[Isolation and culture of mouse marrow mesenchymal stem cells (MSCs) and biological characteristics of MSCs cultured in conditions for spermatogonia in vitro].
OBJECTIVE
To isolate, culture and purify mouse bone marrow mesenchymal stem cells (MSCs) and observe the main biological characteristics of MSCs cultured in conditions for spermatogonia in vitro.


METHODS
The tibias and femurs were dissected from 5 - 6-week old mice and the marrow in the tibias and femurs was flushed out with medium. MSCs were isolated, cultured, purified in vitro by Percoll density gradient centrifugation combined with adherent method and identified by dynamic observation of stem cell characteristics by transmission electron microscope, HE staining, and immunohistochemical detection of cell markers. The quantities of such cytokines as IL-6, IL-8, G-CSF and SCF in culture liquid with MSCs were measured by ELISA, and compared with those of the control group. MSCs of the third generation were divided into two groups to be induced and cultured. MSCs of the control group were cultured with basal medium, while those of the experimental group with conditional medium. The results were analysed by microscopic observation, HE staining and immunohistochemical methods.


RESULTS
Pure MSCs were obtained. The cultured cells, with stem cell characteristics, shuttle-shaped at HE staining, immature under the transmission electron microscope and CD44 and CD90 positive by immunohistochemical detection, could be identified as MSCs. Compared with the control group, the quantities of IL-6, IL-8, G-CSF and SCF in the experimental group increased significantly (P < 0.05). The shapes of MSCs changed and immunohistochemical staining for CD27, CD119 and Oct-4 was positive in the experimental group, but both were just the opposite in the control group.


CONCLUSIONS
Pure MSCs can be obtained by Percoll density gradient centrifugation combined with adherent method and identified by dynamically observing stem cell characteristics, HE staining, observation under the transmission electron microscope and immunohistochemical detection of cell markers. MSCs can secrete cytokines such as IL-6, IL-8, G-CSF, SCF, and so on. MSCs cultured in conditions for spermatogonia may show some biological characteristics of spermatogonia.
Liu
Feng-Hua
FH
Center of Reproductive Medicine, the Second Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510120, China.
Yang
Dong-Zi
DZ
Wang
Yi-Feng
YF
Liang
Xiao-Ping
XP
Peng
Wen-Ming
WM
Cao
Chang-An
CA
Chen
Xi-Gu
XG
Guo
Zhong-Min
ZM
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D016207:Cytokines / Q000032:analysis; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D051379:Mice; D008807:Mice, Inbred BALB C; D013093:Spermatogonia / Q000166:cytology*
D016207:Cytokines