Modeling the cornea: an architectural approach

Nowadays the mechanism regulating the corneal epithelium renewal still remains unclear. Until now it was thought to be the fact of stem cells distributed in the limbus, generating TA cells migrating towards the central cornea and responsible for its renewal. But Majo and Barrandon (Nature Oct 2008) showed the presence of stem cell within the corneal epithelium and the ability of this epithelium to renew without limbal contribution. They prove that the limbal epithelium was involved only in healing processes. To simulate and test the relevance of the different hypothesis a model able to map cells location precisely and analyse or predict their movements in function of the internal forces and tissue biomechanics must be created. In this project I have created a mouse cornea architectural model. It was based on three dimensional reconstruction of a mouse eye using histological cuts. The model include two main parameters; the curvature of the different eye part and their thickness. The techniques developed and used in this project are easily reproducible for the construction of other cornea or eye model of diverse species for instance. This model can be used in clonal analysis to locate cells or groups of cells three-dimensionally. Besides analysis of the corneal structure at the cellular level was performed by the observation of clones (expressing $\beta$ -galactosidase), present in the limbus and cornea epithelium. Their location, form and orientation were investigated. This work provides a useful tool in the study of corneal epithelium renewal. Moreover it establishes the basis for the development of a future biomechanical model that will simulate tissue deformation, internal forces or cell movements. Studies of corneal parameters will be performed on enucleated eyes under normal physiological conditions inside an incubator regulating variables such as temperature, intraocular pressure or humidity level, currently develop in the laboratory

Thymic epithelial cells

Paola Bonfanti

As primary lymphoid organ, the thymus provides the essential environment for maturation and selection of functional T lymphocytes. Thymic epithelial (TE) cells derive from the endoderm, and more specifically, from the third pharyngeal pouch. The thymic epithelium is organized in a three-dimensional architecture that does not easily fit into standard classifications of simple or stratified epithelia. Moreover, the mechanisms that maintain the epithelial compartment in the post-natal thymus remain unclear. Although they have been postulated to exist as in all epithelia, thymic stem cells have not been clearly identified to date. One crucial property of adult multipotent stem cells (SCs) in the skin and other stratified epithelia is clonogenicity, i.e the ability of a single cell to give rise to a cell population. Epidermal clonogenic keratinocytes can be propagated for several generations in vitro and can permanently engraft when transplanted onto patients. In the absence of reliable molecular markers to identify and purify multipotent SCs, the demonstration that they can selfrenew in vitro and in vivo represents, to date, the best method to assess stemness. Likewise, the thymus contains epithelial cells that express genes usually only expressed in the skin or other stratified epithelia, thus suggesting the existence of a possible relationship. In the work described in this thesis, we investigated the potentialities of rat primary TE cells through clonal analysis and functional assays. We demonstrate that embryonic and postnatal rat primary TE cells have a clonogenic growth pattern in vitro. We also describe the molecular signature of subtypes of thymic keratinocytes and address the postnatal role of developmentally regulated genes which are commonly expressed in skin and thymus. Clones of rat TE cells have been transplanted onto developing skin in order to evaluate their response to skin morphogenetic signals. Similarly, the response of these clones, as well as of clones obtained from hair follicles, to a thymic environment has been investigated. We demonstrate that cultured TE cells share morphological, phenotypic and functional characteristics with skin multipotent stem cells. When challenged in thymic or skin in vivo assays the clonal progeny of a single TE cell is able to integrate into a functional thymus or give rise to all skin derivatives respectively, i.e. epidermis, hair follicle and sebaceous gland. Conversely skin cells cannot participate to thymic reconstitution. Uncovering the relationship of TE cells with stem cells of stratified epithelia may provide important insights into the molecular basis of diseases involving both thymus and skin.

