Skip to main content

Advertisement

SpringerLink
Log in

Human hepatocytes derived from pluripotent stem cells: a promising cell model for drug hepatotoxicity screening

  • Review Article
  • Published:
Archives of Toxicology Aims and scope Submit manuscript

Abstract

Drug-induced liver injury (DILI) is a frequent cause of failure in both clinical and post-approval stages of drug development, and poses a key challenge to the pharmaceutical industry. Current animal models offer poor prediction of human DILI. Although several human cell-based models have been proposed for the detection of human DILI, human primary hepatocytes remain the gold standard for preclinical toxicological screening. However, their use is hindered by their limited availability, variability and phenotypic instability. In contrast, pluripotent stem cells, which include embryonic and induced pluripotent stem cells (iPSCs), proliferate extensively in vitro and can be differentiated into hepatocytes by the addition of soluble factors. This provides a stable source of hepatocytes for multiple applications, including early preclinical hepatotoxicity screening. In addition, iPSCs also have the potential to establish genotype-specific cells from different individuals, which would increase the predictivity of toxicity assays allowing more successful clinical trials. Therefore, the generation of human hepatocyte-like cells derived from pluripotent stem cells seems to be promising for overcoming limitations of hepatocyte preparations, and it is expected to have a substantial repercussion in preclinical hepatotoxicity risk assessment in early drug development stages.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

Abbreviations

APAP:

Paracetamol or acetaminophen

BMP:

Bone morphogenic protein

CYP:

P450 enzyme

DILI:

Drug-induced liver injury

ESCs:

Embryonic stem cells

FGF:

Fibroblastic growth factor

HGF:

Hepatocyte growth factor

HLCs:

Hepatocyte-like cells

iPSCs:

Induced pluripotent stem cells

OSM:

Oncostatin

PSCs:

Pluripotent stem cells

SNPs:

Single-nucleotide polymorphisms

References

  • Alexopoulos LG, Saez-Rodriguez J, Cosgrove BD, Lauffenburger DA, Sorger PK (2010) Networks inferred from biochemical data reveal profound differences in toll-like receptor and inflammatory signaling between normal and transformed hepatocytes. Mol Cell Proteomics 9(9):1849–1865. doi:10.1074/mcp.M110.000406

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Anson BD, Kolaja KL, Kamp TJ (2011) Opportunities for use of human iPS cells in predictive toxicology. Clin Pharmacol Ther 89(5):754–758

    Article  CAS  Google Scholar 

  • Antherieu S, Chesne C, Li R, Guguen-Guillouzo C, Guillouzo A (2012) Optimization of the HepaRG cell model for drug metabolism and toxicity studies. Toxicol In Vitro 26(8):1278–1285. doi:10.1016/j.tiv.2012.05.008

    Article  CAS  PubMed  Google Scholar 

  • Asplund A, Pradip A, van Giezen M et al (2016) One standardized differentiation procedure robustly generates homogenous hepatocyte cultures displaying metabolic diversity from a large panel of human pluripotent stem cells. Stem Cell Rev 12(1):90–104. doi:10.1007/s12015-015-9621-9

    Article  CAS  PubMed  Google Scholar 

  • Baxter MA et al (2010) Generating hepatic cell lineages from pluripotent stem cells for drug toxicity screening. Stem Cell Res 5(1):4–22

    Article  CAS  Google Scholar 

  • Baxter M et al (2015) Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes. J Hepatol 62(3):581–589

    Article  CAS  Google Scholar 

  • Berger DR, Ware BR, Davidson MD, Allsup SR, Khetani SR (2015) Enhancing the functional maturity of induced pluripotent stem cell-derived human hepatocytes by controlled presentation of cell-cell interactions in vitro. Hepatology 61(4):1370–1381. doi:10.1002/hep.27621

    Article  CAS  PubMed  Google Scholar 

  • Bjornsson ES (2015) Drug-induced liver injury: an overview over the most critical compounds. Arch Toxicol 89(3):327–334. doi:10.1007/s00204-015-1456-2

    Article  CAS  PubMed  Google Scholar 

  • Bjornsson ES, Bergmann OM, Bjornsson HK, Kvaran RB, Olafsson S (2013) Incidence, presentation, and outcomes in patients with drug-induced liver injury in the general population of Iceland. Gastroenterology 144(7):1419–1425

    Article  Google Scholar 

  • Brolen G et al (2010) Hepatocyte-like cells derived from human embryonic stem cells specifically via definitive endoderm and a progenitor stage. J Biotechnol 145(3):284–294

