Clonage | EPFL Graph Search

Le clonage désigne principalement deux processus. C'est d'une part la multiplication naturelle ou artificielle à l'identique d'un être vivant, c'est-à-dire avec conservation exacte du même génome pour tous les descendants (les clones). C'est donc un synonyme de certaines formes de multiplication asexuée telles que le bouturage. C'est d'autre part la multiplication provoquée d'un fragment d'ADN par l'intermédiaire d'un micro-organisme. Ainsi, en biologie, le mot clonage désigne plusieurs choses :

d'une part, le fait de reproduire des organismes vivants pour obtenir des êtres génétiquement identiques ; ceci peut s'appliquer à de simples cellules (clonage cellulaire, par prélèvement d'une seule cellule, qui est mise en culture de manière individuelle) ou bien à des animaux – donc y compris les êtres humains – et des végétaux (clonage reproductif, bouturage). L'ensemble de ces cellules, ou individus, forme un seul et même clone (tant que le patrimoine génétique est identique) ;
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Molecular and Functional Characterization of Clonogenic Human Thymic Epithelial Cells

Melissa Maggioni

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.

EPFL2012

Exploring the potency of normal and pathological human thymic epithelial cells

Tiphaine Mélodie Chantal Arlabosse

Thymic epithelial cells (TECs) are organised in a unique 3D network that is critical for the development of efficient and self-tolerant T-cells. We report for the first time that the human thymus of all ages, even old and involuted, contains a significant number (up to 30%) of clonogenic TECs with extensive growth potential. All clonogenic TECs derive from a common EpCAM+ precursor, which within 48h in culture undergoes a molecular switch and gives rise to refringent EpCAM- colonies and stratified EpCAM+ colonies. The latter two subpopulations can be distinguished based on their morphology, their expression of genes implicated in epidermal stratification and their expression of genes implicated in epithelia-mesenchymal transition. Clonal analysis revealed that clonogenic cells are hierarchically organised, refringent cells being higher in the hierarchy than stratified cells. Although the thymus normally involutes with age, it can be the site of uncontrolled proliferation, resulting in the formation of thymic tumours including thymomas. Thymomas are classified by the WHO based on histological criteria as type A, B and AB and are frequently associated with Myasthenia Gravis, an autoimmune disease affecting the neuromuscular junction. The biology of thymomas is poorly understood, because of the rarity of the tumours and because of the lack of a reliable in vitro model. We report for the first time, that clonogenic cells can be isolated from all thymoma subtypes as well as from thymic hyperplasia and mediastinal teratoma. The latter cells displayed many similarities with normal TECs but also differences in terms of morphology, gene expression and genetic profile. Importantly, all thymic tumours studied contained refringent-like EpCAM- cells and stratified EpCAM+ cells, supporting the hypothesis that thymic tumour-initiating cells arise from clonogenic TECs. We have not succeeded yet in developing an appropriate assay to support the growth of clonogenic epithelial cells from thymic tumours in vivo. Finally, we report that the SY5 monoclonal antibody raised against human involucrin reproducibly cross-reacts with a molecule present in the Golgi apparatus of refringent cells derived from normal and pathological thymi. Immunocytochemistry and knock-down experiments revealed that the latter molecule is not involucrin. Epitope mapping by phage-display experiments and data base search identified putative candidate proteins, including nesprin-1, a player of the neuromuscular junction, which could be the key to understand the frequent association of thymomas with Myasthenia Gravis.

EPFL2017

Epithelial stem cells and mechanical signal transduction

Vincent Bernard Cattin

Hair follicles are cutaneous structures characteristic of mammals. These sensitive organs cycle and contain multipotent epithelial stem cells which are implicated in hair growth and hair cycle. A single whisker follicle of the rat hosts up to 1500 stem cells, many more than necessary to regenerate the entire follicle throughout the animal's life. These cells are preferentially located in the most innervated region of the hair follicle. We hypothesised that stem cells might have an additional function aside from tissue renewal: stem cells may be involved in the mechanical signal transduction between hair follicle and afferent nervous system. This supplemental role may either take place through the interaction of the multipotent epithelial stem cells with nerve endings, or through neuroepithelial Merkel cells acting as intermediates. Merkel cells are located in the basal layer of the whisker follicle outer root sheath (ORS) and share a close relationship with their neighbouring nerve endings. These cells specifically express the transcription factor Math1 and their function is poorly understood. We demonstrate here by clonal analysis and transplantation assays that Merkel cells are not derived from epithelial stem cells. In addition, Math1-expressing cells isolated from the epidermis are unable to form colonies and to participate in the generation epidermal lineages when transplanted, failing to show stemness properties. Furthermore, when cultured in conditions that favour the growth of neurospheres, they are unable to proliferate, revealing a different behaviour than that of skin-derived neural crest cells. We also demonstrate that multipotent epithelial stem cells express several proteins implicated in glutamate neurotransmission, as well as other molecules usually found in the nervous system. The expression of NMDAR and AMPAR subunits, as well as their scaffolding proteins and neurotransmitter vesicular transport-implicated proteins are uncovered for the first time in the multipotent epithelial stem cells from the bulge. Our results support the mechanical signal transduction hypothesis and confirm the increasingly important role of the recently described neural-like structures of the skin. This work paves the way for a better comprehension of multipotent epithelial stem cell behaviour and stem cell-related pathologies.

EPFL2008