EPFL2008

Oligopotent stem cells are distributed throughout the mammalian ocular surface

Georges Abou Jaoudé, Yann Barrandon, Ariane Rochat

The integrity of the cornea, the most anterior part of the eye, is indispensable for vision. Forty- five million individuals worldwide are bilaterally blind and another 135 million have severely impaired vision in both eyes because of loss of corneal transparency(1); treatments range from local medications to corneal transplants, and more recently to stem cell therapy(2). The corneal epithelium is a squamous epithelium that is constantly renewing, with a vertical turnover of 7 to 14 days in many mammals(3). Identification of slow cycling cells ( label- retaining cells) in the limbus of the mouse has led to the notion that the limbus is the niche for the stem cells responsible for the long- term renewal of the cornea(4); hence, the corneal epithelium is supposedly renewed by cells generated at and migrating from the limbus, in marked opposition to other squamous epithelia in which each resident stem cell has in charge a limited area of epithelium(5,6). Here we show that the corneal epithelium of the mouse can be serially transplanted, is self- maintained and contains oligopotent stem cells with the capacity to generate goblet cells if provided with a conjunctival environment. Furthermore, the entire ocular surface of the pig, including the cornea, contains oligopotent stem cells (holoclones)(7,8) with the capacity to generate individual colonies of corneal and conjunctival cells. Therefore, the limbus is not the only niche for corneal stem cells and corneal renewal is not different from other squamous epithelia. We propose a model that unifies our observations with the literature and explains why the limbal region is enriched in stem cells.

2008

Understanding engraftment of cultured epidermal stem cells

Nicolas Grasset

The epidermis and its appendages protect our body from environmental hazards. Cells generated in the basal layer continuously replace the terminally differentiated keratinocytes that are shed off the epidermal surface. Long-term renewal depends on specific tissue cells, called stem cells. The epidermis can efficiently repair itself when wounded, thanks to the proliferation, migration and differentiation of the stem cells and their progeny. In extensive full-thickness burns, restoration of the epidermal barrier can only be achieved by means of autologous skin grafts. Cell therapy using cultured epithelium autografts (CEA) has been part of the therapeutic arsenal since the early eighties (Gallico et al., 1984; O'Connor et al., 1981) and it has saved the life of many patients worldwide. Paradoxically, little is known on the behavior of the transplanted stem cells and engraftment has remained variable. Unpublished data from our laboratory have shown that the number of stem cells decrease rapidly in transplanted CEA in humans. This further highlights the necessity to thoroughly comprehend the cellular and molecular mechanisms of stem cell engraftment. Several fundamental questions must be addressed, for example: 1) By which mechanisms do stem cells adjust to the stress of transplantation? 2) How many stem cells are required for long-term renewal of the regenerated epidermis? 3) Can one manipulate the niche? To thoroughly investigate the mechanisms of engraftment, it is critical to develop a predictable animal model since it is difficult to experiment on human. We have chosen the pig as a large animal model because clinical procedures used for CEA transplantation cannot be recapitulated in laboratory animals. Firstly, we have demonstrated that pig and human skin share many features. In particular, the skin of the pig contains keratinocyte stem cells that can be characterized by clonal analysis using the same criteria as in the human (holoclone, meroclone, paraclone). Secondly, we have reproducibly produced CEA, including some made from the progeny of a single EGFP-labeled keratinocyte stem cell. Thirdly, we have recapitulated in the pig all surgical procedures used to transplanting CEA in humans. Fourthly, CEA engraftment has been systematically investigated by histology, immunochemistry and clonal analysis. Fifthly, we have demonstrated that the number of stem cells rapidly decreases following CEA transplantation and that there is little apoptosis in transplanted CEA, which suggests that stem cells are rather lost through terminal differentiation. Collectively our results demonstrate that the pig is the best available model system to investigate the mechanisms that govern stem cell engraftment. Superior engraftment may be achieved by improving the grafting bed, by designing smart matrices to better mimic the niche, or by manipulating stem cell behavior. A better comprehension of stem cell engraftment will certainly benefit patients suffering from large burns and those with disabling skin diseases (e.g. dystrophic epidermolysis bullosa). It will also profit stem cell therapy at large since optimal engraftment is also needed for hematopoïetic, muscle and neural stem cells.

EPFL2008

Understanding engraftment of cultured epidermal stem cells

Nicolas Grasset

EPFL2008

Thymic epithelial cells

Paola Bonfanti

EPFL2008