissue engineering, and creation of bio-artificial livers, in addition to their use for drug discovery and regenerative medicine.
      In this review, we discuss the potentials of human-iPS cells and challenges of using these cells in hepatology.

      Methods used for differentiation and enrichment of hepatocyte-like cells from human ES and iPS cells in vitro

      The directed differentiation of human ES cells (iPS cell counterparts) into hepatocytes was first reported in 2003 when Rambhatla et al. used sodium butyrate to generate cells that could express hepatocyte markers [
      • Rambhatla L.
      • Chiu C.P.
      • Kundu P.
      • Peng Y.
      • Carpenter M.K.
      Generation of hepatocyte-like cells from human embryonic stem cells.
      ]. Subsequent reports optimized this method and directed differentiation was achieved in human ES cells by the administration of various growth factors in a time dependent manner (Table 1). For example, it was shown that Activin A and Wnt3a synergistically elicit rapid and highly efficient differentiation of human ES cells into functional hepatic endoderm [
      • Hay D.C.
      • Fletcher J.
      • Payne C.
      • Terrace J.D.
      • Gallagher R.C.
      • Snoeys J.
      • et al.
      Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling.
      ]. Recently, Basma et al. presented a simple and reproducible growth factor-mediated method to generate functional hepatocyte-like cells (HLCs) from human ES cells [
      • Basma H.
      • Soto-Gutierrez A.
      • Yannam G.R.
      • Liu L.
      • Ito R.
      • Yamamoto T.
      • et al.
      Differentiation and transplantation of human embryonic stem cell-derived hepatocytes.
      ]. Transplantation of HLCs which were enriched based on asialoglycoprotein-receptor (ASGPR) expression improved the liver mouse model [
      • Basma H.
      • Soto-Gutierrez A.
      • Yannam G.R.
      • Liu L.
      • Ito R.
      • Yamamoto T.
      • et al.
      Differentiation and transplantation of human embryonic stem cell-derived hepatocytes.
      ]. In addition to growth factors, a variety of protocols have been used for the differentiation of human ES cells towards the hepatic lineage; EB formation, co-culturing with hepatic and non-hepatic cell types, genetic manipulation, and epigenetic modifications (Table 1). Moreover, the application of small molecules has been used for the endodermal differentiation from ES cells [
      • Borowiak M.
      • Maehr R.
      • Chen S.
      • Chen A.E.
      • Tang W.
      • Fox J.L.
      • et al.
      Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells.
      ]. It has been demonstrated that these molecules can regulate specific target(s) in signaling and epigenetic mechanisms and can manipulate cell fate without genetic alterations [
      • Li W.
      • Ding S.
      Small molecules that modulate embryonic stem cell fate and somatic cell reprogramming.
      ,
      • Schugar R.C.
      • Robbins P.D.
      • Deasy B.M.
      Small molecules in stem cell self-renewal and differentiation.
      ,
      • Xu Y.
      • Shi Y.
      • Ding S.
      A chemical approach to stem-cell biology and regenerative medicine.
      ].
      Table 1Differentiation protocols that direct human ES and iPS cells toward hepatocytes.
      Differentiation protocolHepatic featuresMajor result(s)Ref.
      Human ES cells
      Stage 1 (4 days): EB formation, NaBu (5 mM) or DMSO (1%), plated on Matrigel coated plate; Stage 2 (10–11 days): DMSO (1%) (4 days), NaBu (2.5 mM) (6–7 days); Stage 3 (4 days): NaBu (2.5 mM), HGF (2.5 ng/ml)RT-PCR analysis of AFP, ALB, AAT, HNF4, AGPR, GATA4, TAT, C/EBPα, C/EBPβ; IF analysis of ALB, AAT, AFP, CK8, CK18, CK19. PAS staining, EROD assay, BrdU incorporationThe first report on differentiation of human ES cells into functional HLCs
      • Rambhatla L.
      • Chiu C.P.
      • Kundu P.
      • Peng Y.
      • Carpenter M.K.
      Generation of hepatocyte-like cells from human embryonic stem cells.
      Stage 1 (20 days): EB formation and culture in mouse primary hepatocyte CM; Stage 2 (10 days): Plated EB with or without GF including aFGF (100 ng/ml), bFGF (5 ng/ml), HGF (20 ng/ml), BMP4 (50 ng/ml)RT-PCR analysis of AFP, apolipoprotein (A4,B,H,F), fibrinogen α,β,γ, ALB, ADH1C; FACS analysis of ALB-GFPThe first report to demonstrate the possibility of purifying differentiated HLCs by genetic manipulation with further culturing
      • Lavon N.
      • Yanuka O.
      • Benvenisty N.
      Differentiation and isolation of hepatic-like cells from human embryonic stem cells.
      Stage 1 (6 days): EB formation followed by plating on collagen I; Stage 2 (8–43 days): HGF (20 ng/ml), NGF (100 ng/ml), EGF (100 ng/ml), aFGF (100 ng/ml), bFGF (100 ng/ml), RA (1 μM), OSM (10 ng/ml), bovine insulin (0.126 U/ml), Dex (100 nmol), human insulin (0.126 U/ml)qRT-PCR analysis of ALB, AAT; IF analysis of ALB, Western blot analysis of ALB; urea synthesisThe initial protocol for the differentiation of HLCs
      • Shirahashi H.
      • Wu J.
      • Yamamoto N.
      • Catana A.
      • Wege H.
      • Wager B.
      • et al.
      Differentiation of human and mouse embryonic stem cells along a hepatocyte lineage.
      Stage 1 (7–14 days): EB formation followed by plating on collagen 1 or either fibronectin, laminin, Matrigel, or uncoated; Stage 2: Serum-free media with or without HGF, OSM, aFGF, FGF7. (The time and concentrations were not available)qRT-PCR analysis of AFP, ALB, CK18, HNF3β, HNF1, CK19, GATA4, CYP1A1, CYP1A2, CYP2B6, CYP3A4, ASGPR1; IF analysis of ALB, CK18, HNF3B, HNF1, immunoblot analysis of ALB, CK18, HNF1, ASGPR1; urea production, ICG uptake, CYP activityMatrigel and collagen I had greater influence on HLC differentiation compared to laminin and fibronectin
      • Schwartz R.E.
      • Linehan J.L.
      • Painschab M.S.
      • Hu W.S.
      • Verfaillie C.M.
      • Kaufman D.S.
      Defined conditions for development of functional hepatic cells from human embryonic stem cells.
      Stage 1 (5 days): EB formation; Stage 2 (20 days): Plated on collagen I or 3D collagen scaffold, aFGF (100 ng/ml), HGF (20 ng/ml), OSM (10 ng/ml), Dex (0.1 μM), ITSRT-PCR analysis of HNF3β, AFP, TTR, AAT, CK8, CK18, CK19, ALB, CYP7A1, TDO, TAT, G6P; IF analysis of ALB, CK18; AFP & ALB production, urea synthesis, ALB production, EM (ultra structure characteristics), PAS staining, ICG uptake3D differentiation into HLCs made more functional hepatocytes compared to 2D cultures
      • Baharvand H.
      • Hashemi S.M.
      • Kazemi Ashtiani S.
      • Farrokhi A.
      Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro.
      Stage 1 (7 days): DMSO (1%); Stage 2 (9 days): HGF (2.5 or 10 ng/ml). Stage 3 (4 days): HGF (10 ng/ml), OSM (10 ng/ml)RT-PCR analysis of AFP, TTR, HNF4α, AAT, ALB, TDO, C/EBPα, hTERT; IF analysis of AFP, HNF4α, ALB, HepPar1; Western blot analysis of Sox7, Sox17, c-Met, E-cad; ICG uptake, PAS staining, ELISA (ALB, AFP), HPLC: CYP3A4 activityHGF promotes HLC differentiation in a dosedependent manner
      • Hay D.C.
      • Zhao D.
      • Ross A.
      • Mandalam R.
      • Lebkowski J.
      • Cui W.
      Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities.
      Stage 1 (9 days): EB formation; Stage 2 (10–14 days): Plated on collagen I, IMDM, FBS (20%), Dex (100 nM). Stage 3 (7 days): Transduction with AAT-GFP lentivirus. Stage 4: GFP positive cell sortingRT-PCR analysis of ALB, AAT, TF, AFP, CYP1B1, CYP1A1, CYP2B6, CYP3A4, CYP2C9, CYP2E1, ARG, G6P, HNF3|3, HNF4, C/EBPα, C/EBPβ, BMP2, BMP4, GATA4; IF analysis of ALB, AAT, AFP, CK18; PAS staining, ICG uptake, ALB production, transplantation, ELISA (human ALB in mouse blood)The first to perform a successful transplantation of bioluminescence and GFP positive HLCs, to track their fate in the animal liver, and detect human serum albumin in an animal model
      • Duan Y.
      • Catana A.
      • Meng Y.
      • Yamamoto N.
      • He S.
      • Gupta S.
      • et al.
      Differentiation and enrichment of hepatocyte-like cells from human embryonic stem cells in vitro and in vivo.
      Stage 1 (3 days): Activin A (100 ng/ml) + 0% ITS (1 day), Activin A (100 ng/ml) + 0.1% ITS (1 day), Activin A (100 ng/ml) + 1% ITS (1 day); Stage 2 (8 days): FGF4 (30 ng/ml) + BMP2 (20 ng/ml). Stage 3 (5 days): HGF (20 ng/ml). Stage 4: OSM (10 ng/ml), Dex (0.1 μM)RT-PCR analysis of AFP, ALB, CK8, CK18, AAT, HNF4α, PEPCK, TDO, TAT, CYP7A1, CYP2B6, CYP3A4, G6P, qRT-PCR analysis of ALB; IF analysis of Sox17, FoxA2, CK7, CK8, CK18, CK19, Ki67, AAT, ALB, AFP. ALB production, LDL-uptake, PAS staining, ICG uptake, PROD assay, animal model transplantation, virus assayThe first report testing the susceptibility of HLCs by HCV pseudovirus
      • Cai J.
      • Zhao Y.
      • Liu Y.
      • Ye F.
      • Song Z.
      • Qin H.
      • et al.
      Directed differentiation of human embryonic stem cells into functional hepatic cells.
      Stage 1 (5 days): Activin A (100 ng/ml), 0.5% FBS (3 days), Activin A (100 ng/ml), 2% FBS or KOSR (2 days); Stage 2 (6 days): plated on Collagen I-coated plate, FGF4 (1 0 ng/ml),and HGF (10 ng/ml) (3 days), FGF4 (10 ng/ml), HGF (10 ng/ml), MDBK-MM, BSA (0.5 mg/ml) (3 days); Stage 3 (9 days): FGF4 (10 ng/ml), HGF (10 ng/ml), OSM (10 ng/ml), Dex (0.1 μM) (9 days)RT-PCR analysis of AFP, ALB, AAT, CYP3A4, CYP7A1. IF analysis of Sox17, FOXA2, GATA4, Sox7, AFP, ALB, CD26, AAT, HNF4A, Immunoblot analysis of Sox17, FOXA2, GATA4, AFP. FACS analysis of CXCR4. ICG uptake, PAS staining, ALB production, animal model transplantationHighly purified HLCs (70% ALB-positive cells). Transplantation and homing of DE into a mouse model were demonstrated
      • Agarwal S.
      • Holton K.L.
      • Lanza R.
      Efficient differentiation of functional hepatocytes from human embryonic stem cells.
      Stage 1 (3 days): NaBu (1 mM), Activin A (100 ng/ml) (1–2 days), NaBu (0.5 mM), Activin A (100 ng/ml) (2–3 days); Stage 2 (7 days): DMSO (1%); Stage 3 (7 days): HGF (10 ng/ml), OSM (20 ng/ml), L15, FBS (8.3%)RT-PCR analysis of Nanog, hTERT, Brachy, GSC, Sox17, FoxA2, HNF4α, AFP, ALB, TAT, TTR, TDO, CAR, ApoF, PAX6, CXCR4, HNF1α, HNF1β, HNF6, CK7, CK18, CK19, Cyp3A4, CYP3A7, CYP2C19, PXR; IF analysis of FoxA2, HNF4α, AFP, ALB, HepPar, CK18, CK19, CD13, CYP3A, CPR, c-Met. flow cytometry analysis of CXCR4, Western blot analysis of FoxA2, HNF4α, AFP, ALB, AAT, c-Met, E-cad, CYP3A, CYP2D6; Plasma proteins: fibrinogen, fibronectin, A2M; PAS staining, CYP activity measurementReporting of basal metabolism activity and export proteins (alpha 2-macroglobulin, fibrinogen, and fibronectin) by HLCs
      • Hay D.C.
      • Zhao D.
      • Fletcher J.
      • Hewitt Z.A.
      • McLean D.
      • Urruticoechea-Uriguen A.
      • et al.
      Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo.
      Stage 1 (7 days): aFGF (100 ng/ml), FGF4 (10 ng/ml); Stage 2 (7 days): HGF (20 ng/ml), FGF4 (10 ng/ml); Stage 3: HGF (20 ng/ml), OSM (10 ng/ml), Dex (0.1 μM), ITSRT-PCR analysis of Nanog, HNF3β, HNF4β, C/EBPα, C/EBPβ, CK8, CK18, CK19, TTR, AFP, APOB, AAT, ALB, TDO, TAT, G6P, CYP7A1, IF analysis of ALB, CK18, HepPar1; flow cytometry analysis of ALB, CK18; urea, ALB & AFP production, PAS staining, ICG uptake, LDL-uptake, TEMDifferentiation into functional HLCs in a serum-free adherent culture condition
      • Baharvand H.
      • Hashemi S.M.
      • Shahsavani M.
      Differentiation of human embryonic stem cells into functional hepatocyte-like cells in a serum-free adherent culture condition.
      Stage 1 (2 days): EB formation; Stage 2 (3 days): Plated on 5% Matrigel-growth factor reduced, Activin A (100 ng/ml), bFGF (100 ng/ml); Stage 3 (8 days): DMSO (1%), HGF (100 ng/ml); Stage 4: Dex (0.1 μM)qRT-PCR analysis of Nanog, SOX17, SOX7, ALB, AFP, CFV||, ASGR1, PDX1, Nestin, Brachury, Pax6, Nkx 2-5, G6P, UGT, oTc, BSEP; IF analysis of ALB, AFP, FACS analysis of ASGPR, immunohistochemistry analysis of AAT, ALB; AAT & ALB production, animal model transplantation, EM, CYP activity, urea synthesisA simple and reproducible protocol for establishing functional HLCs and transplantation of ASGPR1 – enriched cells into Alb-uPA SCID mice
      • Basma H.
      • Soto-Gutierrez A.
      • Yannam G.R.
      • Liu L.
      • Ito R.
      • Yamamoto T.
      • et al.
      Differentiation and transplantation of human embryonic stem cell-derived hepatocytes.
      Stage 1 (2 days): EB formation; Stage 2 (3 days): Activin A (100 ng/ml), 1% FBS (in suspension). Stage 3 (5 days): Plated on irradiated feeder cells secreting bFGF in HepatoZYME medium; Stage 4 (6 days): HGF (20 ng/ml), OSM (10 ng/ml), Dex (0.1 μM)RT-PCR analysis of Sox17, AFP, bFGF, ALB, CK18, CYP1B1; IF analysis of Sox17, ALB, AFP; ICG uptake and release, PAS staining, ALB production, EMInduction to the early lineage stage of hepatic fate by co-culturing activin-derived endoderm with feeder cells that secreted bFGF
      • Pei H.
      • Yang Y.
      • Xi J.
      • Bai Z.
      • Yue W.
      • Nan X.
      • et al.
      Lineage restriction and differentiation of human embryonic stem cells into hepatic progenitors and zone 1 hepatocytes.
      Stage 1 (3 days): Activin A (100 ng/ml) + 0% ITS. (1 day), Activin A (100 ng/ml) + 0.1% ITS (1 day), Activin A (100 ng/ml) + 1% ITS (1 day); Stage 2 (5 days): FGF4 (30 ng/ml) + BMP2 (20 ng/ml); Stage 3 (5 days): N-CAD+ cell sorting, plated on STO feeder, HGF (20 ng/ml); Stage 4 (5 days): OSM (10 ng/ml), Dex (0.1 μM)RT-PCR analysis of AFP, CK8, CK18, ALB, AAT, TAT, CYP2B6, CYP3A7, PEPCK, CK7; IF analysis of AFP, N-CAD, ALB, HNF4A, GATA4, FOXA2, AAT, CK7; ALB secretion, PAS staining, ICG uptake, LDL uptake, PROD assayThe first proliferative bipotential hepatic progenitor cells that have been reported by sorting N-CAD+ cells
      • Zhao Q.
      • Ren H.
      • Li X.
      • Chen Z.
      • Zhang X.
      • Gong W.
      • et al.
      Differentiation of human umbilical cord mesenchymal stromal cells into low immunogenic hepatocyte-like cells.
      Stage 1: ES cells plated on fibronectin or FBS coated plate, Activin A (10 ng/ml), bFGF (12 ng/ml); Stage 2 (3 days): Ly294002 (1 μM), Activin A (100 ng/ml), BMP4 (10 ng/ml), bFGF (20 ng/ml); Stage 3 (5 days): FGF10 (50 ng/ml) (3 days), FGF10 (50 ng/ml), RA (0.1 μM), SB431542 (1 uM) (2 days); Stage 4 (10 days): HGF (50 ng/ml), FGF4 (30 ng/ml), EGF (50 ng/ml)RT-PCR analysis of Bra, MixL1, Sox17, Sox7, Lhx1, GATA6, CXCR4, Eomes, Hex, microarray analysis; IF analysis of Sox17, FoxA2, GATA4, N-cad, AAT, ALB, CK8, CK18; flow cytometry analysis of CXCR4, ASGPR, LDLR, c-met, CD49f; albumin secretion, CYP activity, PAS staining, LDL uptake, ICG uptake, animal transplantationCombination of high dosages of Activin, BMP4, bFGF in addition to PI3K inhibitor, Ly294002, lead to a more efficient DE. HLC transplantation in a mouse model with transient block of liver growth, thus allowing for better engraftment of the transplanted cells
      • Touboul T.
      • Hannan N.R.
      • Corbineau S.
      • Martinez A.
      • Martinet C.
      • Branchereau S.
      • et al.
      Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development.
      Stage 1 (1-5 days): Activin A (100 ng/ml), bFGF (4 ng/ml) or Wnt3a (50 ng/ml). FBS concentration 0% first day, 0.2% following days; Stage 2 (6–17 days): BMP4 (100 ng/ml), bFGF (4 ng/ml). Some experiments: aFGF (100 ng/ml), bFGF (5 ng/ml), BMP2 (50 ng/ml), BMP4 (200 ng/ml); Stage 3: Dex (0.1 μM), OSM (10 ng/ml), HGF (20 ng/ml), singleQuots (Lonza)RT-PCR analysis of Sox17, HNF3b, CXCR4, AFP, AAT; IF analysis of ALB, AAT, AFP, CK7, CK8, CK18, Ck19. CXCR4, CYP1A2, CYP3A4, EpCAM, HNF3b, HNF1a, HNF4a, Sox7, ICAM1, MRP2, Sox17, LFABP, Western blot analysis of AAT, AFP, CYP3A, MRP2, OATP2; ICG uptake, urea production, PAS staining, CYP activity (Midazelam, Diclofenac, Phenacetin)A more realistic DE by a combination of Activin A and bFGF (excluding extra-embryonic endoderm)
      • Brolen G.
      • Sivertsson L.
      • Bjorquist P.
      • Eriksson G.
      • Ek M.
      • Semb H.
      • et al.
      Hepatocyte-like cells derived from human embryonic stem cells specifically via definitive endoderm and a progenitor stage.
      Stage 1 (3 days): Activin A (100 ng/ml) (2 days), Activin A (100 ng/ml), B27, NaBu (0.5 mM); Stage 2 (11–15 days): Splitting cells in collagen I-coated plate or non-splitting cells, FGF4 (20 ng/ml), HGF (20 ng/ml), BMP2 (10 ng/ml), BMP4 (10 ng/ml) (1 day). The same supplement, DMSO 0.5% (10–14 days); Stage 3: FGF4 (20 ng/ml), HGF (20 ng/ml), OSM (50 ng/ml), Dex (100 nM), 0.5% DMSORT-PCR analyses of CYP1A 1, CYP1B1, CYP2A6, CYP2A7, CYP2B6, CYP2C8, CYP2C19, CYP2B1, CYP7A1, UGT1A3, UGT1A6, UGT1A8, UGT1A10 and IF analyses of CXCR4, SOX17, FOXA2, ALB, AAT, AFP, ASGPR, nuclear receptor, nuclear transporter, MRP1, OATP2, AhR, CAR, PXR, CPR, LXR; flow cytometry analysis of CXCR4, SOX17, FOXA2, AFP, ALB, AAT, ASGPR; Western blot analysis of CYP1A2, CYP3A4, CYP2C9, CYP2D6, UGT1A1, UGT2B7, GST P1-1, GST A1-1; albumin secretion, ICG uptake, metabolic activity, drug metabolismHomogenous population of HLCs (90% albumin positive, 60% ASGPR positive, and 70% AAT positive) which indicates comparable functionality to primary hepatocytes
      • Duan Y.
      • Ma X.
      • Zou W.
      • Wang C.
      • Bahbahan I.S.
      • Ahuja T.P.
      • et al.
      Differentiation and characterization of metabolically functioning hepatocytes from human embryonic stem cells.
      Human-iPS cells
      Stage 1 (3 days): Activin A (100 ng/ml); Stage 2 (4 days): FGF4 (30 ng/ml), BMP2 (20 ng/ml); Stage 3 (6 days): HGF (20 ng/ml), KGF (20 ng/ml); Stage 4 (6 days): OSM (20 ng/ml), Dex (0.1 μM). Stage 5 (3 days): OSM (20 ng/ml), Dex (0.1 μM), N2, B27RT-PCR analysis of AFP, Alb, CK8, CK18, CK19, PEPCK, HNF4α, HNF6, CEBPα, GATA4, HEX; IF analysis of Sox17, Sox7, CDX2, Foxa2, ALB, AFP, AAT, CYP7A4; urea synthesis, PAS staining, ELISA: ALB production, CYP45: inductive activity by drugHepatoblast expansion step by KGF and FGF10
      • Song Z.
      • Cai J.
      • Liu Y.
      • Zhao D.
      • Yong J.
      • Duo S.
      • et al.
      Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells.
      Stage 1 (5 days): Activin A (100 ng/ml), Wnt3 (25 ng/ml) (3 days), Activin A (100 ng/ml) (2 days); Stage 2: DMSO 1%; Stage 3: L15 medium, tryptose phosphate broth (8.3%), FBS (8.3%), hydrocortisone (10 μM), insulin (1 μM), HGF (10 ng/ml), OSM (20 ng/ml)RT-PCR analysis of AFP, Cyp7A1, HNF4a, Cyp1A2, Cyp3A4; IF analysis of albumin, E-cadherin, ELISA: AFP, TTR, fibrinogen, fibronectin; CYP activity: CYP1A2, CYP3A4The first report on the generation of functional HLCs from human-iPS cells
      • Sullivan G.J.
      • Hay D.C.
      • Park I.H.
      • Fletcher J.
      • Hannoun Z.
      • Payne C.M.
      • et al.
      Generation of functional human hepatic endoderm from human induced pluripotent stem cells.
      Stage 1 (5 days): Activin A (100 ng/ml); Stage 2 (5 days): bFGF (10 ng/ml), BMP4 (20 ng/ml); Stage 3 (5 days): HGF (20 ng/ml); Stage 4 (5 days): OSM (20 ng/ml)Oligonucleotide array analysis; IF analysis of FOXA2, SOX17, GATA4, FOXA2, HNF4a, AFP, ALB; flow cytometry analysis of ALB; PAS staining, ICG uptake, LDL uptake, urea synthesisTransplantation of HLCs into the lobe of newborn mice and with demonstration of homing
      • Si-Tayeb K.
      • Noto F.K.
      • Nagaoka M.
      • Li J.
      • Battle M.A.
      • Duris C.
      • et al.
      Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells.
      This protocol was similar to
      • Touboul T.
      • Hannan N.R.
      • Corbineau S.
      • Martinez A.
      • Martinet C.
      • Branchereau S.
      • et al.
      Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development.
      qRT-PCR analysis of HNF4a, AFP, ALB; IF analysis of HNF4a, AFP, CK8, CK18Hepatic lineage differentiation protocol for ES cells that generated HLCs from iPS cells
      • Touboul T.
      • Hannan N.R.
      • Corbineau S.
      • Martinez A.
      • Martinet C.
      • Branchereau S.
      • et al.
      Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development.
      Stage 1 (5 days): Activin A (100 ng/ml), 0.5% FBS or 1% KOSR; Stage 2 (10 days): trypsinized and plated on collagen I-coated plate, FGF4 (10 ng/ml), HGF (10 ng/ml) (2 days), minimal Madin–Darby bovine kidney maintenance medium, FGF4 (10 ng/ml), HGF (10 ng/ml); Stage 3 (10 days): SingleQuotes (lonza), FGF4 (10 ng/ml), HGF (10 ng/ml), OSM (10 ng/ml), Dex (0.1 μM)Flow cytometry analysis of CXCR4; IF analysis of AFP, ALB, AAT, CYP3A4; PAS staining; CYP activity: CYP1A2, CYP3A4Generation of HLCs from hepatocyte-derived iPSc
      • Liu H.
      • Ye Z.
      • Kim Y.
      • Sharkis S.
      • Jang Y.Y.
      Generation of endoderm-derived human induced pluripotent stem cells from primary hepatocytes.
      a2M: alpha-2-Macroglobulin, AAT: Alpha-1-Antitrypsin, aFGF: acidic Fibroblast Growth Factor, AFP: Alpha Feto Protein, AGPR: Aasialo Glyco Protein receptors, ADH1C: Alcohol dehydrogenase 1C, ALB: Albumin, APOB: Apolipoprotein B, ARG: Arginase, bFGF: basic fibroblast growth factor, BrdU: Bromo deoxyuridine, BSEP: Bile salt export pump, BMP: Bone morphogenetic protein, C/EBP: CCAAT enhance binding protein, CM: Conditioned media, CAR: Constitutive androstane receptor, CF VII: Coagulation Factor VII, CK: Cytokeratin, CM: Conditioned media, c-Met: Mesenchymal-epithelial transition factor, CPR: Cytochrome P450 reductase, DE: Definitive endoderm, DEX: Dexamethasone, DMSO: Dimethyl sulfoxide, EB: Embryoid body, E-cad: E-cadherin, EGF: Epidermal growth factor, ELISA: Enzyme linked immunosorbent assay, EM: Electron microscopy, EROD: Ethoxyresorufin-O-deethylase, FACS: Flow cytometry activated cell sorting, FBS: Fetal bovine serum, FGF4: Fibroblast growth factor, GF: Growth factor, G6p: Glucose 6-phosphatase, GFP: Green fluorescence protein, GSC: Goosecoid, HGF: Hepatocyte growth factor, HLC: Hepatocyte-like cell, HNF: Hepatocyte nuclear factor, ICG: Indocyanin green, IF: Immuno fluorescence, ITS: Insulin/transferrin/selenium, KGF: Keratinocyte growth factor, Ly294002: PI3 kinase inhibitor, N-CAD: N-cadherin, NGF: Nerve growth factor, NaBu: Sodium butyrate, OTC: Ornithine transcarbamylase, OSM: OncostatinM, PAS: Periodic acid-Schiff, PEPCK: Phospho enol pyruvate carboxykinase, PROD: Pentoxyresorufin, RA: Retinoc acid, SB431542: TGF-beta inhibitor, TAT: Tyrosine amino transferase, TDO: Tryptophan-2,3-dioxygenase, TEM: Transmission electron microscopy, TERT: Telomerase reverse transcriptase, TF: Transferin, TTR: Transthyretin, UGT: Bilirubin-UDP glucuronosyl transferase.
      It was shown that HLCs could also be generated from human-iPS cells [
      • Liu H.
      • Ye Z.
      • Kim Y.
      • Sharkis S.
      • Jang Y.Y.
      Generation of endoderm-derived human induced pluripotent stem cells from primary hepatocytes.
      ,
      • Si-Tayeb K.
      • Noto F.K.
      • Nagaoka M.
      • Li J.
      • Battle M.A.
      • Duris C.
      • et al.
      Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells.
      ,
      • Song Z.
      • Cai J.
      • Liu Y.
      • Zhao D.
      • Yong J.
      • Duo S.
      • et al.
      Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells.
      ,
      • Sullivan G.J.
      • Hay D.C.
      • Park I.H.
      • Fletcher J.
      • Hannoun Z.
      • Payne C.M.
      • et al.
      Generation of functional human hepatic endoderm from human induced pluripotent stem cells.
      ,
      • Touboul T.
      • Hannan N.R.
      • Corbineau S.
      • Martinez A.
      • Martinet C.
      • Branchereau S.
      • et al.
      Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development.
      ] (Table 1), demonstrating the efficacy of these approaches with pluripotent stem cells of diverse origins. Here, the term in vitro HLCs indicates some of the properties of mature hepatocytes (Table 1). The characteristics of stem cell-derived hepatocytes produced from various differentiation protocols have been critically reviewed [
      • Hengstler J.G.
      • Brulport M.
      • Schormann W.
      • Bauer A.
      • Hermes M.
      • Nussler A.K.
      • et al.
      Generation of human hepatocytes by stem cell technology: definition of the hepatocyte.
      ,
      • Sancho-Bru P.
      • Najimi M.
      • Caruso M.
      • Pauwelyn K.
      • Cantz T.
      • Forbes S.
      • et al.
      Stem and progenitor cells for liver repopulation: can we standardise the process from bench to bedside?.
      ]. The differentiated cells should be assessed by comparing them with primary liver-derived cells for morphology and the expression of a set of proteins such as α-fetoprotein and albumin. However, ultimately, in vitro proof that mature HLCs have been produced need demonstration of functional hepatocyte properties such as nutrient processing, detoxification, plasma protein synthesis, and engraftment, after transplantation into a suitable animal model [
      • Sancho-Bru P.
      • Najimi M.
      • Caruso M.
      • Pauwelyn K.
      • Cantz T.
      • Forbes S.
      • et al.
      Stem and progenitor cells for liver repopulation: can we standardise the process from bench to bedside?.
      ].

