Growth and differentiation of colony-forming human hepatocytes in vitro.
OBJECTIVE
Parenchymal hepatocytes (PHs) of rat contain colony-forming parenchymal hepatocytes (CF-PHs) as a small fraction. We aimed to demonstrate the presence of CF-PHs in humans and characterize them with respect to growth and differentiation potential.


METHODS
Human PHs were co-cultured with Swiss 3T3 cells in the medium containing human serum, EGF, nicontinamide, and ascorbic acid 2-phosphate. To examine differentiation potential hepatocytes were cultured on gels of Matrigel Matrix.


RESULTS
Few PHs formed colonies, the colony-forming efficiency being as low as 0.01-0.09%. The CF-PHs could be subcultured up to 7 passages. They showed a liver epithelial cell-like morphology, and immunocytochemically positive for albumin (ALB), cytokeratin (CK) 7, 8, 18, and 19 in a pre- and early phase-confluence, whereas they showed a typical differentiated hepatocyte-like morphology, and positive for alpha(1)-antitrypsin, but negative for CK7 and 19 in condensed regions at confluence. The CF-PHs at late confluence expressed mRNAs of ALB, HNF4, and isoforms of cytochrome P450 at low levels. However, when cultured on Matrigel, these cells expressed them at high levels comparable to those of original PHs.


CONCLUSIONS
We concluded that the human liver contains highly replicative hepatic progenitor-like cells as a minute population that retain a normal differentiation potential.
Yoshizato Project, Cooperative Link of Unique Science and Technology for Economy Revitalization , CLUSTER, Hiroshima Prefectural Institute of Industrial Science and Technology, Hiroshima 739-0046, Japan.
Chihiro
C
Yamasaki
Chise
C
Tateno
Akio
A
Aratani
Chimoto
C
Ohnishi
Shigeru
S
Katayama
Toshihiko
T
Kohashi
Hiroshi
H
Hino
Hiroyuki
H
Marusawa
Toshimasa
T
Asahara
Katsutoshi
K
Yoshizato
D000293:Adolescent; D000368:Aged; D000418:Albumins / Q000032:analysis / Q000235:genetics; D000818:Animals; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D018920:Coculture Techniques; D003470:Culture Media / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000235:genetics; D005260:Female; D051557:Hepatocyte Nuclear Factor 4 / Q000032:analysis / Q000235:genetics; D022781:Hepatocytes / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D007633:Keratins / Q000032:analysis; D008099:Liver / Q000737:chemistry / Q000166:cytology*; D008297:Male; D051379:Mice; D008858:Microscopy, Phase-Contrast; D008875:Middle Aged; D010641:Phenotype; D012333:RNA, Messenger / Q000032:analysis; D018874:Spheroids, Cellular / Q000737:chemistry / Q000166:cytology; D013234:Stem Cells / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D041701:Swiss 3T3 Cells
D000418:Albumins; D003470:Culture Media; D051557:Hepatocyte Nuclear Factor 4; D012333:RNA, Messenger; D007633:Keratins; D003577:Cytochrome P-450 Enzyme System