    Article  CAS  Google Scholar 

  • Cameron K, Tan R, Schmidt-Heck W et al (2015) Recombinant laminins drive the differentiation and self-organization of hESC-derived hepatocytes. Stem Cell Reports 5(6):1250–1262. doi:10.1016/j.stemcr.2015.10.016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen YF, Tseng CY, Wang HW, Kuo HC, Yang VW, Lee OK (2012) Rapid generation of mature hepatocyte-like cells from human induced pluripotent stem cells by an efficient three-step protocol. Hepatology 55(4):1193–1203. doi:10.1002/hep.24790

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Davidson MD, Ware BR, Khetani SR (2015) Stem cell-derived liver cells for drug testing and disease modeling. Discov Med 19(106):349–358

    PubMed  PubMed Central  Google Scholar 

  • Dianat N, Steichen C, Vallier L, Weber A, Dubart-Kupperschmitt A (2013) Human pluripotent stem cells for modelling human liver diseases and cell therapy. Curr Gene Ther 13(2):120–132

    Article  CAS  Google Scholar 

  • Donato MT, Jover R, Gomez-Lechon MJ (2013) Hepatic cell lines for drug hepatotoxicity testing: limitations and strategies to upgrade their metabolic competence by gene engineering. Curr Drug Metab 14(9):946–968

    Article  CAS  Google Scholar 

  • Du Y et al (2014) Human hepatocytes with drug metabolic function induced from fibroblasts by lineage reprogramming. Cell Stem Cell 14(3):394–403

    Article  CAS  Google Scholar 

  • Gieseck RL 3rd et al (2014) Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture. PLoS One 9(1):e86372. doi:10.1371/journal.pone.0086372

    Article  CAS  PubMed  Google Scholar 

  • Giobbe GG, Michielin F, Luni C et al (2015) Functional differentiation of human pluripotent stem cells on a chip. Nat Methods 12(7):637–640. doi:10.1038/nmeth.3411

    Article  CAS  PubMed  Google Scholar 

  • Godoy P et al (2013) Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 87(8):1315–1530. doi:10.1007/s00204-013-1078-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gomez-Lechon MJ, Donato MT, Castell JV, Jover R (2004) Human hepatocytes in primary culture: the choice to investigate drug metabolism in man. Curr Drug Metab 5(5):443–462

    Article  CAS  Google Scholar 

  • Gomez-Lechon MJ, Castell JV, Donato MT (2008) An update on metabolism studies using human hepatocytes in primary culture. Expert Opin Drug Metab Toxicol 4(7):837–854. doi:10.1517/17425255.4.7.837

    Article  CAS  PubMed  Google Scholar 

  • Gomez-Lechon MJ, Tolosa L, Conde I, Donato MT (2014) Competency of different cell models to predict human hepatotoxic drugs. Expert Opin Drug Metab Toxicol 10(11):1553–1568. doi:10.1517/17425255.2014.967680

    Article  CAS  PubMed  Google Scholar 

  • Grimm FA, Iwata Y, Sirenko O, Bittner M, Rusyn I (2015) High-content assay multiplexing for toxicity screening in induced pluripotent stem cell-derived cardiomyocytes and hepatocytes. Assay Drug Dev Technol 13(9):529–546. doi:10.1089/adt.2015.659

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hannan NR, Segeritz CP, Touboul T, Vallier L (2013) Production of hepatocyte-like cells from human pluripotent stem cells. Nat Protoc 8(2):430–437

    Article  CAS  Google Scholar 

  • Hay DC et al (2008) Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling. Proc Natl Acad Sci USA 105(34):12301–12306

    Article  CAS  Google Scholar 

  • Hay DC et al (2011) Unbiased screening of polymer libraries to define novel substrates for functional hepatocytes with inducible drug metabolism. Stem Cell Res 6(2):92–102

    Article  CAS  Google Scholar 

  • Hewitt NJ et al (2007) Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev 39(1):159–234

    Article  CAS  Google Scholar 

  • Holmgren G et al (2014) Long-term chronic toxicity testing using human pluripotent stem cell-derived hepatocytes. Drug Metab Dispos 42(9):1401–1406

    Article  Google Scholar 

  • Huang P et al (2014) Direct reprogramming of human fibroblasts to functional and expandable hepatocytes. Cell Stem Cell 14(3):370–384

    Article  CAS  Google Scholar 

  • Katsuda T, Sakai Y, Ochiya T (2012) Induced pluripotent stem cell-derived hepatocytes as an alternative to human adult hepatocytes. J Stem Cells 7(1):1–17

    PubMed  Google Scholar 

  • Kia R, Sison RL, Heslop J et al (2013) Stem cell-derived hepatocytes as a predictive model for drug-induced liver injury: are we there yet? Br J Clin Pharmacol 75(4):885–896. doi:10.1111/j.1365-2125.2012.04360.x

    Article  PubMed  Google Scholar 

  • Kia R, Kelly L, Sison-Young RL et al (2015) MicroRNA-122: a novel hepatocyte-enriched in vitro marker of drug-induced cellular toxicity. Toxicol Sci 144(1):173–185. doi:10.1093/toxsci/kfu269

    Article  CAS  PubMed  Google Scholar 

  • Kim JH, Jang YJ, An SY et al (2015) Enhanced metabolizing activity of human ES cell-derived hepatocytes using a 3D culture system with repeated exposures to xenobiotics. Toxicol Sci 147(1):190–206. doi:10.1093/toxsci/kfv121

    Article  CAS  PubMed  Google Scholar 

  • Klein M, Thomas M, Hofmann U, Seehofer D, Damm G, Zanger UM (2015) A systematic comparison of the impact of inflammatory signaling on absorption, distribution, metabolism, and excretion gene expression and activity in primary human hepatocytes and HepaRG cells. Drug Metab Dispos 43(2):273–283. doi:10.1124/dmd.114.060962

    Article  CAS  PubMed  Google Scholar 

  • Kondo Y et al (2014) An efficient method for differentiation of human induced pluripotent stem cells into hepatocyte-like cells retaining drug metabolizing activity. Drug Metab Pharmacokinet 29(3):237–243

    Article  CAS  Google Scholar 

  • Li S et al (2015) Valproic acid-induced hepatotoxicity in Alpers syndrome is associated with mitochondrial permeability transition pore opening-dependent apoptotic sensitivity in an induced pluripotent stem cell model. Hepatology 61(5):1730–1739. doi:10.1002/hep.27712

    Article  CAS  PubMed  Google Scholar 

  • Lu J, Einhorn S, Venkatarangan L et al (2015) Morphological and functional characterization and assessment of iPSC-derived hepatocytes for in vitro toxicity testing. Toxicol Sci 147(1):39–54. doi:10.1093/toxsci/kfv117

    Article  CAS  PubMed  Google Scholar 

  • Mandenius CF et al (2011) Toward preclinical predictive drug testing for metabolism and hepatotoxicity by using in vitro models derived from human embryonic stem cells and human cell lines-a report on the Vitrocellomics EU-project. Altern Lab Anim 39(2):147–171

    CAS  PubMed  Google Scholar 

  • Mann DA (2015) Human induced pluripotent stem cell-derived hepatocytes for toxicology testing. Expert Opin Drug Metab Toxicol 11(1):1–5. doi:10.1517/17425255.2015.981523

    Article  CAS  PubMed  Google Scholar 

  • McGivern JV, Ebert AD (2014) Exploiting pluripotent stem cell technology for drug discovery, screening, safety, and toxicology assessments. Adv Drug Deliv Rev 69–70:170–178

    Article  Google Scholar 

  • Medine CN, Lucendo-Villarin B, Storck C et al (2013) Developing high-fidelity hepatotoxicity models from pluripotent stem cells. Stem Cells Transl Med 2(7):505–509. doi:10.5966/sctm.2012-0138

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mueller D, Kramer L, Hoffmann E, Klein S, Noor F (2014) 3D organotypic HepaRG cultures as in vitro model for acute and repeated dose toxicity studies. Toxicol In Vitro 28(1):104–112. doi:10.1016/j.tiv.2013.06.024

    Article  CAS  PubMed  Google Scholar 

  • Nakamura N, Saeki K, Mitsumoto M et al (2012) Feeder-free and serum-free production of hepatocytes, cholangiocytes, and their proliferating progenitors from human pluripotent stem cells: application to liver-specific functional and cytotoxic assays. Cell Reprogram 14(2):171–185. doi:10.1089/cell.2011.0064

    Article  CAS  PubMed  Google Scholar 

  • Norris W, Paredes AH, Lewis JH (2008) Drug-induced liver injury in 2007. Curr Opin Gastroenterol 24(3):287–297. doi:10.1097/MOG.0b013e3282f9764b00001574-200805000-00005

