3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression.
J Vis Exp
; (101): e52614, 2015 Jul 30.
Article
en En
| MEDLINE
| ID: mdl-26275017
ABSTRACT
Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance. Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology (i) proliferation and terminal differentiation of CD80(lo), Aire-negative into CD80(hi), Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80(hi) mTECs.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Timo
/
Técnicas de Cocultivo
/
Células Epiteliales
/
Timocitos
Tipo de estudio:
Prognostic_studies
Límite:
Animals
/
Pregnancy
Idioma:
En
Revista:
J Vis Exp
Año:
2015
Tipo del documento:
Article