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Clonogenic Culture of Mouse Thymic Epithelial Cells

  • Miho Sekai
  • Jianwei Wang
  • Yoko HamazakiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2048)

Abstract

The thymus plays an essential role in the development and selection of T cells by providing a unique microenvironment that is mainly composed of thymic epithelial cells (TECs). We previously identified stem cells of medullary TECs (mTECs) that are crucial for central tolerance induction using a novel clonogenic culture system. We also found that medullary thymic epithelial stem cells (mTESCs) maintain life-long mTECs regeneration and central T cell self-tolerance in mouse models. The clonogenic efficiency of TECs in vitro is highly correlated to the TEC reconstitution activity in vivo. Here, we describe the clonogenic culture system to evaluate the self-renewing activity of TESCs. The colonies are derived from TESCs, are visualized and quantified by rhodamine-B staining on a feeder layer, and can be passaged in vitro. Thus, our system enables quantitative evaluation of TESC activity and is useful for dissecting the mechanisms that regulate TESC activity in physiological aging as well as in various clinical settings.

Key words

Thymus Thymic epithelial cells Thymic epithelial stem cells Clonogenic assay 

Notes

Acknowledgments

We thank Dr. P. Karagiannis for proofreading. This work was supported by grants from the Japanese Ministry of Education, Culture, Science, Sports, and Technology (24590580, 25111505, 15H01154, 17H05641, 18K19442, and 18H02640 to Y.H. and 15K19125 and 19K16689 to M.S.), iPS Cell Research Fund, AMED under Grant Number (JP19gm5010001 and JP19bm0104001) to Y.H., and the Takeda Science Foundation to Y.H. and M.S.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Immunology and Cell Biology, Graduate School of MedicineKyoto UniversityKyotoJapan
  2. 2.Laboratory of Immunobiology, Graduate School of Medicine, Center for iPS Cell Research and Application (CiRA)Kyoto UniversityKyotoJapan

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