Abstract
This review briefly describes the last decades of experimental work on the thymus. Given the histological complexity of this organ, the multiple embryological origins of its cellular components and its role in carefully regulating T lymphocyte maturation and function, methods to dissect and understand this complexity have been developed through the years. The possibility to study ex vivo the thymus organ function has been achieved by developing Fetal Thymus Organ Cultures (FTOC). Subsequently, the combination of organ disaggregation and reaggregation in vitro represented by Reaggregate Thymus Organ cultures (RTOC) allowed mixing cellular components from different genetic backgrounds. Moreover, RTOC allowed dissecting the different stromal and hematological components to study the interactions between Major Histocompatibility Complex (MHC) molecules and the T-cell receptors during thymocytes selection. In more recent years, prospective isolation of stromal cells and thymocytes at different stages of development made it possible to explore and elucidate the molecular and cellular players in both the developing and adult thymus. Finally, the appearance of novel cell sources such as embryonic stem (ES) cells and more recently induced pluripotent stem (iPS) cells has opened new scenarios in modelling thymus development and regeneration strategies. Most of the work described was carried out in rodents and the current challenge is to develop equivalent or even more informative assays and tools in entirely human model systems.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Miller J (1961) Immunological function of the thymus. Lancet 278:748–749
Mohtashami M, Zúñiga-Pflücker JC (2006) Three-dimensional architecture of the thymus is required to maintain delta-like expression necessary for inducing T cell development. J Immunol 176:730–734
Takahama Y et al. (2017) Generation of diversity in thymic epithelial cells. Nature Reviews Immunology 17:295–305
Robinson JH, Owen JJT (1978) Transplantation tolerance induced in foetal mouse thymus in vitro. Nature 271:758–760
Robinson JH, Owen JJ (1977) Generation of T-cell function in organ culture of foetal mouse thymus. II. Mixed lymphocyte culture reactivity. Clin Exp Immunol 27:322–327
Doherty PC, Zinkernagel RM (1975) Enhanced immunological surveillance in mice heterozygous at the H-2 gene complex. Nature 256:50–52
Zinkernagel RM (1982) Selection of restriction specificities of virus-specific cytotoxic T cells in the thymus: no evidence for a crucial role of antigen-presenting cells. J Exp Med 156:1842–1847
Owen JJ, Jordan RK, Robinson JH et al (1976) In vitro studies on the ontogeny of lymphocyte populations. Ann Immunol 127:951–956
Robinson JH, Owen JJT (1976) Generation of T-cell function in organ culture of foetal mouse thymus. I. Mitogen responsiveness. Clin Exp Immunol 23:347
Röpke C (1997) Thymic epithelial cell culture. Microsc Res Tech 38:276–286
Jenkinson EJ, Van Ewijk W, Owen JJ (1981) Major histocompatibility complex antigen expression on the epithelium of the developing thymus in normal and nude mice. J Exp Med 153:280–292
Jenkinson EJ, Franchi LL, Kingston R et al (1982) Effect of deoxyguanosine on lymphopoiesis in the developing thymus rudiment in vitro: application in the production of chimeric thymus rudiments. Eur J Immunol 12:583–587
Cohen A, Lee JW, Dosch HM et al (1980) The expression of deoxyguanosine toxicity in T lymphocytes at different stages of maturation. J Immunol 125:1578–1582
Fontaine-Perus JC, Calman FM, Kaplan C et al (1981) Seeding of the 10-day mouse embryo thymic rudiment by lymphocyte precursors in vitro. J Immunol 126:2310–2316
Plum J, De Smedt M, Verhasselt B et al (2000) Human T lymphopoiesis. In vitro and in vivo study models. Ann N Y Acad Sci 917:724–731
Martín-Fontecha A, Schuurman HJ, Zapata A (1994) Role of thymic stromal cells in thymocyte education: a comparitive analysis of different models. Thymus 22:201–213
Plum J, De Smedt M, Leclercq G (1993) Exogenous IL-7 promotes the growth of CD3-CD4-CD8-CD44+CD25+/− precursor cells and blocks the differentiation pathway of TCR-alpha beta cells in fetal thymus organ culture. J Immunol 150:2706–2716
Levelt CN, Carsetti R, Eichmann K (1993) Regulation of thymocyte development through CD3. II. Expression of T cell receptor beta CD3 epsilon and maturation to the CD4+8+ stage are highly correlated in individual thymocytes. J Exp Med 178:1867–1875
