2021 Fiscal Year Research-status Report
Understanding the effects of substrate stiffness on rejuvenation of aging MSCs via single cell analysis.
Project/Area Number |
21K12686
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Research Institution | Kyushu University |
Principal Investigator |
久保木 タッサニーヤー 九州大学, 先導物質化学研究所, 助教 (20526834)
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Project Period (FY) |
2021-04-01 – 2024-03-31
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Keywords | MSC aging |
Outline of Annual Research Achievements |
At present, the cellular senescence of the mesenchymal stem cells (MSCs) on various stiffness substrates was investigated by in vitro expansion of the cells on hydrogels in comparison with tissue culture dish. The senescence state was elucidated by quantification of cell, nuclear size and immunofluorescence staining of CSK related proteins. The senescence cells were identified using senescence associated beta-galactosidase (SA-beta-GAL) staining. The cell motility was analyzed by time-lapse observation. The expression of senescence markers such as galactosidase beta-1 (GLB1), and cell cycle inhibitors, p16, p21 and p53 were monitored using real-time PCR. Serially passaged the MSCs on both soft (3 kPa) and stiff (20 kPa) hydrogels decreased the expression of GLB1, p16, p21 p53 and decreased the numbers of SA-beta-GAL positive staining in comparison to the tissue culture dish. On the hydrogels, size of the cells and nucleus were decreased, and their migration properties could also be rescued. These results suggested that the expansion of the MSCs on hydrogel substrates could reverse the aging phenotypes and delayed senescence.
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Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The surface elasticity hydrogels have been shown to delay and reverse aging of the MSCs as expected. This rejuvenation mechanism possibly occur through the mechano-regulation of CSK tension.
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Strategy for Future Research Activity |
The rejuvenation of the MSC aging on gelatinous hydrogels have been confirmed. In the next step, single cell real time PCR approach will be performed to elucidate the rejuvenation mechanism in single cell resolution.
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Causes of Carryover |
The budget was incurred for next fiscal year for rather high running cost of single cell real time PCR analysis.
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