Promoted differentiation of cynomolgus monkey ES cells into hepatocyte-like cells by co-culture with mouse fetal liver-derived cells.
OBJECTIVE
To explore whether a co-culture of cynomolgus monkey embryonic stem (cES) cells with embryonic liver cells could promote their differentiation into hepatocytes.


METHODS
Mouse fetal liver-derived cells (MFLCs) were prepared as adherent cells from mouse embryos on embryonic d (ED) 14, after which undifferentiated cES cells were co-cultured with MFLCs. The induction of cES cells along a hepatic lineage was examined in MFLC-assisted differentiation, spontaneous differentiation, and growth factors (GF) and chemicals-induced differentiations (GF-induced differentiation) using retinoic acid, leukemia inhibitory factor (LIF), FGF2, FGF4, hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone.


RESULTS
The mRNA expression of alpha-fetoprotein, albumin (ALB), alpha-1-antitrypsin, and hepatocyte nuclear factor 4alpha was observed earlier in the differentiating cES cells co-cultured with MFLCs, as compared to cES cells undergoing spontaneous differentiation and those subjected to GF-induced differentiation. The expression of cytochrome P450 7a1, a possible marker for embryonic endoderm-derived mature hepatocytes, was only observed in cES cells that had differentiated in a co-culture with MFLCs. Further, the disappearance of Oct3/4, a representative marker of an undifferentiated state, was noted in cells co-cultured with MFLCs, but not in those undergoing spontaneous or GF-induced differentiation. Immunocytochemical analysis revealed an increased ratio of ALB-immunopositive cells among cES cells co-cultured with MFLCs, while glycogen storage and urea synthesis were also demonstrated.


CONCLUSIONS
MFLCs showed an ability to induce cES cells to differentiate toward hepatocytes. The co-culture system with MFLCs is a useful method for induction of hepatocyte-like cells from undifferentiated cES cells.
Saito
Ko
K
Department of Gastroenterology and Hepatology, Nara Medical University, Nara, Japan
Yoshikawa
Masahide
M
Ouji
Yukiteru
Y
Moriya
Kei
K
Nishiofuku
Mariko
M
Ueda
Shigehiko
S
Hayashi
Noriko
N
Ishizaka
Shigeaki
S
Fukui
Hiroshi
H
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002790:Cholesterol 7-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism; D018920:Coculture Techniques / Q000379:methods; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D006003:Glycogen / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000196:embryology* / Q000378:metabolism; D008252:Macaca fascicularis; D051379:Mice; D008810:Mice, Inbred C57BL; D027701:Organic Cation Transport Proteins / Q000235:genetics / Q000378:metabolism; D014508:Urea / Q000378:metabolism; D000099254:Organic Cation Transporter 3
D002790:Cholesterol 7-alpha-Hydroxylase; D006003:Glycogen; D036341:Intercellular Signaling Peptides and Proteins; D027701:Organic Cation Transport Proteins; D014508:Urea; D000099254:Organic Cation Transporter 3; C502772:Cyp7a1 protein, mouse