Controlled and reversible induction of differentiation and activation of adult human hepatocytes by a biphasic culture technique.
OBJECTIVE
Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC.


METHODS
Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting.


RESULTS
In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel.


CONCLUSIONS
Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome de-differentiation, which occurs during continuous stimulation by means of growth factors.
Auth
Marcus-K H
MK
Universitäts-Kinderklinik Essen,Abteilung für Allgemeine Pädiatrie, Germany. marcus.auth@uni-essen.de
Boost
Kim A
KA
Leckel
Kerstin
K
Beecken
Wolf-Dietrich
WD
Engl
Tobias
T
Jonas
Dietger
D
Oppermann
Elsie
E
Hilgard
Philip
P
Markus
Bernd H
BH
Bechstein
Wolf-Otto
WO
Blaheta
Roman A
RA
D000328:Adult; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism; D006133:Growth Substances / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism
D003470:Culture Media; D006133:Growth Substances; D004815:Epidermal Growth Factor; D019859:Proto-Oncogene Proteins c-met