Generation of hepatocyte-like cells from human induced pluripotent stem (iPS) cells by co-culturing embryoid body cells with liver non-parenchymal cell line TWNT-1

M. Shahid Javed, Naeem Yaqoob, Masaya Iwamuro, Naoya Kobayashi, Toshiyoshi Fujiwara

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Objective: To generate a homogeneous population of patient-specific hepatocyte-like cells (HLCs) from humaniPS cells those show the morphologic and phenotypic properties of primary human hepatocytes. Study Design: An experimental study. Place and Duration of Study: Department of Surgery, Okayama University, Graduate School of Medicine, Japan, from April to December 2011. Methodology: Human iPS cells were generated and maintained on ES qualified matrigel coated plates supplemented with mTeSR medium or alternatively on mitotically inactivated MEF feeder layer in DMEM/F12 medium containing 20% KOSR, 4ng/ml bFGF-2, 1 × 10-4 M 2-mercaptoethanol, 1 mmol/L NEAA, 2mML-glutamine and 1% penicillin-streptomycin. iPS cells were differentiated to HLCs by sequential culture using a four step differentiation protocol: (I) Generation of embryoid bodies (EBs) in suspension culture; (II) Induction of definitive endoderm (DE) from 2 days old EBs by growth in human activin-A (100 ng/ml) and basic fibroblastsgrowth factor (bFGF2) (100 ng/ml) on matrigel coated plates; (III) Induction of hepatic progenitors by co-culture with non-parenchymal human hepatic stellate cell line (TWNT-1); and (IV) Maturation by culture in dexamethasone. Characterization was performed by RT-PCR and functional assays. Results: The generated HLCs showed microscopically morphological phenotype of human hepatocytes, expressed liverspecific genes (ASGPR, Albumin, AFP, Sox17, Fox A2), secreted human liver-specific proteins such as albumin, synthesized urea and metabolized ammonia. Conclusion: Functional HLCs were generated from human iPS cells, which could be used for autologus hepatocyte transplantation for liver failure and as in vitro model for determining the metabolic and toxicological properties ofdrug compounds.

Original languageEnglish
Pages (from-to)91-96
Number of pages6
JournalJournal of the College of Physicians and Surgeons Pakistan
Volume24
Issue number2
Publication statusPublished - Feb 2014

Fingerprint

Embryoid Bodies
Induced Pluripotent Stem Cells
Hepatocytes
Cell Line
Liver
Albumins
Feeder Cells
Hepatic Stellate Cells
Endoderm
Mercaptoethanol
Liver Failure
Streptomycin
Coculture Techniques
Glutamine
Ammonia
varespladib methyl
Penicillins
Toxicology
Dexamethasone
Urea

Keywords

  • Embryoid body cells
  • Hepatocyte-like cells
  • iPS cells
  • Liver failure

ASJC Scopus subject areas

  • Medicine(all)

Cite this

@article{e367717da781486e883d1ad5f301b2c0,
title = "Generation of hepatocyte-like cells from human induced pluripotent stem (iPS) cells by co-culturing embryoid body cells with liver non-parenchymal cell line TWNT-1",
abstract = "Objective: To generate a homogeneous population of patient-specific hepatocyte-like cells (HLCs) from humaniPS cells those show the morphologic and phenotypic properties of primary human hepatocytes. Study Design: An experimental study. Place and Duration of Study: Department of Surgery, Okayama University, Graduate School of Medicine, Japan, from April to December 2011. Methodology: Human iPS cells were generated and maintained on ES qualified matrigel coated plates supplemented with mTeSR medium or alternatively on mitotically inactivated MEF feeder layer in DMEM/F12 medium containing 20{\%} KOSR, 4ng/ml bFGF-2, 1 × 10-4 M 2-mercaptoethanol, 1 mmol/L NEAA, 2mML-glutamine and 1{\%} penicillin-streptomycin. iPS cells were differentiated to HLCs by sequential culture using a four step differentiation protocol: (I) Generation of embryoid bodies (EBs) in suspension culture; (II) Induction of definitive endoderm (DE) from 2 days old EBs by growth in human activin-A (100 ng/ml) and basic fibroblastsgrowth factor (bFGF2) (100 ng/ml) on matrigel coated plates; (III) Induction of hepatic progenitors by co-culture with non-parenchymal human hepatic stellate cell line (TWNT-1); and (IV) Maturation by culture in dexamethasone. Characterization was performed by RT-PCR and functional assays. Results: The generated HLCs showed microscopically morphological phenotype of human hepatocytes, expressed liverspecific genes (ASGPR, Albumin, AFP, Sox17, Fox A2), secreted human liver-specific proteins such as albumin, synthesized urea and metabolized ammonia. Conclusion: Functional HLCs were generated from human iPS cells, which could be used for autologus hepatocyte transplantation for liver failure and as in vitro model for determining the metabolic and toxicological properties ofdrug compounds.",
keywords = "Embryoid body cells, Hepatocyte-like cells, iPS cells, Liver failure",
author = "Javed, {M. Shahid} and Naeem Yaqoob and Masaya Iwamuro and Naoya Kobayashi and Toshiyoshi Fujiwara",
year = "2014",
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language = "English",
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journal = "Journal of the College of Physicians and Surgeons--Pakistan : JCPSP",
issn = "1022-386X",
publisher = "College of Physicians and Surgeons Pakistan",
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TY - JOUR

