[Study on the effects and mechanisms of bone marrow mesenchymal stem cells on porcine primary hepatocyte culture in vitro].
OBJECTIVE
To investigate the expression and distribution of extracellular matrix (ECM) in the co-culture of porcine primary hepatocytes and bone marrow mesenchymal stem cells (MSCs) in vitro.


METHODS
Mononuclear cells were isolated from bone marrow of swine by density gradient centrifugation. MSCs of passage 3 and primary hepatocytes harvested by a two-step in situ collagenase perfusion technique were co-cultured, and the morphological and functional changes of heterotypic interactions were characterized. Immunocytochemical analysis was performed to monitor the expression and distribution of ECM.


RESULTS
The purity of the third passage MSCs and primary hepatocytes was more than 90% and 99%, respectively. More than 95% of the hepatocytes were viable. Compared to hepatocytes culture, co-culture with MSCs significantly enhanced hepatic function: including albumin secretion and urea synthesis (P < 0.01). The best hepatic function level was achieved on day 2 and gradually decreased in the following co-culture days. Immunocytochemical staining suggested that higher amounts of naturally occurring ECM proteins including fibronectin, laminin, and several kinds of collagens were produced in co-culture group compared to hepatocyte homo-culture (P < 0.01). RNAi experiments verified that there was a correlation between ECM and hepatic functions.


CONCLUSIONS
ECM may indeed play a key role in the up-regulation of hepatocyte functions in MSC/hepatocytes co-culture.
Gu
Jin-yang
JY
Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital of Medical School, Nanjing University, Nanjing 210008, China.
Shi
Xiao-lei
XL
Zhang
Yue
Y
Ding
Yi-tao
YT
D000418:Albumins / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D005109:Extracellular Matrix / Q000378:metabolism*; D005260:Female; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D007150:Immunohistochemistry; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D013552:Swine; D014508:Urea / Q000378:metabolism
D000418:Albumins; D014508:Urea