Understanding engraftment of cultured epidermal stem cells

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 Unpublished data from our laboratory have shown that the number of stem cells decrease rapidly in transplanted CEA in humans.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.

Early Fate Choice Determines in vivo Engraftment and in vitro Behavior of Epidermal Stem Cells

François Gorostidi

Regenerative medicine aims at using stem cells to restore or establish lost, damaged, diseased, or aging tissues and organs function. The transplantation of cultured epidermal autograft (CEA) has saved the lives of many burned patients over the last 30 years, but the resulting epidermis is far from perfect, and clinical outcomes remain unpredictable. The engraftment of cultured epidermal stem cells is poorly understood, and, as a result, suboptimal. Using a large animal model, the pig, we recapitulated the clinical settings and outcomes of human CEA transplantation. With this model, we study the engraftment process, and we demonstrate that cultured epidermal stem cells often, but not always, favor the choice of differentiation over self-renewal when transplanted on full thickness wounds. Differences in this early fate choice are likely to explain the variability of clinical outcomes. We developed a system of in vitro live cell imaging (LCI) to study the fate choices of multiple individual cells in a microenvironment that is much more controlled than the grafting bed of a burned patient. We demonstrate that keratinocytes have a very heterogeneous behavior in vitro. With a fluorescent reporter of cell cycle progression (FUCCI), we show the influence of the cell cycle on early fate choices of cultured keratinocytes. By introducing a ROCK inhibitor in the culture medium, we show that keratinocyte’s early fate choices can be impacted so that they show greater clonogenicity and growth potential. The unique combination of large animal CEA transplantation with individual cell LCI demon- strate the critical role of early fate choice, both for epidermal stem cell engraftment and in vitro behavior. The possibility to influence the early fate choices of stem cells by modifying the microenvironment has a great potential to improve the engraftment and the ex vivo ampli- fication of stem cells, the two limiting steps of cell replacement therapy.

EPFL2012

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

Expansion of a VE-Cadherin+CD45+ population in the yolk sac upon Etv2 overexpression in the mouse embryo - Effects of BMP and Wnt signaling inhibition on the differentiation of a Flk1+ extraembryonic mesoderm in vitro model

Matteo Pluchinotta

The difficulty to find compatible donors for bone marrow transplantation makes the need for an alternative source of HSCs urgent. HSC derived from patient-specific iPS cells are ideal candidates for this purpose. Nevertheless, although HSCs are the best characterized adult stem cells, the in vitro generation and expansion of bone fide HSCs still has not been achieved without transgene insertion. Successful derivation of HSCs from ES cells or iPS cells will require a comprehensive understanding of inductive signals and downstream effectors involved in the normal arising of HSCs during embryonic development. BMP and Wnt signaling pathways play a very important role in this differentiation process. Here, we report how BMP and Wnt signaling inhibition cooperatively alter the differentiation of Flk1+PDGFRα- cells after reculture in serum-free, feeder layer-free conditions. This data will be useful in determining the factors that have to be added to a chemically defined culture medium aimed at derivation of bone fide HSC in vitro. In the second part of this master’s project, we studied the spatial and temporal localization requirements of Etv2 expression, a key regulator of the earliest events in the development of the hematopoietic and vascular systems. To gain more insight on this matter, we conditionally knocked-in Etv2 in the early endothelial compartment, using a Tie2Cre system. This leaded to an embryonically lethal phenotype by E11.5, and most interestingly, to the expansion of a VE-Cadherin+CD45+ population in the E10.5 yolk sac. We believe that these cells represent a increased pool of immature pre-definitive hematopoietic stem cells, thus underlining further the importance of Etv2 in the differentiation process towards hematopoietic, and endothelial lineages in the mouse embryo.

2011