T1 - Generation of hepatocyte-like cells from human induced pluripotent stem (iPS) cells by co-culturing embryoid body cells with liver non-parenchymal cell line TWNT-1

AU - Javed, M. Shahid

AU - Yaqoob, Naeem

AU - Iwamuro, Masaya

AU - Kobayashi, Naoya

AU - Fujiwara, Toshiyoshi

PY - 2014/2

Y1 - 2014/2

N2 - Objective: To generate a homogeneous population of patient-specific hepatocyte-like cells (HLCs) from humaniPS cells those show the morphologic and phenotypic properties of primary human hepatocytes. Study Design: An experimental study. Place and Duration of Study: Department of Surgery, Okayama University, Graduate School of Medicine, Japan, from April to December 2011. Methodology: Human iPS cells were generated and maintained on ES qualified matrigel coated plates supplemented with mTeSR medium or alternatively on mitotically inactivated MEF feeder layer in DMEM/F12 medium containing 20% KOSR, 4ng/ml bFGF-2, 1 × 10-4 M 2-mercaptoethanol, 1 mmol/L NEAA, 2mML-glutamine and 1% penicillin-streptomycin. iPS cells were differentiated to HLCs by sequential culture using a four step differentiation protocol: (I) Generation of embryoid bodies (EBs) in suspension culture; (II) Induction of definitive endoderm (DE) from 2 days old EBs by growth in human activin-A (100 ng/ml) and basic fibroblastsgrowth factor (bFGF2) (100 ng/ml) on matrigel coated plates; (III) Induction of hepatic progenitors by co-culture with non-parenchymal human hepatic stellate cell line (TWNT-1); and (IV) Maturation by culture in dexamethasone. Characterization was performed by RT-PCR and functional assays. Results: The generated HLCs showed microscopically morphological phenotype of human hepatocytes, expressed liverspecific genes (ASGPR, Albumin, AFP, Sox17, Fox A2), secreted human liver-specific proteins such as albumin, synthesized urea and metabolized ammonia. Conclusion: Functional HLCs were generated from human iPS cells, which could be used for autologus hepatocyte transplantation for liver failure and as in vitro model for determining the metabolic and toxicological properties ofdrug compounds.

AB - Objective: To generate a homogeneous population of patient-specific hepatocyte-like cells (HLCs) from humaniPS cells those show the morphologic and phenotypic properties of primary human hepatocytes. Study Design: An experimental study. Place and Duration of Study: Department of Surgery, Okayama University, Graduate School of Medicine, Japan, from April to December 2011. Methodology: Human iPS cells were generated and maintained on ES qualified matrigel coated plates supplemented with mTeSR medium or alternatively on mitotically inactivated MEF feeder layer in DMEM/F12 medium containing 20% KOSR, 4ng/ml bFGF-2, 1 × 10-4 M 2-mercaptoethanol, 1 mmol/L NEAA, 2mML-glutamine and 1% penicillin-streptomycin. iPS cells were differentiated to HLCs by sequential culture using a four step differentiation protocol: (I) Generation of embryoid bodies (EBs) in suspension culture; (II) Induction of definitive endoderm (DE) from 2 days old EBs by growth in human activin-A (100 ng/ml) and basic fibroblastsgrowth factor (bFGF2) (100 ng/ml) on matrigel coated plates; (III) Induction of hepatic progenitors by co-culture with non-parenchymal human hepatic stellate cell line (TWNT-1); and (IV) Maturation by culture in dexamethasone. Characterization was performed by RT-PCR and functional assays. Results: The generated HLCs showed microscopically morphological phenotype of human hepatocytes, expressed liverspecific genes (ASGPR, Albumin, AFP, Sox17, Fox A2), secreted human liver-specific proteins such as albumin, synthesized urea and metabolized ammonia. Conclusion: Functional HLCs were generated from human iPS cells, which could be used for autologus hepatocyte transplantation for liver failure and as in vitro model for determining the metabolic and toxicological properties ofdrug compounds.

KW - Embryoid body cells

KW - Hepatocyte-like cells

KW - iPS cells

KW - Liver failure

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