Clonogenic Culture of Mouse Thymic Epithelial Cells

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


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 



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.


  1. 1.
    Anderson KL, Moore NC, McLoughlin DE, Jenkinson EJ, Owen JJ (1998) Studies on thymic epithelial cells in vitro. Dev Comp Immunol 22(3):367–377CrossRefGoogle Scholar
  2. 2.
    Mohtashami M, Zuniga-Pflucker JC (2006) Three-dimensional architecture of the thymus is required to maintain delta-like expression necessary for inducing T cell development. J Immunol 176(2):730–734CrossRefGoogle Scholar
  3. 3.
    Petrie HT, Zuniga-Pflucker JC (2007) Zoned out: functional mapping of stromal signaling microenvironments in the thymus. Annu Rev Immunol 25:649–679. Scholar
  4. 4.
    Takahama Y, Ohigashi I, Baik S, Anderson G (2017) Generation of diversity in thymic epithelial cells. Nat Rev Immunol 17(5):295–305. Scholar
  5. 5.
    Boehm T (2008) Thymus development and function. Curr Opin Immunol 20(2):178–184. Scholar
  6. 6.
    Vaidya HJ, Briones Leon A, Blackburn CC (2016) FOXN1 in thymus organogenesis and development. Eur J Immunol 46(8):1826–1837. Scholar
  7. 7.
    Gray DH, Chidgey AP, Boyd RL (2002) Analysis of thymic stromal cell populations using flow cytometry. J Immunol Methods 260(1–2):15–28CrossRefGoogle Scholar
  8. 8.
    Seach N, Wong K, Hammett M, Boyd RL, Chidgey AP (2012) Purified enzymes improve isolation and characterization of the adult thymic epithelium. J Immunol Methods 385(1–2):23–34. Scholar
  9. 9.
    Jain R, Gray DH (2014) Isolation of thymic epithelial cells and analysis by flow cytometry. Curr Protoc Immunol 107(3):26 21–26 15. Scholar
  10. 10.
    Gray D, Abramson J, Benoist C, Mathis D (2007) Proliferative arrest and rapid turnover of thymic epithelial cells expressing Aire. J Exp Med 204(11):2521–2528. Scholar
  11. 11.
    Gray DH, Seach N, Ueno T, Milton MK, Liston A, Lew AM, Goodnow CC, Boyd RL (2006) Developmental kinetics, turnover, and stimulatory capacity of thymic epithelial cells. Blood 108(12):3777–3785. Scholar
  12. 12.
    Ucar A, Ucar O, Klug P, Matt S, Brunk F, Hofmann TG, Kyewski B (2014) Adult thymus contains FoxN1(−) epithelial stem cells that are bipotent for medullary and cortical thymic epithelial lineages. Immunity 41(2):257–269. Scholar
  13. 13.
    Wong K, Lister NL, Barsanti M, Lim JM, Hammett MV, Khong DM, Siatskas C, Gray DH, Boyd RL, Chidgey AP (2014) Multilineage potential and self-renewal define an epithelial progenitor cell population in the adult thymus. Cell Rep 8(4):1198–1209. Scholar
  14. 14.
    Sekai M, Hamazaki Y, Minato N (2014) Medullary thymic epithelial stem cells maintain a functional thymus to ensure lifelong central T cell tolerance. Immunity 41(5):753–761. Scholar
  15. 15.
    Kim MJ, Miller CM, Shadrach JL, Wagers AJ, Serwold T (2015) Young, proliferative thymic epithelial cells engraft and function in aging thymuses. J Immunol 194(10):4784–4795. Scholar
  16. 16.
    Meireles C, Ribeiro AR, Pinto RD, Leitao C, Rodrigues PM, Alves NL (2017) Thymic crosstalk restrains the pool of cortical thymic epithelial cells with progenitor properties. Eur J Immunol 47(6):958–969. Scholar

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© 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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