Molecular and Functional Characterization of Clonogenic Human Thymic Epithelial Cells

The thymus is a primary lymphoid organ where bone marrow derived T-cell progenitors come in contact with a unique microenvironment able to sustain their maturation into functional T-cells. This fundamental immunological function of the thymus is supported by thymic epithelial cells, which represent the main component of the thymic stroma. The thymic stroma is organized in a characteristic tridimensional structure that can be distinguished into two main regions, namely, the cortex and medulla. These two regions are responsible for two fundamental processes in the thymus: the positive- and negative- selection of T-cells. The first one involves the selection of T-cells able to react with antigens presented mainly on the surface of cortical cells. T-cell negative selection, meanwhile, is responsible for the elimination of self-reactive T-cells and it is mainly carried out by medullary thymic epithelial cells. The thymic epithelium derives from the primitive endoderm and cortical and medullary thymic epithelial cells have been demonstrated to originate from a common embryonic progenitor population. Currently, the presence of such population has not been described in adult animals and the mechanisms responsible for the maintenance of the post-natal thymus have yet to be clarified. Recently, Bonfanti and colleagues demonstrated that the rat thymus contains a population of clonogenic epithelial cells (rTECs), able to self-renew in the culture system developed in 1975 for epidermal stem cells [Bonfanti et al., 2010, Rheinwald and Green, 1975]. As clonogenicity is a property of skin and other stratified epithelia stem cells, the clonogenic capacity of TECs suggests them as potential thymic epithelial stem cells. Bonfanti and colleagues also demonstrated that rTECs displayed the capacity to integrate into a thymic microenvironment and to express thymic functional markers. The work described in this thesis aims to investigate the stem cell potency and the regenerative capacity of the human thymus. We demonstrated that the human thymus also contains a population of clonogenic thymic epithelial cells (hTECs) able to self-renew and to maintain a general thymic phenotype in the culture system used for skin stem cells and for rTECs. By clonal analysis, we showed that hTECs can be distinguished in three sub-populations, which displayed different molecular phenotypes and different growth and differentiation potentials in vitro. We then investigated the role of Foxn1 transcription factor in hTECs, as Foxn1 is know to be involved in thymic epithelial cells differentiation during development and also in the maintenance of the adult thymus in mice. By experimentally down-regulating Foxn1, we demonstrated that Foxn1 expression is required to sustain the growth of one of the described hTEC sub-populations. We next investigated the differentiation capacity of clonogenic hTECs. Currently, additional experiments are still required to assess hTECs ability to integrate and differentiate in a functional thymic environment. Bonfanti and colleagues also demonstrated that rTECs, if challenged in a hair follicle morphogenetic assay, can be reprogrammed into skin stem cells. Therefore, we decided to investigate the potential of hTECs in an epidermal differentiation assay. In these conditions, hTECs did not show the ability to differentiate into epidermis, probably due to the absence in this assay of the strong morphogenetic signals responsible for the reprogramming process of rTECs into skin stem cells.