| Project/Area Number |
22KJ1841
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| Project/Area Number (Other) |
22J13683 (2022)
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| Research Category |
Grant-in-Aid for JSPS Fellows
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| Allocation Type | Multi-year Fund (2023)
Single-year Grants (2022) |
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| Section | 国内 |
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| Review Section |
Basic Section 44020:Developmental biology-related
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| Research Institution | Kyoto University |
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Principal Investigator |
PRETEMER YANN (2023) 京都大学, iPS細胞研究所, 特別研究員(PD)
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| Research Fellow |
PRETEMER YANN (2022) 京都大学, 医学研究科, 特別研究員(DC2)
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| Project Period (FY) |
2023-03-08 – 2024-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2023: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2022: ¥900,000 (Direct Cost: ¥900,000)
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| Keywords | iPS cell / thymic epithelial cell / development / thymus |
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| Outline of Research at the Start |
To better understand and study human thymus development, establishing a faithful in vitro model of the thymus is crucial. Here, I aim to use human iPS cells to induce thymic epithelial cells (TEC) - the stromal cells responsible for T cell maturation - by precisely recapitulating the signaling at each developmental stage. Using this model, it will become possible to observe thymus development in real time and elucidate many of the unknowns in early thymus formation and lineage determination.
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| Outline of Annual Research Achievements |
In the past fiscal year, I have succeeded in improving the stability and efficiency of an original thymic epithelial cell (TEC) induction system from human iPSCs. This was made possible by exploring the impact of activating or suppressing major developmental signaling pathways. Optimization of concentration, duration, and time point of addition of factors that regulate these pathways led to efficient fate determination from the anterior foregut endoderm to the pharyngeal endoderm and eventually TEC progenitor stages.
In consequence, it became possible to perform robust and long-term induction to mature TEC expressing key functional markers, even in 2D culture conditions. Using scRNA-seq, I was able to distinguish different TEC sublineages in separate clusters, each with characteristic gene expression profiles resembling cortical and medullary TEC (cTEC and mTEC) lineages. These results were confirmed at the protein level, as well, using ICC and flow cytometry. Cells positive for the TEC master regulator FOXN1, confirmed using a FOXN1 reporter iPSC line, also expressed markers specific to cTEC and mTEC. Currently, I am further evaluating the functionality of the induced TEC in a co-culture system with other cell types.
These results show that human thymus development can be faithfully modeled in vitro, with induced TEC serving as a promising platform to deepen our understanding of the signaling and mechanisms of fate determination at each stage.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
While the original plan was to incorporate decellularized human thymus scaffolds, organoid culture, or co-culture with mesenchymal cells into the induction system to provide signals conducive to TEC maturation, I found that even in the absence of these additional materials, it is possible to induce mature TEC in 2D culture conditions by chemically modulating specific developmental pathways that then allow mature TEC to autonomously differentiate from pharyngeal endoderm. Therefore, the resulting optimized TEC induction system is simple to perform and does not require complex techniques or additional materials. This unexpected success has allowed the research to proceed on a faster schedule than originally foreseen.
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| Strategy for Future Research Activity |
Going forward, I will expand on this year's results and validate the robustness of the induction system using several iPSC lines, some of which have already shown preliminary results similar to the main iPSC line used to establish the TEC induction system. I will test the clone-to-clone variability and assess the potential of this system to be more widely applied in the future, such as to the modeling of diseases of thymus development for example, as well as determine potential shortcomings where it can be further optimized.
Most importantly, I will apply it to the exploration of the developmental signaling and mechanisms of fate determination at both the pharyngeal endoderm to TEC progenitor, as well as the TEC progenitor to cTEC or mTEC stages, using a timecourse of scRNA-seq. By tracking and analyzing the details of the cell populations at key stages from anterior foregut endoderm to mature TEC, I aim to find clues of the precise signals required to proceed to each stage, which have not yet been elucidated in the human thymus. Furthermore, the cell populations at each stage will be compared to primary fetal or neonatal TEC to determine the similarity of the induced cells to actual primary TEC and ascertain the reliability of the results obtained from this in vitro induction system.
Together, these analyses will exploit the advantage of this induction system to observe human thymus development in real time in simple 2D culture to shed light on yet unknown developmental processes.
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