      Human-iPS cells as disease models

      The use of animal models is one of the established ways to study the mechanisms of liver diseases and possible therapeutic applications. Several genetic disorders which involve hepatocytes have been modeled in rodents and large animals (Supplementary Table 1). Although these models of human congenital and acquired diseases are invaluable, they provide a limited representation of human pathophysiology. Animal models do not always faithfully mimic human diseases, particularly those for human contiguous gene syndromes. For example, mice carrying the same genetic deficiencies as Fanconi’s anemia patients do not develop spontaneous bone marrow failure which is the hallmark of the human disease [
      • Chen M.
      • Tomkins D.J.
      • Auerbach W.
      • McKerlie C.
      • Youssoufian H.
      • Liu L.
      • et al.
      Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia.
      ]. Huntington’s disease patients show dyskinesia (involuntary movements), whereas mice do not [
      • Rosenthal N.
      • Brown S.
      The mouse ascending: perspectives for human-disease models.
      ]. Moreover, many genetic diseases within the liver do not have hot spots nor do they arise from mutations within a single gene. For example, the gene for α-1 antitrypsin has been mapped on the long arm of chromosome 14 and, to date, although as many as 100 alleles have been found for α-1 antitrypsin deficiency, only a small number of them are associated with liver disease [
      • Fairbanks K.D.
      • Tavill A.S.
      Liver disease in alpha 1-antitrypsin deficiency: a review.
      ]. In addition, it is difficult to undertake genetic studies of human diseases in animal models when the genes involved are unknown [
      • Alini M.
      • Eisenstein S.M.
      • Ito K.
      • Little C.
      • Kettler A.A.
      • Masuda K.
      • et al.
      Are animal models useful for studying human disc disorders/degeneration?.
      ].
      Researchers have tried to overcome these problems by using human cell cultures along with animal models as essential complements to human disease studies. Primary human cells have a limited life span in culture. As a result, most human cell lines in wide use today carry genetic and epigenetic artifacts which arise from their accommodations to tissue cultures and are derived either from malignant tissues or have been genetically modified in order to drive immortal growth [
      • Grimm S.
      The art and design of genetic screens: mammalian culture cells.
      ]. Indeed, many human cell types have never faithfully been adapted for growth in vitro. Human embryos which have been shown to carry genetic diseases by virtue of preimplantation genetic diagnoses can yield ES cell lines that model single-gene disorders [
      • Verlinsky Y.
      • Strelchenko N.
      • Kukharenko V.
      • Rechitsky S.
      • Verlinsky O.
      • Galat V.
      • et al.
      Human embryonic stem cell lines with genetic disorders.
      ]. For example, human ES cell lines have been generated for disorders such as cystic fibrosis [
      • Mateizel I.
      • De Temmerman N.
      • Ullmann U.
      • Cauffman G.
      • Sermon K.
      • Van de Velde H.
      • et al.
      Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders.
      ], Huntington’s disease [
      • Mateizel I.
      • De Temmerman N.
      • Ullmann U.
      • Cauffman G.
      • Sermon K.
      • Van de Velde H.
      • et al.
      Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders.
      ], and Fragile X syndrome [
      • Eiges R.
      • Urbach A.
      • Malcov M.
      • Frumkin T.
      • Schwartz T.
      • Amit A.
      • et al.
      Developmental study of fragile X syndrome using human embryonic stem cells derived from preimplantation genetically diagnosed embryos.
      ]. However, the vast majority of diseases that show more complex genetic patterns of inheritance are not represented in this pool.
      The ability to create pluripotent stem cell lines from patients exhibiting specific diseases may facilitate the construction of a library of iPSCs. As such, disease-specific human-iPS cells provide an unprecedented opportunity to recapitulate and investigate human pathologies in vitro. Several groups have successfully derived a wide range of iPS cells from patients with diseases (for review see [

      Seifinejad A, Tabebordbar M, Baharvand H, Boyer LA, Hosseini Salekdeh G. Progress and promise towards safe induced pluripotent stem cells for therapy. Stem Cell Rev, 2010. [Epub ahead of print].

