[Protocols for cloning human bone marrow-derived hepatic stem cells in vitro].
OBJECTIVE
To explore practical protocols for cloning bone marrow-derived hepatic stem cells in vitro.


METHODS
The cell fraction rich in CD117(+) cells and CD184(+) cells was separated from fresh bone marrow by density gradient centrifugation and cultured for 0, 7 and 14 days in high-glucose DMEM supplemented with or without 10% autologous serum or in serum-free high-glucose DMEM. All the media were supplemented with different concentrations of hepatocyte growth promoting factors (HGPF), thrombopoietin (TPO) and interleukin-3 (IL-3). The quantitative changes of CD117(+) cells and CD184(+) cells were measured by flow cytometry.


RESULTS
The optimal effect for cell cloning was achieved with high-glucose DMEM with 10% autologous serum group supplemented with 40 microg/ml HGPF, 50 ng/ml TPO, and 10 ng/ml IL-3. At day 7 of cell culture in this media, the quantity of CD117(+) cells and CD184(+) cells increased by 6.55 and 6.20 folds, and by 11.62 and 20.57 folds at day 14, respectively.


CONCLUSIONS
It is practical for cloning bone marrow-derived hepatic stem cells in high-glucose DMEM with 10% autologous serum supplemented with 40 microg/ml HGPF, 50 ng/ml TPO, and 10 ng/ml IL-3.
Yuan
Jie
J
Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. candlewjy@163.com
Liao
Cai-xian
CX
Qin
An-cheng
AC
Liao
Xin-xin
XX
Huang
Yong-ping
YP
Gong
Zu-yuan
ZY
Liao
Hui
H
D001854:Bone Marrow Cells / Q000166:cytology*; D018929:Cell Culture Techniques; D002999:Clone Cells; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology; D006801:Humans; D008099:Liver / Q000166:cytology*; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology*; D013926:Thrombopoietin / Q000494:pharmacology
D017228:Hepatocyte Growth Factor; D013926:Thrombopoietin; D019009:Proto-Oncogene Proteins c-kit