Müller KP, Kyewski BA (1993) T cell receptor targeting to thymic cortical epithelial cells in vivo induces survival, activation and differentiation of immature thymocytes. Eur J Immunol 23:1661–1670
Jones LA, Izon DJ, Nieland JD et al (1993) CD28-B7 interactions are not required for intrathymic clonal deletion. Int Immunol 5:503–512
Jenkinson EJ, Anderson G, Moore NC et al (1994) Positive selection by purified MHC class II+ thymic epithelial cells in vitro: costimulatory signals mediated by B7 are not involved. Dev Immunol 3:265–271
Jenkinson EJ, Anderson G (1994) Fetal thymic organ cultures. Curr Opin Immunol 6:293–297
Hogquist KA, Gavin MA, Bevan MJ (1993) Positive selection of CD8+ T cells induced by major histocompatibility complex binding peptides in fetal thymic organ culture. J Exp Med 177:1469–1473
Ashton-Rickardt PG, Van Kaer L, Schumacher TN et al (1993) Peptide contributes to the specificity of positive selection of CD8+ T cells in the thymus. Cell 73:1041–1049
Ashton-Rickardt PG (2007) Studying T-cell repertoire selection using fetal thymus organ culture. Methods Mol Biol 380:171–184
Anderson G, Jenkinson EJ (2001) Lymphostromal interactions in thymic development and function. Nat Rev Immunol 1:31–40
Sugawara T, Di Bartolo V, Miyazaki T et al (1998) An improved retroviral gene transfer technique demonstrates inhibition of CD4-CD8- thymocyte development by kinase-inactive ZAP-70. J Immunol 161:2888–2894
Owens BM, Hawley RG, Spain LM (2005) Retroviral transduction in fetal thymic organ culture. Methods Mol Med 105:311–322
Travers H, Anderson G, Gentle D et al (2001) Protocols for high efficiency, stage-specific retroviral transduction of murine fetal thymocytes and thymic epithelial cells. J Immunol Methods 253:209–222
Takahama Y (2001) Genetic modulation of immature T lymphocytes and its application. J Med Investig 48:25–30
Seet CS et al. (2017) Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids. Nature Methods 14:521–530
Anderson G, Jenkinson EJ, Moore NC et al (1993) MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus. Nature 362:70–73
Deng Z, Liu H, Rui J, Liu X (2016) Reconstituted Thymus Organ Culture. Methods in Molecular Biology 1323:151–8
Xing Y, Hogquist KA (2014) Isolation, identification, and purification of murine thymic epithelial cells. J. Vis. Exp. (Issue 90), e51780
Stoeckle C, Rota IA, Tolosa E et al (2013) Isolation of myeloid dendritic cells and epithelial cells from human thymus. J. Vis. Exp. (Issue 79), e50951
Seach N, Hammett M, Chidgey A (2013) Epithelial cell culture protocols, vol 945. Humana Press, Totowa, NJ, pp 251–272
Wong K, Lister NL, Barsanti M et al (2014) Multilineage potential and self-renewal define an epithelial progenitor cell population in the adult thymus. Cell Rep 8(4):1198–1209
Bonfanti P, Claudinot S, Amici AW et al (2010) Microenvironmental reprogramming of thymic epithelial cells to skin multipotent stem cells. Nature 466:978–982
Poznansky MC, Evans RH, Foxall RB et al (2000) Efficient generation of human T cells from a tissue-engineered thymic organoid. Nat Biotechnol 18:729–734
Lannes-Vieira J, Dardenne M, Savino W (1991) Extracellular matrix components of the mouse thymus microenvironment: ontogenetic studies and modulation by glucocorticoid hormones. J Histochem Cytochem 39:1539–1546
Emre Y, Irla M, Dunand-Sauthier I et al (2013) Thymic epithelial cell expansion through matricellular protein CYR61 boosts progenitor homing and T-cell output. Nat Commun 4:2842
Ocampo JSP, de Brito JM, Corrêa-de-Santana E et al (2008) Laminin-211 controls thymocyte--thymic epithelial cell interactions. Cell Immunol 254:1–9
Macchiarini P, Jungebluth P, Go T et al (2008) Clinical transplantation of a tissue-engineered airway. Lancet 372:2023–2030
Benders KEM, van Weeren PR, Badylak SF et al (2013) Extracellular matrix scaffolds for cartilage and bone regeneration. Trends Biotechnol 31:169–176
Fan Y, Tajima A, Goh SK et al (2015) Bioengineering thymus organoids to restore thymic function and induce donor-specific immune tolerance to allografts. Mol Ther 23(7):1262–1277
Gordon J, Wilson VA, Blair NF et al (2004) Functional evidence for a single endodermal origin for the thymic epithelium. Nat Immunol 5:546–553
Rossi SW, Jenkinson WE, Anderson G et al (2006) Clonal analysis reveals a common progenitor for thymic cortical and medullary epithelium. Nature 441:988–991
Amici AW, Onikoyi FO, Bonfanti P (2014) Lineage potential, plasticity and environmental reprogramming of epithelial stem/progenitor cells. Biochem Soc Trans 42:637–644