      ]). Therefore, it is possible to generate iPS cells from patients who have inherited liver diseases. These cells can be used as instruments to study the pathogenesis, disease mechanism(s) and possible cures for inherited liver disorders. However, the demonstration of disease-related phenotypes and the ability to model pathogenesis and treatment of disease remain key challenges in this field. It should be noted that many diseases might be non-autonomous with the involvement of more than one cell type in the disease process. One possible solution would be the simultaneous culture of several cell types. Another problem is the time frame which disease symptoms are manifested. For example, amyotrophic lateral sclerosis (ALS) or Parkinson’s disease (PD) symptoms may take years to develop.

      Role of human-iPS cells in drug development and toxicity evaluation

      According to a report by the US Food and Drug Administration, “a 10-percent improvement in predicting failures before clinical trials could save $100 million in development costs per drug.” [

      FDA Report: Innovation and Stagnation-Challenge and Opportunity on the Critical Path to New Medical Products. Available from: http://wwwfdagov/oc/initiatives/criticalpath/whitepaperhtml, March 2004.

      ]. Therefore, there is increasing demand for new in vitro models to improve drug development by narrowing selected target drugs or eliminating toxic and non-effective drugs during the first stages of development. A cell-based assay should be simple, fast, reproducible, and cost effective with a consistent supply. Traditionally, whole embryo cultures and cells for drug research and analysis have been derived from animal or human tissues; either as primary cultures in which batch-to-batch variability is a common problem or as immortalized lines which often harbor uncharacterized genetic abnormalities [
      • Spielmann H.
      Reproduction and development.
      ]. The mouse ES cell test [
      • Buesen R.
      • Genschow E.
      • Slawik B.
      • Visan A.
      • Spielmann H.
      • Luch A.
      • et al.
      Embryonic stem cell test (EST) remastered: comparison between the validated EST and the new molecular FACS-EST for assessing developmental toxicity in vitro.
      ,
      • Paquette J.A.
      • Kumpf S.W.
      • Streck R.D.
      • Thomson J.J.
      • Chapin R.E.
      • Stedman D.B.
      Assessment of the Embryonic Stem Cell Test and application and use in the pharmaceutical industry.
      ] is also one of the most recently developed tests used to assess the embryotoxic potentials of test chemicals, however, it may not entirely mimic human pharmacological risk assessment due to differences in xenobiotic biotransformation pathways and capacities. Human ES cells can be utilized as in vitro models for drug development and toxicology analyses, as well [
      • Hannoun Z.
      • Fletcher J.
      • Greenhough S.
      • Medine C.
      • Samuel K.
      • Sharma R.
      • et al.
      The comparison between conditioned media and serum-free media in human embryonic stem cell culture and differentiation.
      ,
      • Jensen J.
      • Hyllner J.
      • Bjorquist P.
      Human embryonic stem cell technologies and drug discovery.
      ]. Today approximately 70% of the top 20 pharmaceutical companies utilize stem cells in their research and among these, 64% use human ES cells or their derivatives [
      • Crook J.M.
      • Kobayashi N.R.
      Human stem cells for modeling neurological disorders: accelerating the drug discovery pipeline.
      ,
      • Harding S.E.
      • Ali N.N.
      • Brito-Martins M.
      • Gorelik J.
      The human embryonic stem cell-derived cardiomyocyte as a pharmacological model.
      ,
      • Hay D.C.
      • Zhao D.
      • Fletcher J.
      • Hewitt Z.A.
      • McLean D.
      • Urruticoechea-Uriguen A.
      • et al.
      Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo.
      ,
      • Jensen J.
      • Hyllner J.
      • Bjorquist P.
      Human embryonic stem cell technologies and drug discovery.
      ]. Although of value, human ES cells and their derivatives do not encompass all the variances within a population or all ethnicities. The generation of iPS cells from individuals will more faithfully represent the highly polymorphic variants in metabolic genes of human populations and provide the pharmaceutical industry with a unique opportunity to revolutionize toxicological assays [
      • Calvo E.
      • Baselga J.
      Ethnic differences in response to epidermal growth factor receptor tyrosine kinase inhibitors.
      ,
      • Sekine I.
      • Yamamoto N.
      • Nishio K.
      • Saijo N.
      Emerging ethnic differences in lung cancer therapy.
      ,
      • Sullivan G.J.
      • Hay D.C.
      • Park I.H.
      • Fletcher J.
      • Hannoun Z.
      • Payne C.M.
      • et al.
      Generation of functional human hepatic endoderm from human induced pluripotent stem cells.
      ]. In support of this, human iPS cell-derived cardiomyocytes have recently been used to study the effect of cardioactive drugs [
      • Tanaka T.
      • Tohyama S.
      • Murata M.
      • Nomura F.
      • Kaneko T.
      • Chen H.
      • et al.
      In vitro pharmacologic testing using human induced pluripotent stem cell-derived cardiomyocytes.
      ,
      • Yokoo N.
      • Baba S.
      • Kaichi S.
      • Niwa A.
      • Mima T.
      • Doi H.
      • et al.
      The effects of cardioactive drugs on cardiomyocytes derived from human induced pluripotent stem cells.
      ]. Moreover, Lee et al. have used neural disease familial dysautonomia (FD)-iPS cells for validating the potency of candidate drugs in reversing aberrant splicing and ameliorating neuronal differentiation and migration of FD [
      • Lee G.
      • Papapetrou E.P.
      • Kim H.
      • Chambers S.M.
      • Tomishima M.J.
      • Fasano C.A.
      • et al.
      Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs.
      ].
      The liver is the primary organ involved in drug metabolism and therefore one of the most common tissues affected by drug toxicity [
      • Hay D.C.
      • Zhao D.
      • Fletcher J.
      • Hewitt Z.A.
      • McLean D.
      • Urruticoechea-Uriguen A.
      • et al.
      Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo.
      ]. Although there are no current studies, iPS cell-derived hepatocytes could soon replace hepatocytes or hepatoma cell lines in drug toxicity screening assays [
      • Guguen-Guillouzo C.
      • Corlu A.
      • Guillouzo A.
      Stem cell-derived hepatocytes and their use in toxicology.
      ,
      • Hay D.C.
      • Zhao D.
      • Fletcher J.
      • Hewitt Z.A.
      • McLean D.
      • Urruticoechea-Uriguen A.
      • et al.
      Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo.
      ]. Hepatocytes generated from iPS cells that have been derived from individuals with different cytochrome p450 polymorphisms would be of immense value for predicting potential liver toxicities of new drugs in patients [
      • Yamanaka S.
      A fresh look at iPS cells.
      ]. Additionally, liver disease-specific iPS derived hepatocytes can be utilized to discover the effects of new drugs on specific disorders before proceeding to animal studies and clinical trials.

      Human iPS cell-derived hepatocytes in a bio-artificial liver design

      Extracorporeal bio-artificial liver (BAL) systems aim to bridge patients until a suitable donor organ becomes available for whole organ or cell transplantation, or liver regeneration. BAL systems consist of viable hepatocytes in different perfusion bioreactor conformations. The patient’s blood perfuses through one of these systems, theoretically compensating for the liver’s vital functions.
      Different BAL systems are currently undergoing clinical trials [
      • Fiegel H.C.
      • Kaufmann P.M.
      • Bruns H.
      • Kluth D.
      • Horch R.E.
      • Vacanti J.P.
      • et al.
      Hepatic tissue engineering: from transplantation to customized cell-based liver directed therapies from the laboratory.
      ,
      • Mazariegos G.V.
      • Kramer D.J.
      • Lopez R.C.
      • Shakil A.O.
      • Rosenbloom A.J.
      • DeVera M.
      • et al.
      Safety observations in phase I clinical evaluation of the Excorp Medical Bioartificial Liver Support System after the first four patients.
      ,
      • Millis J.M.
      • Cronin D.C.
      • Johnson R.
      • Conjeevaram H.
      • Conlin C.
      • Trevino S.
      • et al.
      Initial experience with the modified extracorporeal liver-assist device for patients with fulminant hepatic failure: system modifications and clinical impact.
      ,
      • Sauer I.M.
      • Kardassis D.
      • Zeillinger K.
      • Pascher A.
      • Gruenwald A.
      • Pless G.
      • et al.
      Clinical extracorporeal hybrid liver support – phase I study with primary porcine liver cells.
      ,
      • van de Kerkhove M.P.
      • Di Florio E.
      • Scuderi V.
      • Mancini A.
      • Belli A.
      • Bracco A.
      • et al.
      Phase I clinical trial with the AMC-bioartificial liver.
      ]. Although these devices have bridged a number of patients to transplantation, [
      • Demetriou A.A.
      • Brown Jr., R.S.
      • Busuttil R.W.
      • Fair J.
      • McGuire B.M.
      • Rosenthal P.
      • et al.
      Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure.
      ,
      • Sauer I.M.
      • Kardassis D.
      • Zeillinger K.
      • Pascher A.
      • Gruenwald A.
      • Pless G.
      • et al.
      Clinical extracorporeal hybrid liver support – phase I study with primary porcine liver cells.
      ,
      • van de Kerkhove M.P.
      • Di Florio E.
      • Scuderi V.
      • Mancini A.
      • Belli A.
      • Bracco A.
      • et al.
      Phase I clinical trial with the AMC-bioartificial liver.
      ] their efficiency should be enhanced. Among the considerations that are necessary for the development of BAL devices (for review see [
      • Park J.K.
      • Lee D.H.
      Bioartificial liver systems: current status and future perspective.
      ]), the cellular component plays a critical role. To date, the different cell types that have been used in various BALs include primary porcine or human hepatocytes [
      • Pascher A.
      • Sauer I.M.
      • Hammer C.
      • Gerlach J.C.
      • Neuhaus P.
      Extracorporeal liver perfusion as hepatic assist in acute liver failure: a review of world experience.
      ], cell lines [
      • Roberts E.A.
      • Letarte M.
      • Squire J.
      • Yang S.
      Characterization of human hepatocyte lines derived from normal liver tissue.
      ], fetal liver cells [
      • Monga S.P.
      • Hout M.S.
      • Baun M.J.
      • Micsenyi A.
      • Muller P.
      • Tummalapalli L.
      • et al.
      Mouse fetal liver cells in artificial capillary beds in three-dimensional four-compartment bioreactors.
      ], and ES cell-derived cells [
      • Soto-Gutierrez A.
      • Kobayashi N.
      • Rivas-Carrillo J.D.
      • Navarro-Alvarez N.
      • Zhao D.
      • Okitsu T.
      • et al.
      Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell-derived hepatocytes.
      ]. Primary hepatocytes often retain their differentiated functions for a short duration in vitro. In addition, immunogenic reactions resulting from the xenogenecity of porcine hepatocyte products and the possibility of xenozoonotic retroviral infection of patients with porcine endogenous retrovirus (PERV) [
      • Wilson C.A.
      • Wong S.
      • Muller J.
      • Davidson C.E.
      • Rose T.M.
      • Burd P.
      Type C retrovirus released from porcine primary peripheral blood mononuclear cells infects human cells.
      ] are major drawbacks to using these cells. Primary human hepatocytes, on the other hand, are not available in sufficient amounts needed for clinical BAL usage. Continuous cell lines, as the other cell source, often lose functions that cells possess in vivo [
      • Nyberg S.L.
      • Remmel R.P.
      • Mann H.J.
      • Peshwa M.V.
      • Hu W.S.
      • Cerra F.B.
      Primary hepatocytes outperform Hep G2 cells as the source of biotransformation functions in a bioartificial liver.
      ].
      Human ES cells and iPS cells, however, show great promise as cell sources for BAL devices. Self-renewal and the high potential to differentiate into all cell types, including hepatocytes, make them good candidates. Optimization of the current differentiation protocols can allow these cells to substitute for presently used cell sources.

      Human-iPS cells and tissue engineering: founders of personalized regenerative medicine

      Tissue engineering approaches facilitate the pathway from laboratory to clinic. The tissue engineering technique, which mimics in vivo conditions, leads to maximum cellular function in vitro [
      • Lutolf M.P.
      • Hubbell J.A.
      Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.
      ] and eases the scale-up cultures which are important for transplantation strategies or extracorporeal bio-artificial livers.
      Under monolayer culture conditions hepatocytes lose their liver specific functions within a few days [
      • Crane L.J.
      • Miller D.L.
      Plasma protein synthesis by isolated rat hepatocytes.
      ]. Therefore, researchers have studied various extracellular matrix (ECM) compositions, scaffolds and bioreactor designs, such as galactosylated polymers [
      • Cho C.S.
      • Seo S.J.
      • Park I.K.
      • Kim S.H.
      • Kim T.H.
      • Hoshiba T.
      • et al.
      Galactose-carrying polymers as extracellular matrices for liver tissue engineering.
      ,
      • Yang J.
      • Goto M.
      • Ise H.
      • Cho C.S.
      • Akaike T.
      Galactosylated alginate as a scaffold for hepatocytes entrapment.
      ], spheroid cultures [
      • Tong J.Z.
      • De Lagausie P.
      • Furlan V.
      • Cresteil T.
      • Bernard O.
      • Alvarez F.
      Long-term culture of adult rat hepatocyte spheroids.
      ] and perfusion bioreactors [
      • De Bartolo L.
      • Salerno S.
      • Curcio E.
      • Piscioneri A.
      • Rende M.
      • Morelli S.
      • et al.
      Human hepatocyte functions in a crossed hollow fiber membrane bioreactor.
      ,
      • Shvartsman I.
      • Dvir T.
      • Harel-Adar T.
      • Cohen S.
      Perfusion cell seeding and cultivation induce the assembly of thick and functional hepatocellular tissue-like construct.
      ] to enhance primary hepatocyte function in vitro [
      • Ng S.
      • Wu Y.N.
      • Zhou Y.
      • Toh Y.E.
      • Ho Z.Z.
      • Chia S.M.
      • et al.
      Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices.
      ,
      • Sullivan J.P.
      • Gordon J.E.
      • Bou-Akl T.
      • Matthew H.W.
      • Palmer A.F.
      Enhanced oxygen delivery to primary hepatocytes within a hollow fiber bioreactor facilitated via hemoglobin-based oxygen carriers.
      ]. Differentiation of human ES and iPS cells into hepatocytes in spheroid cultures [
      • Mizumoto H.
      • Aoki K.
      • Nakazawa K.
      • Ijima H.
      • Funatsu K.
      • Kajiwara T.
      Hepatic differentiation of embryonic stem cells in HF/organoid culture.
      ] or in perfusion bioreactors and culturing ES/iPS cell-derived HLCs onto galactosylated polymers could one approach to enhance the yield and functionality of differentiated cells. Additionally, tissue engineering may assist in deriving more mature HLCs from ES and iPS cells within rationally tailored three-dimensional microenvironments [
      • Martínez E.
      • Lagunas A.
      • Mills C.
      • Rodríguez-Seguí S.
      • Estévez M.
      • Oberhansl S.
      • et al.
      Stem cell differentiation by functionalized micro- and nanostructured surfaces.
      ] which simulate the natural ECM and maintain mature, functional hepatocytes [
      • Fan J.
      • Shang Y.
      • Yuan Y.
      • Yang J.
      Preparation and characterization of chitosan/galactosylated hyaluronic acid scaffolds for primary hepatocytes culture.
      ]. In recent years, studies have demonstrated the effects of different ECMs or bioreactor configurations on the hepatic differentiation of ES cells [
      • Baharvand H.
      • Hashemi S.M.
      • Kazemi Ashtiani S.
      • Farrokhi A.
      Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro.
      ,
      • Ishii T.
      • Fukumitsu K.
      • Yasuchika K.
      • Adachi K.
      • Kawase E.
      • Suemori H.
      • et al.
      Effects of extracellular matrixes and growth factors on the hepatic differentiation of human embryonic stem cells.
      ,
      • Mizumoto H.
      • Aoki K.
      • Nakazawa K.
      • Ijima H.
      • Funatsu K.
      • Kajiwara T.
      Hepatic differentiation of embryonic stem cells in HF/organoid culture.
      ], however additional research should be undertaken to determine the optimum conditions necessary for maturation of ES cell-derived hepatocytes.
      The combination of tissue engineering with iPS cells as a personalized and easily expandable cell type shows great potential for the treatment of multiple liver diseases. By using this approach, hepatic induction of iPS cells would be the first step in personalized treatment for end stage liver diseases either through cell therapy, BAL treatment, or by personalized drug screening; all of which have been discussed in detail in this review.

      Human iPS cell transplantation for the treatment of liver disease

      Hepatocyte transplantation has recently been used as an alternative to orthotopic liver transplantation (OLT) in patients with liver-based congenital metabolic disorders such as: alpha-1 antitrypsin deficiency [
      • Strom S.C.
      • Fisher R.A.
      • Thompson M.T.
      • Sanyal A.J.
      • Cole P.E.
      • Ham J.M.
      • et al.
      Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure.
      ], urea cycle defects [
      • Horslen S.P.
      • McCowan T.C.
      • Goertzen T.C.
      • Warkentin P.I.
      • Cai H.B.
      • Strom S.C.
      • et al.
      Isolated hepatocyte transplantation in an infant with a severe urea cycle disorder.
      ,
      • Strom S.C.
      • Chowdhury J.R.
      • Fox I.J.
      Hepatocyte transplantation for the treatment of human disease.
      ], Crigler–Najjar I [
      • Ambrosino G.
      • Varotto S.
      • Strom S.C.
      • Guariso G.
      • Franchin E.
      • Miotto D.
      • et al.
      Isolated hepatocyte transplantation for Crigler–Najjar syndrome type 1.
      ,
      • Fox I.J.
      • Chowdhury J.R.
      • Kaufman S.S.
      • Goertzen T.C.
      • Chowdhury N.R.
      • Warkentin P.I.
      • et al.
      Treatment of the Crigler–Najjar syndrome type I with hepatocyte transplantation.
      ,
      • Khan A.A.
      • Parveen N.
      • Mahaboob V.S.
      • Rajendraprasad A.
      • Ravindraprakash H.R.
      • Venkateswarlu J.
      • et al.
      Treatment of Crigler–Najjar syndrome type 1 by hepatic progenitor cell transplantation: a simple procedure for management of hyperbilirubinemia.
      ], glycogenosis type Ia [
      • Muraca M.
      • Gerunda G.
      • Neri D.
      • Vilei M.T.
      • Granato A.
      • Feltracco P.
      • et al.
      Hepatocyte transplantation as a treatment for glycogen storage disease type 1a.
      ], Refsum disease (heredopathia atactica polyneuritiformis), coagulation factor deficiency [
      • Sokal E.M.
      • Smets F.
      • Bourgois A.
      • Van Maldergem L.
      • Buts J.P.
      • Reding R.
      • et al.
      Hepatocyte transplantation in a 4-year-old girl with peroxisomal biogenesis disease: technique, safety, and metabolic follow-up.
      ], argininosuccinate-lyase deficiency [
      • Stephenne X.
      • Najimi M.
      • Sibille C.
      • Nassogne M.C.
      • Smets F.
      • Sokal E.M.
      Sustained engraftment and tissue enzyme activity after liver cell transplantation for argininosuccinate lyase deficiency.
      ], and factor VII deficiency [
      • Dhawan A.
      • Mitry R.R.
      • Hughes R.D.
      • Lehec S.
      • Terry C.
      • Bansal S.
      • et al.
      Hepatocyte transplantation for inherited factor VII deficiency.
      ]. Promising results and partial correction of disease symptoms have been obtained from most of these studies [
      • Ambrosino G.
      • Varotto S.
      • Strom S.C.
      • Guariso G.
      • Franchin E.
      • Miotto D.
      • et al.
      Isolated hepatocyte transplantation for Crigler–Najjar syndrome type 1.
      ,
      • Dhawan A.
      • Mitry R.R.
      • Hughes R.D.
      • Lehec S.
      • Terry C.
      • Bansal S.
      • et al.
      Hepatocyte transplantation for inherited factor VII deficiency.
      ,
      • Fox I.J.
      • Chowdhury J.R.
      • Kaufman S.S.
      • Goertzen T.C.
      • Chowdhury N.R.
      • Warkentin P.I.
      • et al.
      Treatment of the Crigler–Najjar syndrome type I with hepatocyte transplantation.
      ,
      • Khan A.A.
      • Parveen N.
      • Mahaboob V.S.
      • Rajendraprasad A.
      • Ravindraprakash H.R.
      • Venkateswarlu J.
      • et al.
      Treatment of Crigler–Najjar syndrome type 1 by hepatic progenitor cell transplantation: a simple procedure for management of hyperbilirubinemia.
      ,
      • Muraca M.
      • Gerunda G.
      • Neri D.
      • Vilei M.T.
      • Granato A.
      • Feltracco P.
      • et al.
      Hepatocyte transplantation as a treatment for glycogen storage disease type 1a.
      ,
      • Sokal E.M.
      • Smets F.
      • Bourgois A.
      • Van Maldergem L.
      • Buts J.P.
      • Reding R.
      • et al.
      Hepatocyte transplantation in a 4-year-old girl with peroxisomal biogenesis disease: technique, safety, and metabolic follow-up.
      ,
      • Stephenne X.
      • Najimi M.
      • Sibille C.
      • Nassogne M.C.
      • Smets F.
      • Sokal E.M.
      Sustained engraftment and tissue enzyme activity after liver cell transplantation for argininosuccinate lyase deficiency.
      ]. However, the metabolic improvement does not persist and therefore it is necessary to repeat hepatocyte infusions [
      • Stephenne X.
      • Najimi M.
      • Sibille C.
      • Nassogne M.C.
      • Smets F.
      • Sokal E.M.
      Sustained engraftment and tissue enzyme activity after liver cell transplantation for argininosuccinate lyase deficiency.
      ,
      • Yannaki E.
      • Anagnostopoulos A.
      • Kapetanos D.
      • Xagorari A.
      • Iordanidis F.
      • Batsis I.
      • et al.
      Lasting amelioration in the clinical course of decompensated alcoholic cirrhosis with boost infusions of mobilized peripheral blood stem cells.
      ].
      The routine clinical deployment of cell transplantation is further complicated by the scarcity of transplantable allogeneic hepatocytes, their variable engraftment rates and the difficulty in monitoring allograft rejection. Moreover, in vitro expansion of mature hepatocytes is not feasible because long term cultivation of hepatocytes results in a reduction in hepatocyte metabolism.
      Although limited clinical success has been reported with banking cryopreserved hepatocytes [
      • Schneider A.
      • Attaran M.
      • Meier P.N.
      • Strassburg C.
      • Manns M.P.
      • Ott M.
      • et al.
      Hepatocyte transplantation in an acute liver failure due to mushroom poisoning.
      ,
      • Stephenne X.
      • Najimi M.
      • Smets F.
      • Reding R.
      • de Goyet J.
      • Sokal E.M.
      Cryopreserved liver cell transplantation controls ornithine transcarbamylase deficient patient while awaiting liver transplantation.
      ], this may not alleviate cell shortages since human hepatocytes are easily damaged during the freeze-thaw procedure. Hence, there is a need for additional cell sources such as stem or progenitor cells that can be expanded and differentiated into hepatocytes. Many studies have shown the therapeutic potential of various stem cells (for review see [
      • Duncan A.W.
      • Dorrell C.
      • Grompe M.
      Stem cells and liver regeneration.
      ] and Supplementary Table 2). The iPS cells are merging as the most promising source for the derivation of truly isogenic grafts. Differentiated derivatives of iPS cells that ameliorate a range of diseases in animal models have been reported. For instance, human iPS cell-derived neural progenitors transplanted into the brains of rats with PD generated functional dopamine neurons [
      • Cai J.
      • Yang M.
      • Poremsky E.
      • Kidd S.
      • Schneider J.S.
      • Iacovitti L.
      Dopaminergic neurons derived from human induced pluripotent stem cells survive and integrate into 6-OHDA lesioned rats.
      ] and human iPS cell-derived cardiomyocytes have demonstrated in vivo functional integration in animals with infarcted hearts [
      • Nelson T.J.
      • Martinez-Fernandez A.
      • Yamada S.
      • Perez-Terzic C.
      • Ikeda Y.
      • Terzic A.
      Repair of acute myocardial infarction by human stemness factors induced pluripotent stem cells.
      ]. Moreover, mouse iPS cells have been used as sources for transplants to restore auditory spiral ganglion neurons [
      • Nishimura K.
      • Nakagawa T.
      • Ono K.
      • Ogita H.
      • Sakamoto T.
      • Yamamoto N.
      • et al.
      Transplantation of mouse induced pluripotent stem cells into the cochlea.
      ]. Transplantation of mouse iPS cell-derived endothelial cells and endothelial progenitor cells into the livers of irradiated hemophilia A mice have increased survival rates and plasma factor VIII [
      • Xu D.
      • Alipio Z.
      • Fink L.M.
      • Adcock D.M.
      • Yang J.
      • Ward D.C.
      • et al.
      Phenotypic correction of murine hemophilia A using an iPS cell-based therapy.
      ]. To date, there is no report on the transplantation of iPS cell-derived hepatocytes into animal models, but successful differentiation of human-iPS cells into hepatocytes [
      • Si-Tayeb K.
      • Noto F.K.
      • Nagaoka M.
      • Li J.
      • Battle M.A.
      • Duris C.
      • et al.
      Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells.
      ,
      • Song Z.
      • Cai J.
      • Liu Y.
      • Zhao D.
      • Yong J.
      • Duo S.
      • et al.
      Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells.
      ,
      • Sullivan G.J.
      • Hay D.C.
      • Park I.H.
      • Fletcher J.
      • Hannoun Z.
      • Payne C.M.
      • et al.
      Generation of functional human hepatic endoderm from human induced pluripotent stem cells.
      ] has paved the way for the future application of patient-specific iPS cells to be utilized as cell therapies for liver diseases.

