2017 Fiscal Year Research-status Report
Mesenchymal stem cell function to prevent regulated cell death
| Project/Area Number |
17K15729
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| Research Institution | Kochi University |
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Principal Investigator |
NAJI ABDERRAHIM 高知大学, 教育研究部医療学系連携医学部門, 講師 (90650903)
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| Project Period (FY) |
2017-04-01 – 2019-03-31
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| Keywords | Mesenchymal stem cells / Cell death / Functional potency / Cellular therapy / Clinical translation / Selection technologies / Mitochondria / Cell Communications |
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| Outline of Annual Research Achievements |
Mesenchymal stem cells (MSCs) embody a biological material for cell therapy, which is safe, barely immunogenic and of immediate applicability for certain fatal diseases. Today, MSC therapy is expected to develop rapidly in more advanced-phases of clinical trials. Yet, obstacles exist to promote MSC therapy. To overcome difficulties there are needs to develop reliable in vitro and in vivo MSC potency assays and biomarkers predictive of response of any MSC product intended for use in cell therapy.
To achieve this, we developed in vitro assays to measure MSC potency to prevent regulated cell death (RCD), including apoptosis, necroptosis and pyroptosis, of a third-party cell that is either an immune or an epithelial cell. We have established human derived MSC products from different tissue, including bone marrow (BM-MSCs), and adipose (ASCs), obtained from different donors. We established a traceable MSC Master Bank for all of our donors of biological tissue obtained from Etablissement Francais du Sang (EFS), Toulouse, France.
We comparatively analyzed in vitro the capacity of BM-MSCs and ASCs to prevent RCD, we found that our MSC potency test allow to compare MSC efficiency in vitro between different MSC products from our Master Bank, and as well as of primary MSC products provided by ATCC. Regarding the mechanism of action, we uncovered the critical role of mitochondria transfer from MSC to cells undergoing RCD. Further, we found that MSCs themselves are resistant to cell death signaling, and that correlate with their potency to prevent RCD in a moribund third-party cell.
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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
Our research plan for FY2017 was to establish novel in vitro potency tests for MSCs, and to determine the mechanism of action used by MSC to modulate RCD.
To this end, we executed the production of MSCs. MSCs were expanded in vitro and established as CD14-CD34-CD45- non-hematopoietic cells, and phenotypically characterized as CD73+CD90+CD105+ cells. Cell bank of MSCs was established for each donor with unique identification. We conducted co-culture experiments with macrophages and epithelial cells, challenged with specific RCD inducers (FasL, Etoposide, Shikonin, Bortezomib, FTY720, ATP, Nigericin), and different molecular and cellular biological analyses have been performed.
Yet, we had to expand analyses in order to avoid RCD overlaps by testing different conditions for RCD inducers according to each target cell. We tested RCD inducers for each of MSC product generated in the laboratory.The mechanism of action of MSCs to modulate RCD has been characterized. Transfer of mitochondria from MSCs to target cells has been examined by fluorescent microscopy and TEM imaging.Flow cytometry analyses are being now undertaken. Further, we have implemented and designed novel experiments to redefine the preferential pathways and biological structures used , e.g. Mitochondria transfer via TNT, or EVs, or Cx43, in MSC potency to modulate RCD. To this end, we are developing techniques to knockdown Miro-1, Miro-2, nSmase2 and Cx43, in samples of each MSC products, we have generated in the lab. Therefore, we can consider that the current status of our research is progressing rather smoothly.
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| Strategy for Future Research Activity |
We developed novel in vitro MSC potency tests aimed at measuring MSC capabilities to modulate regulated cell death pathways (RCD).However,we lack specific biomarkers to exclusively distinguish MSCs with therapeutic interests in vitro.
To address this complexity, we are elaborating experiments using a combination of cell surface markers that are associated with functional evaluation of MSCs (Naji et al.,Stem Cells Translational Medicine, 2017; 6 (3): 713-719).An advanced format for cytometry combined with mass spectrometry has been developed. The fusion of the two technologies, called mass cytometry (CyTOF), makes it possible to measure more than 40 parameters simultaneously within single cells and thus to analyze with precision the cells of interest within a population of MSCs.
This development aims to increase the resolution at which our observations are possible. Our technological goals evolved to refine our perception on identity of MSCs obtained from samples of our different donors and from different tissue sources. We plan to use the CyTOF technology to better identify cell subsets in our MSC product, in order to determine specific markers associated with biological functions of MSCs to modulate RCD that is measured in our MSC potency tests.These implementations are being developed and are progressing in collaboration with Dr. Benoit Favier, IDMIT, University of Paris, France. These experiments are justified to provide the scientific and medical community with the means to identify MSCs with potent therapeutic efficacy, and with implications in translational medicine.
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| Causes of Carryover |
The subsidy (BA) 635,104 JPY occurred for FY2017,as we have further optimized our priorities in our current research,and through beneficiating in expanding our international collaborations.The usage of the subsidy (BA) 635,104 JPY combined with the grant we requested as for FY2018 would allow us to manage further costs that are associated to expenditure for implementing our innovative plan for performing advanced experiments using CYTOF analysis.The funding that will be available during the budget-period FY2018, combined to budget (BA) of FY2017, will cover a part of the research associated to CYTOF technology. This may benefit to conduct successfully our research plan, including the development of our laboratory main research interests.
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Research Products
(4 results)