Copelan EA (2006) Hematopoietic stem-cell transplantation. N Engl J Med 354:1813–1826
Ronfard V, Rives JM, Neveux Y et al (2000) Long-term regeneration of human epidermis on third degree burns transplanted with autologous cultured epithelium grown on a fibrin matrix. Transplantation 70:1588–1598
Rama P, Matuska S, Paganoni G et al (2010) Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med 363:147–155
Daley GQ (2012) The promise and perils of stem cell therapeutics. Cell Stem Cell 10:740–749
Gurdon JB, Elsdale TR, Fischberg M (1958) Sexually mature individuals of Xenopus laevis from the transplantation of single somatic nuclei. Nature 182:64–65
Thomson JA (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147
Takahashi K, Tanabe K, Ohnuki M et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872
Savino W, Dardenne M, Velloso LA et al (2007) The thymus is a common target in malnutrition and infection. Br J Nutr 98(Suppl 1):S11–S16
Holländer G a, Krenger W, Blazar BR (2010) Emerging strategies to boost thymic function. Curr Opin Pharmacol 10:443–453
Baksh D, Davies JE, Zandstra PW (2003) Adult human bone marrow-derived mesenchymal progenitor cells are capable of adhesion-independent survival and expansion. Exp Hematol 31:723–732
Markert ML, Devlin BH, McCarthy EA (2010) Thymus transplantation. Clin Immunol 135:236–246
Davies EG (2013) Immunodeficiency in Digeorge syndrome and options for treating cases with complete athymia. Front Immunol 4:322
Lai L, Jin J (2009) Generation of thymic epithelial cell progenitors by mouse embryonic stem cells. Stem Cells 27:3012–3020
Lai L, Cui C, Jin J et al (2011) Mouse embryonic stem cell-derived thymic epithelial cell progenitors enhance T-cell reconstitution after allogeneic bone marrow transplantation. Blood 118:3410–3418
Parent AV, a Russ H, Khan IS et al (2013) Generation of functional thymic epithelium from human embryonic stem cells that supports host T cell development. Cell Stem Cell 13:219–229
Sun X, Xu J, Lu H et al (2013) Directed differentiation of human embryonic stem cells into thymic epithelial progenitor-like cells reconstitutes the thymic microenvironment in vivo. Cell Stem Cell 13:230–236
Soh C-L, Giudice A, Jenny RA et al (2014) FOXN1GFP/w reporter hESCs enable identification of integrin-β4, HLA-DR, and EpCAM as markers of human PSC-derived FOXN1+ thymic epithelial progenitors. Stem Cell Rep 2:1–13
Jenkinson WE, Rossi SW, Jenkinson EJ et al (2005) Development of functional thymic epithelial cells occurs independently of lymphostromal interactions. Mech Dev 122:1294–1299
Zuklys S, Balciunaite G, Agarwal A et al (2000) Normal thymic architecture and negative selection are associated with Aire expression, the gene defective in the autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). J Immunol 165:1976–1983
Holländer G, Gill J, Zuklys S et al (2006) Cellular and molecular events during early thymus development. Immunol Rev 209:28–46
Tajima A, Pradhan I, Trucco M, Fan Y (2016) Restoration of Thymus Function with Bioengineered Thymus Organoids. Current stem cell reports 2(2):128–139
Discher DE, Mooney DJ, Zandstra PW (2009) Growth factors, matrices, and forces combine and control stem cells. Science 324:1673–1677
Di Maggio N, Piccinini E, Jaworski M et al (2011) Toward modeling the bone marrow niche using scaffold-based 3D culture systems. Biomaterials 32:321–329
Gosselin EA, Haleigh EB, Jonathan BS, Christopher JM (2018) Designing natural and synthetic immune tissues. Nature Materials 17(6): 484–498
Acknowledgments
We thank Giulio Cossu, Peter W. Zandstra, Anna Cariboni and Sara Campinoti for critical reading.
P.B. is supported by the European Research Council (ERC-2014-Stg), the Rosetrees Trust Foundation, the UCL Excellence Fellowship Program and the NIHR Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust; E.P. was supported by the New Ideas Grant provided by the Ontario Institute for Regenerative Medicine (OIRM). S.C. is supported by a GOSH Charity studentship (V6116).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Piccinini, E., Bonfanti, P. (2019). Disassembling and Reaggregating the Thymus: The Pros and Cons of Current Assays. In: Boyd, A. (eds) Immunological Tolerance. Methods in Molecular Biology, vol 1899. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8938-6_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8938-6_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8936-2
Online ISBN: 978-1-4939-8938-6
eBook Packages: Springer Protocols