Purification of fetal mouse hepatoblasts by magnetic beads coated with monoclonal anti-e-cadherin antibodies and their in vitro culture.
A simple, rapid, and reproducible method of fetal hepatoblast purification was established to investigate mechanisms controlling interactions between hepatoblasts and nonparenchymal cells during liver development. Because E-cadherin is exclusively expressed on the cell membrane of hepatoblasts, magnetic beads coated with monoclonal antibodies to an extracellular epitope of its molecule were used to purify hepatoblasts from a cell suspension prepared from 12.5-day fetal mouse livers. The purity and yield in the hepatoblast fraction prepared in our protocol were more than 90% and approximately 30%, respectively. The nonparenchymal fraction rarely contained hepatoblasts; the rate of hepatoblast contamination in this fraction was less than 1%. Separate cultures of these two fractions were compared with cocultures of both fractions. In culture of the hepatoblast fraction, hepatoblasts formed aggregates similar to a bunch of grapes via their loose adhesion, floating in the medium after 24 h, and dissociated into single cells from the aggregates after 120 h of culture. By contrast, in the mixed culture, the majority of hepatoblasts formed multicellular spheroids after 24 h, and these spheroids changed into monolayer cell sheets after 120 h of culture. The cells comprising these monolayer sheets abundantly expressed albumin and carbamoylphosphate synthase I. In the mixed culture, fibroblastic cells also proliferated extensively with spreading on glass slides and surrounded the hepatoblast or hepatocyte colonies. On the other hand, fibroblastic cells spreading on glass slides decreased gradually in cultures of the nonparenchymal cell fraction alone. These findings indicated that the coexistence of hepatoblasts and nonparenchymal cells may be essential for their mutual survival, proliferation, differentiation, and morphogenesis. The conditioned medium of fetal liver cell cultures could partially replace the effects of the nonparenchymal cells on hepatoblasts in vitro. Our isolation protocol for fetal mouse hepatoblasts using immunobeads can greatly facilitate studies on mechanisms of cell-cell interactions during liver development.
Nitou
Miho
M
Department of Biology, Faculty of Science, Shizuoka University, Oya, Shizuoka, 422-8529, Japan.
Sugiyama
Yoshinori
Y
Ishikawa
Katsutoshi
K
Shiojiri
Nobuyoshi
N
D000418:Albumins / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D015820:Cadherins / Q000276:immunology* / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods*; D048429:Cell Size; D002478:Cells, Cultured; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D003893:Desmin / Q000378:metabolism; D005333:Fetus / Q000033:anatomy & histology; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology*; D018189:Immunomagnetic Separation; D007633:Keratins / Q000378:metabolism; D051379:Mice
D000418:Albumins; D000911:Antibodies, Monoclonal; D015820:Cadherins; D017077:Culture Media, Conditioned; D003893:Desmin; D007633:Keratins