In vitro screening of exogenous factors for human neural stem/progenitor cell proliferation using measurement of total ATP content in viable cells.
One of the newest and most promising methods for treating intractable neuronal diseases and injures is the transplantation of ex vivo-expanded human neural stem/progenitor cells (NSPCs). Human NSPCs are selectively expanded as free-floating neurospheres in serum-free culture medium containing fibroblast growth factor 2 (FGF2) and/or epidermal growth factor (EGF); however, the culture conditions still need to be optimized for performance and cost before the method is used clinically. Here, to improve the NSPC culture method for clinical use, we used an ATP assay to screen the effects of various reagents on human NSPC proliferation. Human NSPCs responded to EGF, FGF2, and leukemia inhibitory factor (LIF) in a dose-dependent manner, and the minimum concentrations eliciting maximum effects were 10 ng/ml EGF, 10 ng/ ml FGF2, and 5 ng/ml LIF. EGF and LIF were stable in culture medium without NSPCs, although FGF2 was degraded. In the presence of human NSPCs, however, FGF2 and LIF were both degraded very rapidly, to below the estimated minimum concentration on day 3, but EGF remained above the minimum concentration for 5 days. Adding supplemental doses of each growth factor during the incubation promoted human NSPC proliferation. Among other supplements, insulin and transferrin promoted human NSPC growth, but progesterone, putrescine, selenite, D-glucose, and lactate were not effective and were cytotoxic at higher concentrations. Supplementing with conditioned medium from human NSPCs significantly increased human NSPC proliferation, but using a high percentage of the medium had a negative effect. These findings suggest that human NSPC culture is regulated by a balance in the culture medium between decreasing growth factor levels and increasing positive or negative factors derived from the NSPCs. Thus, in designing culture conditions for human NSPCs, it is useful to take the individual properties of each factor into consideration.
Kanemura
Yonehiro
Y
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Amagasaki, Hyogo, Japan. kanemura@onh.go.jp
Mori
Hideki
H
Nakagawa
Atsuyo
A
Islam
Mohammed Omedul
MO
Kodama
Eri
E
Yamamoto
Atsuyo
A
Shofuda
Tomoko
T
Kobayashi
Satoshi
S
Miyake
Jun
J
Yamazaki
Tomohiko
T
Hirano
Shun-ichiro
S
Yamasaki
Mami
M
Okano
Hideyuki
H
D000255:Adenosine Triphosphate / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004622:Embryo, Mammalian / Q000166:cytology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D019344:Lactic Acid / Q000378:metabolism; D053605:Leukemia Inhibitory Factor; D009474:Neurons / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism
D015850:Interleukin-6; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D016222:Fibroblast Growth Factor 2; D019344:Lactic Acid; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D005947:Glucose