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.
OBJECTIVE
To generate a homogeneous population of patient-specific hepatocyte-like cells (HLCs) from human iPS cells those show the morphologic and phenotypic properties of primary human hepatocytes.


METHODS
An experimental study.


METHODS
Department of Surgery, Okayama University, Graduate School of Medicine, Japan, from April to December 2011.


METHODS
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 x 10-4 M 2-mercaptoethanol, 1 mmol/L NEAA, 2mM L-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 fibroblasts growth 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 liver-specific genes (ASGPR, Albumin, AFP, Sox17, Fox A2), secreted human liver-specific proteins such as albumin, synthesized urea and metabolized ammonia.


CONCLUSIONS
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 of drug compounds.
Department of Physiology, Sargodha Medical College, University of Sargodha, Sargodha.
M Shahid
MS
Javed
Department of Physiology, Sargodha Medical College, University of Sargodha, Sargodha.
Naeem
N
Yaqoob
Department of Medicine, Okayama University Graduate School of Medicine, Okayama, Japan.
Masaya
M
Iwamuro
Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Okayama, Japan.
Naoya
N
Kobayashi
Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Okayama, Japan.
Toshiyoshi
T
Fujiwara
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D058732:Embryoid Bodies; D004627:Embryonic Induction / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000502:physiology; D051379:Mice; D021141:Nucleic Acid Amplification Techniques; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction