In vitro screening of exogenous factors for human neural stem/progenitor cell proliferation using measurement of total ATP content in viable cells

Yonehiro Kanemura, Hideki Mori, Atsuyo Nakagawa, Mohammed Omedul Islam, Eri Kodama, Atsuyo Yamamoto, Tomoko Shofuda, Satoshi Kobayashi, Jun Miyake, Tomohiko Yamazaki, Shun Ichiro Hirano, Mami Yamasaki, Hideyuki Okano

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Abstract

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.

Original languageEnglish
Pages (from-to)673-682
Number of pages10
JournalCell Transplantation
Volume14
Issue number9
DOIs
Publication statusPublished - 2005

Keywords

  • ATP assay
  • Culture condition
  • Human neural stem/progenitor cell
  • Neuropshere

ASJC Scopus subject areas

  • Biomedical Engineering
  • Cell Biology
  • Transplantation

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    Kanemura, Y., Mori, H., Nakagawa, A., Islam, M. O., Kodama, E., Yamamoto, A., Shofuda, T., Kobayashi, S., Miyake, J., Yamazaki, T., Hirano, S. I., Yamasaki, M., & Okano, H. (2005). In vitro screening of exogenous factors for human neural stem/progenitor cell proliferation using measurement of total ATP content in viable cells. Cell Transplantation, 14(9), 673-682. https://doi.org/10.3727/000000005783982701