      Human iPS cell therapies for hereditary metabolic liver diseases

      Cell therapy of hereditary liver diseases (Supplementary Table 3) with patient-specific iPS cells would require ex vivo gene correction before (somatic cell) or after reprogramming (Fig. 1). There are reports of successful gene therapies with different cell types, including ES cells and hepatocytes, that can overcome genetic disorders such as familial hypercholesterolemia, Gligler–Najjar, hypercholestasis and albumin deficiency [
      • Ishibashi S.
      • Brown M.S.
      • Goldstein J.L.
      • Gerard R.D.
      • Hammer R.E.
      • Herz J.
      Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery.
      ,
      • Kiwaki K.
      • Kanegae Y.
      • Saito I.
      • Komaki S.
      • Nakamura K.
      • Miyazaki J.I.
      • et al.
      Correction of ornithine transcarbamylase deficiency in adult spf(ash) mice and in OTC-deficient human hepatocytes with recombinant adenoviruses bearing the CAG promoter.
      ,
      • Kobayashi K.
      • Oka K.
      • Forte T.
      • Ishida B.
      • Teng B.
      • Ishimura-Oka K.
      • et al.
      Reversal of hypercholesterolemia in low density lipoprotein receptor knockout mice by adenovirus-mediated gene transfer of the very low density lipoprotein receptor.
      ]. In a liver-directed gene therapy study of patients with homozygous familial hypercholesterolemia, Grossman et al. [
      • Grossman M.
      • Raper S.E.
      • Kozarsky K.
      • Stein E.A.
      • Engelhardt J.F.
      • Muller D.
      • et al.
      Successful ex vivo gene therapy directed to liver in a patient with familial hypercholesterolaemia.
      ] have demonstrated the feasibility of engrafting limited numbers of retroviral-transduced hepatocytes. In this study, the results have shown a lack of toxicity and persistent gene expression lasting at least four months following gene therapy. Successful reports of human hepatocyte genetic correction, using lentiviral vectors, followed by transplantation in the Gunn rat [
      • Nguyen T.H.
      • Oberholzer J.
      • Birraux J.
      • Majno P.
      • Morel P.
      • Trono D.
      Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes.
      ] [
      • Birraux J.
      • Menzel O.
      • Wildhaber B.
      • Jond C.
      • Nguyen T.H.
      • Chardot C.
      A step toward liver gene therapy: efficient correction of the genetic defect of hepatocytes isolated from a patient with Crigler–Najjar syndrome type 1 with lentiviral vectors.
      ,
      • Nguyen T.H.
      • Birraux J.
      • Wildhaber B.
      • Myara A.
      • Trivin F.
      • Le Coultre C.
      • et al.
      Ex vivo lentivirus transduction and immediate transplantation of uncultured hepatocytes for treating hyperbilirubinemic Gunn rat.
      ] and nonhuman primates [
      • Menzel O.
      • Birraux J.
      • Wildhaber B.E.
      • Jond C.
      • Lasne F.
      • Habre W.
      • et al.
      Biosafety in ex vivo gene therapy and conditional ablation of lentivirally transduced hepatocytes in nonhuman primates.
      ] have been published. These findings confirm the potential feasibility of gene therapy by using lentiviral vectors to treat liver-based inborn errors of metabolism, which are a prerequisite to clinical applications (for review see [
      • Nguyen T.H.
      • Mainot S.
      • Lainas P.
      • Groyer-Picard M.T.
      • Franco D.
      • Dagher I.
      • et al.
      Ex vivo liver-directed gene therapy for the treatment of metabolic diseases: advances in hepatocyte transplantation and retroviral vectors.
      ]).
      Patient-specific iPS cells are considered a promising alternative for an ex vivo gene therapy approach. With this strategy, mouse iPS cells have been successfully derived from a mouse model of sickle cell anemia. The defective gene was replaced by homologous recombination at the beta-globin locus prior to the generation of hematopoietic stem cells. Treated iPS cells subsequently differentiated into hematopoietic precursors which have been successfully used in the sickle cell mouse [
      • Hanna J.
      • Wernig M.
      • Markoulaki S.
      • Sun C.W.
      • Meissner A.
      • Cassady J.P.
      • et al.
      Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin.
      ]. Raya et al. corrected the genetic defect existent in somatic cells of Fanconi’s anemia patients and generated iPS cells that have the potential to differentiate into hematopoietic progenitors of phenotypically normal disease-free myeloid and erythroid lineages [
      • Raya A.
      • Rodriguez-Piza I.
      • Guenechea G.
      • Vassena R.
      • Navarro S.
      • Barrero M.J.
      • et al.
      Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells.
      ]. These data offer proof of concept that iPS cell technology can be used to generate patient-specific cells with the potential value for cell therapy applications and curing diseases.

      Induced liver progenitor cells/hepatocytes

      To generate iPS cells, somatic cells must first be completely dedifferentiated into pluripotent stem cells and then subsequently redifferentiated into the adult cell type of interest; a time-consuming procedure that risks teratoma formation of pluripotent stem cells. If the generation of either somatic stem or progenitor cells, or fully differentiated cells directly from fibroblasts or other types of somatic cells could be achieved, it might not be necessary to produce iPS cells and therefore the risk of teratoma formation would be eliminated. The reprogramming of mouse and human fibroblasts into iPS cells with a combination of reprogramming factors has raised the question of whether transcription factors could directly induce other defined somatic cell fates. A remarkable breakthrough that addressed this question was achieved when the transient activation of three transcription factors (Ngn3, Pdx1, and Mafa) induced the direct reprogramming of adult mouse pancreatic exocrine cells into insulin-producing endocrine cells with robust (20%) efficiency [
      • Zhou Q.
      • Brown J.
      • Kanarek A.
      • Rajagopal J.
      • Melton D.A.
      In vivo reprogramming of adult pancreatic exocrine cells to beta-cells.
      ]. Motoyama et al. showed reprogramming of adult hepatocytes into insulin-producing cells by nucleofection of non-viral bisictronic vectors including Pdx1 and Ngn3 [
      • Motoyama H.
      • Ogawa S.
      • Kubo A.
      • Miwa S.
      • Nakayama J.
      • Tagawa Y.
      • et al.
      In vitro reprogramming of adult hepatocytes into insulin-producing cells without viral vectors.
      ]. However, in these and most other studies, the reprogramming occurred only between closely related cell lineages. Recently, Veirbuchen et al. set out to determine whether specific transcription factors could directly reprogram fibroblasts into functional neurons [
      • Vierbuchen T.
      • Ostermeier A.
      • Pang Z.P.
      • Kokubu Y.
      • Sudhof T.C.
      • Wernig M.
      Direct conversion of fibroblasts to functional neurons by defined factors.
      ]. They began with a set of 19 candidate transcription factors involved in neuronal development or function. Eventually a combination of only three transcription factors (Ascl1, Brn2, and Myt1l) that rapidly and efficiently converted mouse embryonic and postnatal fibroblasts into functional neurons in vitro was discovered.
      Although transdifferentiation may eventually replace current technologies for generating iPS cells and ES cells, future studies will be necessary to determine whether induced differentiated cells could represent an alternative method to generate patient-specific hepatocytes.

      Challenges in the application of human-iPS cells for therapy

      Although iPS technology offers multiple treatment opportunities, substantial technical advances are necessary before clinical applications can be considered.

      Collection and establishment of iPS cells under GMP guidelines

      The starting cell material should be obtained and processed in conditions that are acceptable under good manufacturing practice (GMP) guidelines from rather young donors in order to avoid accumulation of environmental DNA damage. A recent report indicates that banked cord blood samples might offer a solution to this problem and provide “naive” cells as starting material for the future generation of clinical grade iPS cells [
      • Giorgetti A.
      • Montserrat N.
      • Aasen T.
      • Gonzalez F.
      • Rodriguez-Piza I.
      • Vassena R.
      • et al.
      Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2.
      ,
      • Haase A.
      • Olmer R.
      • Schwanke K.
      • Wunderlich S.
      • Merkert S.
      • Hess C.
      • et al.
      Generation of induced pluripotent stem cells from human cord blood.
      ]. Furthermore, the maintenance, expansion, and differentiation of iPS cells will require GMP compatible cell culture conditions which need to avoid cells, chemicals and proteins that are from animal sources. Recent reports have shown that iPS cells can be generated in serum and feeder-free conditions and maintained in chemically defined and xenobiotic-free media and supplements [
      • Sun N.
      • Panetta N.J.
      • Gupta D.M.
      • Wilson K.D.
      • Lee A.
      • Jia F.
      • et al.
      Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells.
      ,
      • Totonchi M.
      • Taei A.
      • Seifinejad A.
      • Tabebordbar M.
      • Rassouli H.
      • Farrokhi A.
      • et al.
      Feeder- and serum-free establishment and expansion of human induced pluripotent stem cells.
      ].
      However, whether there are physiological differences between human-iPS cells established and grown under standard conditions versus feeder- and serum-free conditions remains unknown.

