2022 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 |
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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 / cellular senescence / rejuvenation / substrate elasticity / gelatinous hydrogel / redox homeostasis / DNA damage response / cell cycle |
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| Outline of Annual Research Achievements |
Mesenchymal stem cells (MSCs) from bone marrow are widely used in clinical applications due to their therapeutic properties. However, in vitro expansion of MSCs on tissue culture dish (TC) induce aging (replicative senescence), which reduce their quantities and qualities with undefined mechanism.
The first-year report demonstrated that the early passage MSCs cultured on hydrogels exhibited the reverse senescence phenotypes. In this second-year report, to confirm the rejuvenation capacity of the hydrogels, the late passage MSCs that already undergone cell cycle arrest were evaluated. In agreement with the results obtained from the young MSCs, down-regulation of senescence markers was also observed on the gels. Treating the MSCs on TC with CSK tension inhibitors downregulated these markers, emphasizing the importance of the CSK tension on cellular senescence. To gain insight into the effect of mechanical stimuli on redox homeostasis, premature senescence was induced using hydrogen peroxide and the cellular stress response was evaluated. On TC, the treated cells increased ROS, antioxidant genes and senescence markers. The hydrogels could attenuate the effects of hydrogen peroxide induced oxidative stress and suppressed senescence, indicating that the MSCs cultured on the hydrogels possess higher radical scavenging capacity and could better maintain redox homeostasis. Knocking down the master of redox regulator enhanced the senescence, suggesting the interconnection between mechanical signaling, redox homeostasis and cellular 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 results obtained so far support the proposed hypothesis that the compliance substrate could delay or reverse aging. As expected, several evidence indicated the crosstalk between mechanical stimuli mediated cellular tension on cellular redox homeostasis. Down-regulation of cell cycle inhibitors in the MSCs culture on gels indicated the stimulation of cell cycle progression and proliferation. However, the proliferation potential of the cells could not be rescued, as demonstrated by the decreased expression of proliferation marker and less numbers of cells recovered from the gels compared with that of the TC. This evidence suggested that further activation of the downstream signaling pathways is required. For this reason, prior to moving forward to the single analysis as in the original plan, various stiffness and culture conditions were extensively evaluated until the regenerative potential of the MSCs is achieved. Recently, the surface elasticity conditions that could promote the cell proliferation and suppress senescence are successfully established and therapeutic properties are being investigated.
DNA damage has been proposed as a primary cause of aging and consequently induce cell cycle arrest. In comparison to the TC, the MSCs on gels exhibited less formation of DNA damage foci with increased numbers of cell proliferation, indicating the implication of substrate stiffness on DNA damage response. Further investigation is being confirmed to explain the correlation of these findings.
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| Strategy for Future Research Activity |
The micro-engineered surface elasticity tunable hydrogels platform for in vitro expansion of the MSCs have successfully established. The hydrogels provide a suitable environment to maintain proper redox homeostasis, decrease cellular tension and stress and delay/reverse aging. In addition to DNA damage response and proliferation potential, evaluation of other therapeutic properties of the MSCs such as the secretion of therapeutic cytokines/growth factors or differentiation potential also require. The senescence cells produce and secrete inflammatory cytokine and growth factors known as senescence-associated secretory phenotype (SASP), which associated with negative effects to the neighboring cells such as an acceleration senescence or promotion cancer cell metastasis. In this study, expression of SASP molecules in the late passage MSCs was suppressed in the cells cultured on gels. Further investigation of SAPS modulation via substrate stiffness will be performed.
For the research plan in the final year, the recovery of the MSC therapeutic properties including DNA damage repair, proliferation, differentiation potential and SASP production will be investigated. After these responses were confirmed, single cell real time PCR analysis will be performed to gain a better understanding of substrate stiffness induced rejuvenation of the MSC aging in single cell resolution. For the final experiment, direct correlation of various regulators of the related signaling pathways will be elucidated using knockdown approach in order to clarify the underlying mechanism of rejuvenation.
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| Causes of Carryover |
The main concept of this research is to understand the heterogeneity of the stem cell aging on substrate stiffness. Special technique of single cell analysis is required for completion of this project. In our laboratory, we have facility for single separation and cDNA synthesis. However, for downstream analysis of gene expression, we have to send the samples to the outsource company for single cell real time PCR analysis. The budget for this analysis is more than million yen so several steps are being confirmed prior to the final single cell analysis approach. For this reason, rather large amount of budget is secured for this final experiment.
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