    Article  CAS  PubMed  Google Scholar 

  • O’Brien PJ, Chan K, Silber PM (2004) Human and animal hepatocytes in vitro with extrapolation in vivo. Chem Biol Interact 150(1):97–114

    Article  Google Scholar 

  • Park BK, Laverty H, Srivastava A, Antoine DJ, Naisbitt D, Williams DP (2011) Drug bioactivation and protein adduct formation in the pathogenesis of drug-induced toxicity. Chem Biol Interact 192(1–2):30–36

    Article  CAS  Google Scholar 

  • Rashid ST et al (2010) Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest 120(9):3127–3136

    Article  CAS  Google Scholar 

  • Roberts M et al (2014) The global intellectual property landscape of induced pluripotent stem cell technologies. Nat Biotechnol 32(8):742–748

    Article  CAS  Google Scholar 

  • Saez-Rodriguez J, Alexopoulos LG, Zhang M, Morris MK, Lauffenburger DA, Sorger PK (2011) Comparing signaling networks between normal and transformed hepatocytes using discrete logical models. Cancer Res 71(16):5400–5411. doi:10.1158/0008-5472.CAN-10-4453

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schwartz RE, Fleming HE, Khetani SR, Bhatia SN (2014) Pluripotent stem cell-derived hepatocyte-like cells. Biotechnol Adv 32(2):504–513

    Article  CAS  Google Scholar 

  • Sengupta S, Johnson BP, Swanson SA, Stewart R, Bradfield CA, Thomson JA (2014) Aggregate culture of human embryonic stem cell-derived hepatocytes in suspension are an improved in vitro model for drug metabolism and toxicity testing. Toxicol Sci 140(1):236–245

    Article  CAS  Google Scholar 

  • Simeonov KP, Uppal H (2014) Direct reprogramming of human fibroblasts to hepatocyte-like cells by synthetic modified mRNAs. PLoS One 9(6):e100134. doi:10.1371/journal.pone.0100134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sirenko O, Hesley J, Rusyn I, Cromwell EF (2014) High-content assays for hepatotoxicity using induced pluripotent stem cell-derived cells. Assay Drug Dev Technol 12(1):43–54. doi:10.1089/adt.2013.520

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Si-Tayeb K et al (2010) Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 51(1):297–305. doi:10.1002/hep.23354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sivertsson L, Synnergren J, Jensen J, Bjorquist P, Ingelman-Sundberg M (2013) Hepatic differentiation and maturation of human embryonic stem cells cultured in a perfused three-dimensional bioreactor. Stem Cells Dev 22(4):581–594. doi:10.1089/scd.2012.0202

    Article  CAS  PubMed  Google Scholar 

  • Sjogren AK, Liljevald M, Glinghammar B et al (2014) Critical differences in toxicity mechanisms in induced pluripotent stem cell-derived hepatocytes, hepatic cell lines and primary hepatocytes. Arch Toxicol 88(7):1427–1437. doi:10.1007/s00204-014-1265-z

    Article  CAS  PubMed  Google Scholar 

  • Soldatow VY, Lecluyse EL, Griffith LG, Rusyn I (2013) Models for liver toxicity testing. Toxicol Res (Camb) 2(1):23–39. doi:10.1039/C2TX20051A

    Article  CAS  Google Scholar 

  • Song Z et al (2009) Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Res 19(11):1233–1242

    Article  Google Scholar 

  • Stine JG, Lewis JH (2011) Drug-induced liver injury: a summary of recent advances. Expert Opin Drug Metab Toxicol 7(7):875–890. doi:10.1517/17425255.2011.577415

    Article  PubMed  Google Scholar 

  • Subba Rao M, Sasikala M, Nageshwar Reddy D (2013) Thinking outside the liver: induced pluripotent stem cells for hepatic applications. World J Gastroenterol 19(22):3385–3396. doi:10.3748/wjg.v19.i22.3385

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Subramanian K et al (2014) Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells. Stem Cells Dev 23(2):124–131. doi:10.1089/scd.2013.0097

    Article  CAS  PubMed  Google Scholar 

  • Szkolnicka D, Farnworth SL, Lucendo-Villarin B, Hay DC (2014) Deriving functional hepatocytes from pluripotent stem cells. Curr Protoc Stem Cell Biol 30:1G–5. doi:10.1002/9780470151808.sc01g05s30

