Established Thymic Epithelial Progenitor/Stem Cell-Like Cell Lines Differentiate into Mature Thymic Epithelial Cells and Support T Cell Development

et al. (2013) Established Thymic Epithelial Progenitor/Stem Cell-Like Cell Lines Differentiate into Mature Thymic Epithelial Cells and Support T Cell Development. PLoS ONE 8(9): e75222. doi:10.1371/journal.pone.0075222 Established Thymic Epithelial Progenitor/Stem Cell-Like Cell Lines Differentiate into Mature Thymic Epithelial Cells and Support T Cell Development Pengfei Chen 0 Jun Zhang 0 Yu Zhan 0 Juanjuan Su 0 Yarui Du 0 Guoliang Xu 0 Yufang Shi 0 Ulrich 0 Siebenlist 0 Xiaoren Zhang 0 Elias T. Zambidis, Johns Hopkins School of Medicine, United States of America 0 1 Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine , Shanghai , China , 2 Department of Transfusion, First Affiliated Hospital of Bengbu Medical College , Anhui , China , 3 The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai , China , 4 Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health , Bethesda, Maryland , United States of America Common thymic epithelial progenitor/stem cells (TEPCs) differentiate into cortical and medullary thymic epithelial cells (TECs), which are required for the development and selection of thymocytes. Mature TEC lines have been widely established. However, the establishment of TEPC lines is rarely reported. Here we describe the establishment of thymic epithelial stomal cell lines, named TSCs, from fetal thymus. TSCs express some of the markers present on tissue progenitor/stem cells such as Sca-1. Gene expression profiling verifies the thymic identity of TSCs. RANK stimulation of these cells induces expression of autoimmune regulator (Aire) and Aire-dependent tissue-restricted antigens (TRAs) in TSCs in vitro. TSCs could be differentiated into medullary thymic epithelial cell-like cells with exogenously expressed NF-B subunits RelB and p52. Importantly, upon transplantation under the kidney capsules of nude mice, TSCs are able to differentiate into mature TEC-like cells that can support some limited development of T cells in vivo. These findings suggest that the TSC lines we established bear some characteristics of TEPC cells and are able to differentiate into functional TEC-like cells in vitro and in vivo. The cloned TEPC-like cell lines may provide useful tools to study the differentiation of mature TEC cells from precursors. - Funding: This work was supported by grants from the National Basic Research program (2011CB946102), the National Natural Science Foundation of China (90919017, 30972695 and 31270937), Knowledge Innovation Project of Chinese Academy of Sciences (KSCX1-YW-22), National Key Programs on Infectious Disease (2008ZX10002-014) and with support from the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. These authors contributed equally to this work. The epithelial architecture in the thymus acts as a shelter fostering the expansion, maturation and selection of T lymphocytes [1,2]. The thymus contains thymic epithelial cells that form a complex three-dimensional meshwork structure organized in anatomically distinct cortical and medullary compartments. Cortical thymic epithelial cells (cTECs) provide the differentiation signal, regulate the directional migration and population expansion of immature T lymphocytes, and positively select CD4+CD8+ thymocytes, which are capable of recognizing self-major histocompatibility complex (MHC). Medullary thymic epithelial cells (mTECs) play a vital role in establishing central immunological tolerance [3,4]. The promiscuous expression of tissue-restricted self-antigens (TRAs) in mTECs under the control of Aire and other unknown transcription factors contributes to the negative selection of self-reactive T cells and the generation of regulatory T cells through direct antigen presentation by mTECs and crosspresentation by thymic dendritic cells [2,5]. Patterns of keratin expression have been described as markers of TEC subsets [1]. Keratin 5 (K5) is prominently expressed on mTECs and a small subset of cTECs. Keratin 14 (K14) is only observed on mTECs. A majority of cTECs expresses Keratin 8 (K8) but not K5 [6]. In addition to keratins, other markers are used to distinguish mTECs from cTECs. mTECs are positive for Ulex europaeus agglutinin-1 (UEA-1), Aire, ER-TR5 and MTS10, whereas cTECs are positive for ERTR4, Ly51 and CD205 [7-9]. While the factors and downstream signaling that control the development of cTECs are unknown, it is becoming clear that members of the TNFR family, such as RANK, CD40, and lymphotoxin receptor, and the activation of the downstream alternative NF-B signaling pathway are required for the development and differentiation of mTECs [10-13]. Mice in which these members of the TNFR superfamily, their ligands and tumor necrosis factor-receptor-associated factor 6 (TRAF6) [14], a critical downstream molecule of RANK and CD40 signaling, were deleted were reported to be deficient in mTECs and to exhibit disorders. Mice that were deficient in components of the alternative NF-B signaling pathway, such as NF-B-inducing kinase (NIK), IB-kinase (IKK), RelB, and NF-B2, also exhibited defects in the development and function of mTECs, albeit to varying degrees. MicroRNAs have recently been shown to help regulate the program of TEC differentiation and survival [15]. However, the program for the development and differentiation of mTECs that is controlled by signals acting via the alternative NF-B signaling pathway remains unknown. The thymus originates from the endoderm of the third pharyngeal pouch of the anterior gut. Plet-1+ founder cells in this region develop until day 11.5 of embryonic development in the mouse [1,16-20]. The generation of thymic epithelial cells is accompanied by thymocytes development. By day 12.5 of embryonic development hematopoietic progenitor cells enter the thymic primordia and promote the generation of the EpCAM1 + Plet-1+ epithelial population. Accumulating evidence indicates that these cells are thymic epithelial progenitor cells (TEPCs), which can give rise to both mTECs and cTECs [21]. TEPCs are also reportedly found within the rudiment of the thymus [22] and at the cortico-medullary junction in the adult thymus. The TEPC population within the fetal thymus has been defined with several surface markers, including K5+K8+ TECs, pan-cytokartin +EpCAM+ or pan-cytokartin+MTS24+ or EpCAM1+ MTS24+ [21,23,24], MTS20+ [25], EpCAM1+CD205+CD40- [26] and Claudin3/4loUEA1- [27]. Further studie (...truncated)