2004 Fiscal Year Final Research Report Summary
The elucidation of the physiochemical factor of the mouse ES cell on the ambryoid body formation.
| Project/Area Number |
15560675
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biofunction/Bioprocess
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| Research Institution | University of Yamanashi |
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Principal Investigator |
KUROSAWA Hiroshi University of Yamanashi, Department of Research Interdisciplinary Graduate School of Medicine and Engineering, Associate Professor, 大学院・医学工学総合研究部, 助教授 (10225295)
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| Project Period (FY) |
2003 – 2004
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| Keywords | embryoid body / ES cell / undifferentiated / alkaline phosphatase / low-oxygen / high-oxygen / differentiation / cardiomyocyte |
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| Research Abstract |
Embryonic stem (ES) cell is a highly expected candidate for the cell source of cell therapy. Therefore, it is desired that the establishment of the technology for controlling proliferation and differentiation of ES cell. In this study, it was investigated that the effect of physicochemical factors on proliferation and embryoid body (EB) formation of ES cells.
Mouse ES cells of the cell line 129SV were cultured under various oxygen atmospheres (5%, 20%, and 40%). 2-Methacryloyloxyethyl Phosphorylcholine (MPC) coated U-bottom 96-well plate was suitable for EB formation. A single EB was formed from 1000 ES cells per well in the high formation rate. It was found that the non-adhesive property of culture vessel is an important factor for EB formation. The culture vessels that have U-and V-bottom were more desirable to form EB than flat-bottom. We have established a novel culture method for EB formation using MPC coated U-bottom 96-well plate.
The cultivation in low-oxygen (5% O_2) atmosphere
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lowered the proliferation of ES. High-oxygen (40% O_2) atmosphere slightly inhibited the proliferation, but ES cells cultured in 40% O_2 retained specific AP activity relatively high when compared to the cultures in 5% O_2 and 20% O_2. These results suggested that high-oxygen atmosphere suppressed the differentiation of ES cells.
The cell number consisted of an EB increased to about 30,000 cells/EB by day 4 in 20% O_2 and 40% O_2, but it increased to about 20,000 cells/EB by day 5 in 5% O_2. The size of EB formed in 5% O_2 was the smallest (diameter : 280±20 μm), and the size of EB formed in 20% O_2 was the largest (diameter : 400±20 μm). The appearance of the EB formed in 40% O_2 was more compact (diameter : 340±20 μm), and its contour was relatively clear. All EBs, which were formed under various oxygen tensions, had the capacity for differentiation into various cell types. For example, the beating of cardiac muscle was microscopically observed in the outgrowth of attached EBs formed in 20% O_2 by day 3 of culture for the differentiation. However, the progress of the cardiomyogenic differentiation in the EB formed in 40% O_2 was retarded for about 1 day in comparison with the EB formed in 20% O_2. This suggested that 40% O_2 suppressed the differentiation of ES cells. Less
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Research Products
(4 results)