    Article  PubMed  Google Scholar 

  • Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4):663–676

    Article  CAS  Google Scholar 

  • Takayama K, Inamura M, Kawabata K et al (2012) Efficient generation of functional hepatocytes from human embryonic stem cells and induced pluripotent stem cells by HNF4alpha transduction. Mol Ther 20(1):127–137. doi:10.1038/mt.2011.234

    Article  CAS  PubMed  Google Scholar 

  • Takayama K et al (2013) 3D spheroid culture of hESC/hiPSC-derived hepatocyte-like cells for drug toxicity testing. Biomaterials 34(7):1781–1789

    Article  CAS  Google Scholar 

  • Takayama K et al (2014) Prediction of interindividual differences in hepatic functions and drug sensitivity by using human iPS-derived hepatocytes. Proc Natl Acad Sci USA 111(47):16772–16777

    Article  CAS  Google Scholar 

  • Tasnim F, Phan D, Toh YC, Yu H (2015) Cost-effective differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules. Biomaterials 70:115–125. doi:10.1016/j.biomaterials.2015.08.002

    Article  CAS  PubMed  Google Scholar 

  • Terry C, Dhawan A, Mitry RR, Lehec SC, Hughes RD (2010) Optimization of the cryopreservation and thawing protocol for human hepatocytes for use in cell transplantation. Liver Transpl 16(2):229–237. doi:10.1002/lt.21983

    Article  PubMed  Google Scholar 

  • Tolosa L, Caron J, Hannoun Z et al (2015) Transplantation of hESC-derived hepatocytes protects mice from liver injury. Stem Cell Res Ther 6(1):246. doi:10.1186/s13287-015-0227-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Touboul T et al (2010) Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development. Hepatology 51(5):1754–1765. doi:10.1002/hep.23506

    Article  CAS  PubMed  Google Scholar 

  • Ulvestad M et al (2013) Drug metabolizing enzyme and transporter protein profiles of hepatocytes derived from human embryonic and induced pluripotent stem cells. Biochem Pharmacol 86(5):691–702

    Article  CAS  Google Scholar 

  • Vinoth KJ et al (2014) Evaluation of human embryonic stem cells and their differentiated fibroblastic progenies as cellular models for in vitro genotoxicity screening. J Biotechnol 184:154–168

    Article  CAS  Google Scholar 

  • Vunjak-Novakovic G, Bhatia S, Chen C, Hirschi K (2013) HeLiVa platform: integrated heart–liver–vascular systems for drug testing in human health and disease. Stem Cell Res Ther 4(Suppl 1):S8. doi:10.1186/scrt369

    Article  PubMed  PubMed Central  Google Scholar 

  • Ware BR, Berger DR, Khetani SR (2015) Prediction of drug-induced liver injury in micropatterned co-cultures containing iPSC-derived human hepatocytes. Toxicol Sci 145(2):252–262

    Article  CAS  Google Scholar 

  • Wobus AM, Boheler KR (2005) Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev 85(2):635–678

    Article  CAS  Google Scholar 

  • Yao R et al (2014) Hepatic differentiation of human embryonic stem cells as microscaled multilayered colonies leading to enhanced homogeneity and maturation. Small 10(21):4311–4323. doi:10.1002/smll.201401040

    Article  CAS  PubMed  Google Scholar 

  • Yi F, Liu GH, Izpisua Belmonte JC (2012) Human induced pluripotent stem cells derived hepatocytes: rising promise for disease modeling, drug development and cell therapy. Protein Cell 3(4):246–250. doi:10.1007/s13238-012-2918-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yildirimman R et al (2011) Human embryonic stem cell derived hepatocyte-like cells as a tool for in vitro hazard assessment of chemical carcinogenicity. Toxicol Sci 124(2):278–290

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors wish to thank financial support from the ALIVE Foundation.

Author information

Authors and Affiliations

  1. Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe, Hospital Universitario y Politécnico La Fe de Valencia, Torre A, 6ª Planta, Avenida Fernando Abril Martorell 106, 46026, Valencia, Spain

    María José Gómez-Lechón & Laia Tolosa

  2. CIBERehd, FIS, 08036, Barcelona, Spain

    María José Gómez-Lechón

Authors
  1. María José Gómez-Lechón
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Laia Tolosa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Laia Tolosa.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gómez-Lechón, M.J., Tolosa, L. Human hepatocytes derived from pluripotent stem cells: a promising cell model for drug hepatotoxicity screening. Arch Toxicol 90, 2049–2061 (2016). https://doi.org/10.1007/s00204-016-1756-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00204-016-1756-1

Keywords

Search

Navigation