Microenvironmental reprogramming of thymic epithelial cells to skin multipotent stem cells

The thymus develops from the third pharyngeal pouch of the anterior gut and provides the necessary environment for thymopoiesis (the process by which thymocytes differentiate into mature T lymphocytes) and the establishment and maintenance of self-tolerance(1-3). It contains thymic epithelial cells (TECs) that form a complex three-dimensional network organized in cortical and medullary compartments, the organization of which is notably different from simple or stratified epithelia(4). TECs have an essential role in the generation of self-tolerant thymocytes through expression of the autoimmune regulator Aire(5,6), but the mechanisms involved in the specification and maintenance of TECs remain unclear(7-9). Despite the different embryological origins of thymus and skin (endodermal and ectodermal, respectively), some cells of the thymic medulla express stratified-epithelium markers(10-12), interpreted as promiscuous gene expression. Here we show that the thymus of the rat contains a population of clonogenic TECs that can be extensively cultured while conserving the capacity to integrate in a thymic epithelial network and to express major histocompatibility complex class II (MHC II) molecules and Aire. These cells can irreversibly adopt the fate of hair follicle multipotent stem cells when exposed to an inductive skin microenvironment; this change in fate is correlated with robust changes in gene expression. Hence, microenvironmental cues are sufficient here to re-direct epithelial cell fate, allowing crossing of primitive germ layer boundaries and an increase in potency(13).

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

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

A Novel 3D hydrogel based microfluidic cell culture platform

Keshav Krishnamani

Breakthroughs in health care over the coming decades may well consist of cell based therapeutics, potentially allowing the repair and replacement of damaged tissues and organs. Recently developed induced pluripotency methods as well as advances in stem cell biology show hope of such a reality. But such prospects come with the need to have a robust understanding and control over cell development and differentiation processes. Cells in the body develop in tightly controlled and specialized micro-environments that provide many complex cues and interactions that guide cell fate by directing gene expression. But current cell culture techniques that are most widely used to study the cell outside the body have not significantly changed from when first started over a century ago, using simple containers and flasks with plastic surfaces. Many studies have shown that three-dimensions (3D) over conventional 2D cell culture alone, showed to have increased levels of cell organization, morphology and function. By combining novel biomaterials and microfabrication methods, this project aims to create a tool that allows the study of cells within a simplified 3D environment. With the potential to build up the physical and soluble complexity of this environment in a tailor made fashion, a "dissection" of the functions and influences of external factors may be understood. Such a tool can also be used for the development of cell based models, highly relevant for drug discovery application as well as potentially for the development of cell based therapeutics. We have developed a novel microfluidic 3D cell culture platform that allows controlled soluble factor perfusion in an enzymatically formed and cell responsive PEG-based hydrogel. Soft lithographic processes were used to mold microchannels on a layer of hydrogel. In a second step, a flat hydrogel layer is placed on top to enclose and form the microchannels . The system allows the control over soluble factor delivery and it is able to perform long-term cell culture and generate user controlled gradients of soluble factors. With continuous perfusion, metabolic waste is constantly removed and cells are supplied with fresh medium. The platform also can allow creation of tailor made environments building up complexity in a controlled fashion. Higher throughput is also achieved by arraying individual culture chambers. The device was applied towards the culture of mouse embryonic stem cells (mESC) in order to demonstrate their self-renewal capacity on the microfluidic platform. The effects of some cytokine factors were also studied. Meaningful readouts such as fluorescence are easily used for analysis and cells can possibly be extracted from the device for further downstream analysis and processing. Through this device we show that microfluidics technologies combined with biomaterials enables the creation of platforms that are biologically relevant for the study and understanding of cell processes from cancer to stem cell development, processes which are fundamentally regulated by interactions with the microenvironment. The platform developed may also be used to develop human tissue models perhaps more relevant than current animal based testing, providing new tools for drug development and also potentially new approaches for developing cell based therapeutics

2009

Understanding engraftment of cultured epidermal stem cells

Nicolas Grasset

EPFL2008

A Novel 3D hydrogel based microfluidic cell culture platform

Keshav Krishnamani

2009

Thymic epithelial cells

Paola Bonfanti

EPFL2008