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In Vitro Screening of Exogenous Factors for Human Neural Stem/Progenitor Cell Proliferation Using Measurement of Total ATP Content in Viable Cells

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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.

Keywords: ATP assay; Culture condition; Human neural stem/progenitor cell; Neuropshere

Document Type: Research Article

DOI: http://dx.doi.org/10.3727/000000005783982701

Affiliations: 1: *Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 3-11-46 Nakoji, Amagasaki, Hyogo 661-0974, Japan 2: Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 3-11-46 Nakoji, Amagasaki, Hyogo 661-0974, Japan, Institute of Biomedical Research and Innovation, Kobe, Hyogo 650-0047, Japan 3: Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 3-11-46 Nakoji, Amagasaki, Hyogo 661-0974, Japan 4: Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 3-11-46 Nakoji, Amagasaki, Hyogo 661-0974, Japan, New Energy and Industrial Technology Development Organization, Tokyo 170-6028, Japan 5: Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, Osaka 540-0006, Japan, Department of Neurosurgery, Osaka National Hospital, National Hospital Organization, Osaka 540-0006, Japan 6: Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan

Publication date: September 1, 2005

More about this publication?
  • Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.
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