In vitro expansion and functional recovery of mature hepatocytes from mouse adult liver.
BACKGROUND
Mature hepatocytes retain the ability to regenerate the liver lobule fully in vivo following injury. Several cytokines and soluble factors (hepatocyte growth factors, epidermal growth factors, insulin and nicotinamide) are known to be important for proliferation of mature hepatocytes in vitro. However, hepatocytes monolayer-cultured on extracellular matrices have gradually lost their specific functions, particularly those in drug metabolism.


OBJECTIVE
We have explored and established a new culture system for expansion of functional hepatocytes.


METHODS
We evaluated two approaches for efficient expansion of mature hepatocytes: (i) Co-culture with mouse embryonic fibroblasts (MEF); (ii) Addition to culture of inhibitors of cell signals involved in liver regeneration. After expansion steps, 3-dimensional spheroid-forming culture was used to re-induce mature hepatocellular function.


RESULTS
The addition of inhibitors for tumour growth factor (TGF) β and glycogen synthase kinase (GSK) 3β efficiently induced in vitro expansion of mature hepatocytes. Although expression of hepatocellular functional genes decreased after expansion in monolayer culture, their expression and the activity of cytochrome P450 enzymes significantly increased with spheroid formation. Furthermore, when hepatocytes were co-cultured with MEF, addition of a MAPK/ERK kinase (MEK) inhibitor at the spheroid formation step enhanced drug-metabolism-related gene expression.


CONCLUSIONS
Combination of the MEF co-culture system with the addition of inhibitors of TGFβ and GSK3β induced in vitro expansion of hepatocytes. Moreover, expression of mature hepatocellular genes and the activity of drug-metabolism enzymes in expanded hepatocytes were re-induced after spheroid culture.
Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Hidenori
H
Ito
Akihide
A
Kamiya
Keiichi
K
Ito
Ayaka
A
Yanagida
Ken
K
Okada
Hiromitsu
H
Nakauchi
D000818:Animals; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors; D000071679:Glycogen Synthase Kinase 3 beta; D049452:Green Fluorescent Proteins; D022781:Hepatocytes / Q000166:cytology* / Q000502:physiology*; D007150:Immunohistochemistry; D008099:Liver / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D012038:Regeneration / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015626:Tissue Expansion / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors
D017931:DNA Primers; D016212:Transforming Growth Factor beta; D049452:Green Fluorescent Proteins; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3