      Generation of iPS cells without genetic modifications

      One major challenge is to design methods that involve minimal modification to the genome and generate safer iPS cells for cell therapy (for review see [

      Seifinejad A, Tabebordbar M, Baharvand H, Boyer LA, Hosseini Salekdeh G. Progress and promise towards safe induced pluripotent stem cells for therapy. Stem Cell Rev, 2010. [Epub ahead of print].

      ]). Virus-mediated delivery of reprogramming factors could result in potential harmful genomic alterations. Several groups have reported the derivation of iPS cells by integration of the four reprogramming factors with the use of plasmids [
      • Kaji K.
      • Norrby K.
      • Paca A.
      • Mileikovsky M.
      • Mohseni P.
      • Woltjen K.
      Virus-free induction of pluripotency and subsequent excision of reprogramming factors.
      ] and lentiviruses [
      • Soldner F.
      • Hockemeyer D.
      • Beard C.
      • Gao Q.
      • Bell G.W.
      • Cook E.G.
      • et al.
      Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors.
      ] followed by removing transgene sequences from the host genome using Cre-lox mediated excision (Fig. 1). The combination of a single-vector system and piggyBac transposon has been utilized for reprogramming mouse embryonic fibroblasts which resulted in the seamless elimination of vector and transgene sequences from iPS cells by transposase re-expression [
      • Kaji K.
      • Norrby K.
      • Paca A.
      • Mileikovsky M.
      • Mohseni P.
      • Woltjen K.
      Virus-free induction of pluripotency and subsequent excision of reprogramming factors.
      ,
      • Woltjen K.
      • Michael I.P.
      • Mohseni P.
      • Desai R.
      • Mileikovsky M.
      • Hamalainen R.
      • et al.
      PiggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.
      ] (Fig. 1).
      Removal of the reprogramming vector increased the similarity of iPS cells to ES cells [
      • Soldner F.
      • Hockemeyer D.
      • Beard C.
      • Gao Q.
      • Bell G.W.
      • Cook E.G.
      • et al.
      Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors.
      ] and markedly improved the developmental potential and differentiation capacity of iPS cells [
      • Sommer C.A.
      • Stadtfeld M.
      • Murphy G.J.
      • Hochedlinger K.
      • Kotton D.N.
      • Mostoslavsky G.
      Induced pluripotent stem cell generation using a single lentiviral stem cell cassette.
      ]. iPS cells were also generated without viral integration by exploiting non-integrating viruses or plasmids to introduce reprogramming factors [
      • Okita K.
      • Nakagawa M.
      • Hyenjong H.
      • Ichisaka T.
      • Yamanaka S.
      Generation of mouse induced pluripotent stem cells without viral vectors.
      ,
      • Stadtfeld M.
      • Nagaya M.
      • Utikal J.
      • Weir G.
      • Hochedlinger K.
      Induced pluripotent stem cells generated without viral integration.
      ,
      • Yu J.
      • Hu K.
      • Smuga-Otto K.
      • Tian S.
      • Stewart R.
      • Slukvin I.I.
      • et al.
      Human induced pluripotent stem cells free of vector and transgene sequences.
      ].
      The protein transduction approach has been suggested as an alternative to a nucleic acid-based approach for the reprogramming of somatic cells into iPS cells without genetic manipulation. It has been demonstrated that protein transduction domains, also called transduction peptides or cell-penetrating peptides (CPP), allow the cargo protein to enter the cell when fused to them. The generation of stable iPS cells from mouse fibroblasts has been reported by protein transduction of Oct4, Sox2, Klf4, and c-Myc fused with a CPP in combination with a histone deacetylase inhibitor, valproic acid [
      • Zhou H.
      • Wu S.
      • Joo J.Y.
      • Zhu S.
      • Han D.W.
      • Lin T.
      • et al.
      Generation of induced pluripotent stem cells using recombinant proteins.
      ]. However, human-iPS cells have been generated by exposing human neonatal fibroblasts to cell extracts from HEK293 cell lines that expressed high levels of the four reprogramming factors in the absence of additional chemicals [
      • Kim D.
      • Kim C.H.
      • Moon J.I.
      • Chung Y.G.
      • Chang M.Y.
      • Han B.S.
      • et al.
      Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.
      ] (Fig. 1). In contrast to the prior study, the efficiency of iPS cell generation is low in the protein transduction approach and requires further optimization. The application of small molecules to increase reprogramming efficiency may also facilitate reprogramming with a virus-free approach [
      • Huangfu D.
      • Osafune K.
      • Maehr R.
      • Guo W.
      • Eijkelenboom A.
      • Chen S.
      • et al.
      Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2.
      ,
      • Shi Y.
      • Do J.T.
      • Desponts C.
      • Hahm H.S.
      • Scholer H.R.
      • Ding S.
      A combined chemical and genetic approach for the generation of induced pluripotent stem cells.
      ] (Fig. 1). Using a high-throughput screening of exogenous factors and small molecules, Jaenisch and colleagues identified a small molecule, kenpaullone, that can replace Klf4 in the formation of mouse iPS cells [
      • Lyssiotis C.A.
      • Foreman R.K.
      • Staerk J.
      • Garcia M.
      • Mathur D.
      • Markoulaki S.
      • et al.
      Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4.
      ,
      • Sekine I.
      • Yamamoto N.
      • Nishio K.
      • Saijo N.
      Emerging ethnic differences in lung cancer therapy.
      ,
      • Lavon N.
      • Yanuka O.
      • Benvenisty N.
      Differentiation and isolation of hepatic-like cells from human embryonic stem cells.
      ]. The screening of small molecules that can contribute to the generation of iPS cells may eventually lead to the development of fully chemically defined conditions for the production of iPS cells.
      The application of protein transduction and small molecular approaches possibly represent a significant advance in generating iPS cells, effectively eliminating any risk of modifying the target cell genome by exogenous genetic sequences which have been associated with all previous iPS cell methods, and consequently offer a method for generating safer human-iPS cells. Moreover, removal of the reprogramming vector has increased the similarity of iPS cells to ES cells [
      • Soldner F.
      • Hockemeyer D.
      • Beard C.
      • Gao Q.
      • Bell G.W.
      • Cook E.G.
      • et al.
      Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors.
      ] thus markedly improving their developmental potential and differentiation capacity [
      • Sommer C.A.
      • Stadtfeld M.
      • Murphy G.J.
      • Hochedlinger K.
      • Kotton D.N.
      • Mostoslavsky G.
      Induced pluripotent stem cell generation using a single lentiviral stem cell cassette.
      ]. Although promising, the efficiency of iPS cell generation is low in both methods and requires further optimization. In particular, the concentrations of the individual factors need to be calibrated to approximate normal endogenous levels. Additionally, strategies for exploiting endogenous gene expression in certain cell types has also allowed for easier reprogramming and/or fewer required exogenous genes [
      • Kim D.
      • Kim C.H.
      • Moon J.I.
      • Chung Y.G.
      • Chang M.Y.
      • Han B.S.
      • et al.
      Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.
      ,
      • Kim J.B.
      • Zaehres H.
      • Wu G.
      • Gentile L.
      • Ko K.
      • Sebastiano V.
      • et al.
      Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors.
      ,
      • Shi Y.
      • Do J.T.
      • Desponts C.
      • Hahm H.S.
      • Scholer H.R.
      • Ding S.
      A combined chemical and genetic approach for the generation of induced pluripotent stem cells.
      ].

      Target cell purification before clinical application

      Assuming that ES and iPS cell-derived progenitors of mature cells or the final differentiated cells used for transplantation are successfully derived in a quantity and quality appropriate for transplantation; it will still be necessary to remove undifferentiated iPS cells and other cell types that have the potential to form tumors in vivo (for review see [
      • Kiuru M.
      • Boyer J.L.
      • O’Connor T.P.
      • Crystal R.G.
      Genetic control of wayward pluripotent stem cells and their progeny after transplantation.
      ,
      • Knoepfler P.S.
      Deconstructing stem cell tumorigenicity: a roadmap to safe regenerative medicine.
      ,
      • Lowry W.E.
      • Quan W.L.
      Roadblocks en route to the clinical application of induced pluripotent stem cells.
      ]).
      The most direct strategy is to purify cells using fluorescence activated cell sorting (FACS). The application of this method relies on the expression of a cell-type-specific reporter (e.g. promoter driving expression of a fluorescent protein) or by cell-surface molecules recognized by antibodies such as the anti-cell membrane hepatocyte marker, ASGPR [
      • Basma H.
      • Soto-Gutierrez A.
      • Yannam G.R.
      • Liu L.
      • Ito R.
      • Yamamoto T.
      • et al.
      Differentiation and transplantation of human embryonic stem cell-derived hepatocytes.
      ]. The cells could be sorted via progenitor cell specific markers (positive sorting) or stem cell specific markers (negative sorting). Consequently, it is highly desirable to identify suitable markers for the purification of human cells for clinical applications. In addition, antibody production and transfection reagents will require standardization to comply with FDA regulations. The cell-sorting procedures will also need to be carried out with dedicated flow cytometers that are free from exposure to xenobiotics. Introduction of a stem cell specific suicide gene to eliminate stem cells is another strategy to purify a given cell type [
      • Kiuru M.
      • Boyer J.L.
      • O’Connor T.P.
      • Crystal R.G.
      Genetic control of wayward pluripotent stem cells and their progeny after transplantation.
      ,
      • Knoepfler P.S.
      Deconstructing stem cell tumorigenicity: a roadmap to safe regenerative medicine.
      ,
      • Lowry W.E.
      • Quan W.L.
      Roadblocks en route to the clinical application of induced pluripotent stem cells.
      ].

      Increasing the efficiency of reprogramming

      Within three years of publication of the first iPS cells in 2006 [
      • Takahashi K.
      • Yamanaka S.
      Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.
      ], much progress has been made towards their generation and applications. However, since the generation of the first iPS cells, attempts have been made to increase the efficiency of fully-reprogrammed iPS cells for therapeutic applications.
      These include the application of various sets of reprogramming factors [
      • Liao J.
      • Wu Z.
      • Wang Y.
      • Cheng L.
      • Cui C.
      • Gao Y.
      • et al.
      Enhanced efficiency of generating induced pluripotent stem (iPS) cells from human somatic cells by a combination of six transcription factors.
      ,
      • Mali P.
      • Ye Z.
      • Hommond H.H.
      • Yu X.
      • Lin J.
      • Chen G.
      • et al.
      Improved efficiency and pace of generating induced pluripotent stem cells from human adult and fetal fibroblasts.
      ], reprogramming different somatic cell types [
      • Aasen T.
      • Raya A.
      • Barrero M.J.
      • Garreta E.
      • Consiglio A.
      • Gonzalez F.
      • et al.
      Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.
      ,
      • Eminli S.
      • Foudi A.
      • Stadtfeld M.
      • Maherali N.
      • Ahfeldt T.
      • Mostoslavsky G.
      • et al.
      Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells.
      ,
      • Utikal J.
      • Polo J.M.
      • Stadtfeld M.
      • Maherali N.
      • Kulalert W.
      • Walsh R.M.
      • et al.
      Immortalization eliminates a roadblock during cellular reprogramming into iPS cells.
      ], application of chemicals/small molecules to increase efficiency, replacing reprogramming factors [
      • Huangfu D.
      • Maehr R.
      • Guo W.
      • Eijkelenboom A.
      • Snitow M.
      • Chen A.E.
      • et al.
      Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds.
      ,
      • Motoyama H.
      • Ogawa S.
      • Kubo A.
      • Miwa S.
      • Nakayama J.
      • Tagawa Y.
      • et al.
      In vitro reprogramming of adult hepatocytes into insulin-producing cells without viral vectors.
      ,
      • Shi Y.
      • Desponts C.
      • Do J.T.
      • Hahm H.S.
      • Scholer H.R.
      • Ding S.
      Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds.
      ], and the use of hypoxia conditions [
      • Kobayashi H.
      • Yamada Y.
      • Kanayama S.
      • Furukawa N.
      • Noguchi T.
      • Haruta S.
      • et al.
      The role of hepatocyte nuclear factor-1beta in the pathogenesis of clear cell carcinoma of the ovary.
      ]. Nevertheless, the generation of iPS cells has, so far, been an inefficient process (<2%) resulting in a heterogeneous population of cells which makes the identification of fully-reprogrammed iPS cells challenging. More recently, several groups have shown that the inactivation of p53 remarkably increased the efficiency of iPS cellular generation [
      • Hong H.
      • Takahashi K.
      • Ichisaka T.
      • Aoi T.
      • Kanagawa O.
      • Nakagawa M.
      • et al.
      Suppression of induced pluripotent stem cell generation by the p53-p21 pathway.
      ,
      • Kawamura T.
      • Suzuki J.
      • Wang Y.V.
      • Menendez S.
      • Morera L.B.
      • Raya A.
      • et al.
      Linking the p53 tumour suppressor pathway to somatic cell reprogramming.
      ,
      • Marion 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.
      ,
      • Utikal J.
      • Polo J.M.
      • Stadtfeld M.
      • Maherali N.
      • Kulalert W.
      • Walsh R.M.
      • et al.
      Immortalization eliminates a roadblock during cellular reprogramming into iPS cells.
      ]. However, the inactivation of p53 may result in low quality iPS cells, thus causing genomic abnormalities. Given the rapid advancements within the iPS cell field, it is most likely that highly efficient, safe iPS cells will be routinely generated in the near future.

      Patient-specific iPS cells: custom- versus ready-made iPS cells

      A potential benefit would be the ability to generate patient-specific iPS cells that can be transplanted without the concern of rejection or the need for immunosuppressive drugs. However, custom-made iPS cells may not be available any time soon. Even if the capability to generate safe iPS cells becomes available, the generation and expansion of iPS cells would take a couple of months. Moreover, several more months will be needed to differentiate iPS cells into the required cell types and to subsequently expand them. Subsequently, several steps should be taken to ensure the cells are safe for transplantation and do not form tumors.
      All in all, the generation of custom-made iPS cells will be a very costly process and therefore only available to a subset of the population. Additionally the process is presently too slow for the treatment of disorders such as spinal cord injuries which would need prompt treatment. Therefore, ready-made iPS cells have been proposed as a solution. Japanese scientists are preparing a national library of therapy-ready iPS cell lines. It has been estimated that 50 well-chosen cell lines could provide close immunological matches for 90% of the Japanese population.
      Therefore, patients who require urgent treatment could use the best immunological match, whereas people with chronic disorders might chose to have an iPS cell line that is patient specific, provided they could afford it [
      • Cyranoski D.
      Stem cells: 5 things to know before jumping on the iPS bandwagon.
      ].

      Generation of fully-reprogrammed iPS cells

      It has been shown that some of the iPS clones are only partially reprogrammed into an ES cell state [
      • Chan E.M.
      • Ratanasirintrawoot S.
      • Park I.H.
      • Manos P.D.
      • Loh Y.H.
      • Huo H.
      • et al.
      Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells.
      ,
      • Lowry W.E.
      • Richter L.
      • Yachechko R.
      • Pyle A.D.
      • Tchieu J.
      • Sridharan R.
      • et al.
      Generation of human induced pluripotent stem cells from dermal fibroblasts.
      ]. Shutdown of the exogenously expressed transcription factors which can be detected early during reprogramming is one of the most specific single markers of reprogramming success. Lowry et al. have reported in their research, that some of the human iPS clones generated were only partially reprogrammed to an ES cell state whereas other clones appeared faithfully reprogrammed, as measured by their gene expression program and their inability to form embryoid bodies [
      • Lowry W.E.
      • Richter L.
      • Yachechko R.
      • Pyle A.D.
      • Tchieu J.
      • Sridharan R.
      • et al.
      Generation of human induced pluripotent stem cells from dermal fibroblasts.
      ]. Recently, Chen et al. identified three distinct colony types which morphologically resembled ES cells but differed in molecular phenotype and differentiation potential [
      • Chan E.M.
      • Ratanasirintrawoot S.
      • Park I.H.
      • Manos P.D.
      • Loh Y.H.
      • Huo H.
      • et al.
      Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells.
      ]. They demonstrated that incompletely reprogrammed colonies exhibited some features of reprogramming; including down-regulation of fibroblast markers, expression of pluripotency markers, changes in the histone modification state, formation of ES cell–like morphology, and the ability to differentiate into teratoma-like tumors. According to Chen et al., SSEA-4, alkaline phosphatase, hTERT or GDF-3 were insufficient to reliably distinguish a fully reprogrammed from a partially reprogrammed state whereas proviral silencing and expression of TRA-1-60, DNMT3B and REX1 were suggested as markers to distinguish between fully and partially reprogrammed iPS cells. Clearly more work should be undertaken for the rigorous characterization and standardization of putative iPS cells [
      • Chan E.M.
      • Ratanasirintrawoot S.
      • Park I.H.
      • Manos P.D.
      • Loh Y.H.
      • Huo H.
      • et al.
      Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells.
      ].

      Are iPSCs and ESCs functionally equivalent?

      An important outstanding question is the extent to which iPS cells and ES cells are functionally equivalent. The first mouse iPS cells generated by Yamanaka and Takahashi were similar to ES cells in the expression of certain ES cell marker genes, morphology, proliferation, and the formation of teratomas but these cells failed to produce adult chimeric mice and showed a different global gene expression [
      • Takahashi K.
      • Yamanaka S.
      Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.
      ]. Germline transmission with mouse iPS cells has been reported [
      • Boland M.J.
      • Hazen J.L.
      • Nazor K.L.
      • Rodriguez A.R.
      • Gifford W.
      • Martin G.
      • et al.
      Adult mice generated from induced pluripotent stem cells.
      ,
      • Zhao X.Y.
      • Li W.
      • Lv Z.
      • Liu L.
      • Tong M.
      • Hai T.
      • et al.
      IPS cells produce viable mice through tetraploid complementation.
      ]. However, several studies have shown that although human-iPS cells are quite similar to human ES cells, they are clearly not identical. Yamanaka and his colleagues reported at least 1267 genes that display a greater than five-fold difference in expression levels in iPS cells when compared with human ES cells [
      • 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.
      ]. Chan et al. applied genome-wide methods to compare mouse and human-iPS cells with ES cells’ subkaryotypic genome alteration, mRNA, small noncoding RNA expression and histone modification profiling [
      • Chan E.M.
      • Ratanasirintrawoot S.
      • Park I.H.
      • Manos P.D.
      • Loh Y.H.
      • Huo H.
      • et al.
      Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells.
      ]. They showed that iPS cells represented a unique type of pluripotent cell as defined by gene expression and should be considered as a unique subtype of pluripotent cell.
      Recently, it has been shown that removal of the reprogramming vector increased the similarity of iPS cells to ES cells [
      • Soldner F.
      • Hockemeyer D.
      • Beard C.
      • Gao Q.
      • Bell G.W.
      • Cook E.G.
      • et al.
      Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors.
      ] and markedly improved the developmental potential and differentiation capacity of iPS cells [
      • Sommer C.A.
      • Stadtfeld M.
      • Murphy G.J.
      • Hochedlinger K.
      • Kotton D.N.
      • Mostoslavsky G.
      Induced pluripotent stem cell generation using a single lentiviral stem cell cassette.
      ]. The detailed implications of these differences between iPS cells and ES cells and their functional role in self-renewal or differentiation are unknown and need extensive investigation.

      Cell source for reprogramming

      Another important issue facing researchers concerns the varied origins of iPS cells which may affect their propensities to differentiate and safety. Certain cell types may be better suited for complete reprogramming with reduced risks of teratoma formation [
      • Aoi T.
      • Yae K.
      • Nakagawa M.
      • Ichisaka T.
      • Okita K.
      • Takahashi K.
      • et al.
      Generation of pluripotent stem cells from adult mouse liver and stomach cells.
      ,
      • Miura K.
      • Okada Y.
      • Aoi T.
      • Okada A.
      • Takahashi K.
      • Okita K.
      • et al.
      Variation in the safety of induced pluripotent stem cell lines.
      ]. It has been assumed that the production of pancreatic-β cells and hepatocytes from somatic cell-derived iPS cells of an endodermal origin, such as gastric epithelial cells or hepatocytes, may be more effective; however, direct evidence for this assumption is lacking. Recently, Miura et al. have reported that hepatocyte and gastric epithelial-iPS cell clones fail to generate neurospheres while fibroblast-iPS cells are efficient neurosphere producers [
      • Miura K.
      • Okada Y.
      • Aoi T.
      • Okada A.
      • Takahashi K.
      • Okita K.
      • et al.
      Variation in the safety of induced pluripotent stem cell lines.
      ]. Notably, iPS cells derived from epithelial cells and tissues such as the liver, gastric mucosa [
      • Aoi T.
      • Yae K.
      • Nakagawa M.
      • Ichisaka T.
      • Okita K.
      • Takahashi K.
      • et al.
      Generation of pluripotent stem cells from adult mouse liver and stomach cells.
      ], and skin [
      • Aasen T.
      • Raya A.
      • Barrero M.J.
      • Garreta E.
      • Consiglio A.
      • Gonzalez F.
      • et al.
      Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.
      ] have less retroviral integration than iPS cells derived from fibroblasts. Those iPS cells which have been derived from mouse hepatocytes or human keratinocytes show fewer retroviral integration sites when compared with iPS cells derived from fibroblasts. More recently, the generation of iPS cells from human hepatocytes was reported [
      • Liu H.
      • Ye Z.
      • Kim Y.
      • Sharkis S.
      • Jang Y.Y.
      Generation of endoderm-derived human induced pluripotent stem cells from primary hepatocytes.
      ], however, more studies should examine whether hepatocytes from these iPS cells offer any advantages over differentiated cells from human ES cells or from iPS cells of other origins.

      Concluding remarks

      Human-iPS cells bypass the ethical and immunological concerns associated with human ES cells. They represent an unlimited resource for in vitro modeling and the development of patient-specific medicines suitable for treating a number of deficiency states, including human liver disease. That being said, human iPS cell technology is still in its infancy and a number of hurdles need to be overcome before cell therapies become a reality. These include: (i) generation of iPS cells without viral integration, (ii) increasing the efficiency of iPS cell production such that enough cells will be generated for iPS cell screening and quality evaluation, (iii) differentiation of iPS cells into desired cell types that are comparable to their in vivo counterparts, (iv) isolation of differentiated cells with sufficient purity, and (v) the proper homing and function of iPS cells post-transplantation.
      Given the rapid pace of developments within the iPS cell field, it is likely that the generation of safe and effective iPS cells for use in cell therapy will be achieved in the near future.

      Conflict of interest

      The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The authors who have taken part in this study do not have a relationship with the manufacturers of the drugs involved either in the past or present and did not receive funding from the manufacturers to carry out their research.

      Acknowledgements

      This study was supported by a grant from Royan Institute . M. Ott was supported by the Excellence Cluster “Rebirth” of the German Research Foundation .

      Supplementary data

      • Supplementary data 1

        Supplementary Table 1. Animal models of liver genetic disorders; Supplementary Table 2. Potential transplantable cell sources for liver failure therapy; Supplementary Table 3. Liver genetic disorders and their related genes; and References.

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