Selection, proliferation and differentiation of bone marrow-derived liver stem cells with a culture system containing cholestatic serum in vitro.
OBJECTIVE
To explore the feasibility of direct separation, selective proliferation and differentiation of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro.


METHODS
Whole bone marrow cells of rats cultured in routine medium were replaced with conditioning selection media containing 20 mL/L, 50 mL/L, 70 mL/L, and 100 mL/L cholestatic sera, respectively, after they attached to the plates. The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures. Then the selected BDLSC were induced to proliferate and differentiate with the addition of hepatocyte growth factor (HGF). The morphology and phenotypic markers of BDLSC were characterized using immunohistochemistry, RT-PCR and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.


RESULTS
Bone marrow cells formed fibroblast-like but not hepatocyte-like colonies in the presence of 20 mL/L cholestatic serum. In 70 mL/L cholestatic serum, BDLSC colonies could be selected but could not maintain good growth status. In 100 mL/L cholestatic serum, all of the bone marrow cells were unable to survive. A 50 mL/L cholestatic serum was the optimal concentration for the selection of BDLSC at which BDLSC could survive while the other populations of the bone marrow cells could not. The selected BDLSC proliferated and differentiated after HGF was added. Hepatocyte-like colony-forming units (H-CFU) then were formed. H-CFU expressed markers of embryonic hepatocytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and cytochrome P450-2b1), and hepatocyte nuclear factors (HNF-1alpha and HNF-3beta). They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.


CONCLUSIONS
The selected medium containing cholestatic serum can select BDLSC from whole bone marrow cells. It will be a new way to provide a readily available alternate source of cells for clinical hepatocyte therapy.
Cai
Yun-Feng
YF
Department of Liver-biliary Surgery, the First Hospital of Foshan City, 1 Dafu Nanlu, Foshan 528000, Guangdong Province, China. yfcai70@yahoo.com.cn
Zhen
Zuo-Jun
ZJ
Min
Jun
J
Fang
Tian-Ling
TL
Chu
Zhong-Hua
ZH
Chen
Ji-Sheng
JS
D000818:Animals; D001798:Blood Proteins / Q000494:pharmacology*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002779:Cholestasis / Q000097:blood*; D003135:Common Bile Duct; D017077:Culture Media, Conditioned / Q000494:pharmacology; D006003:Glycogen / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D008026:Ligation; D008099:Liver / Q000166:cytology*; D010641:Phenotype; D051381:Rats; D017207:Rats, Sprague-Dawley
D001798:Blood Proteins; D017077:Culture Media, Conditioned; D006003:Glycogen