A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3) can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes.

Hsp27, a small heat-shock protein, has important roles in many cellular processes, including cytoskeleton dynamics, cell differentiation, and apoptosis. Its expression in normal epidermis correlates with differentiation; however, little is known about the regulatory mechanisms involved. In this study, we report that Hsp27 undergoes upregulation, phosphorylation, and redistribution to the cytoskeleton during the late phase of epidermal keratinocyte differentiation. Our results also show that the expression of the dual leucine zipper-bearing kinase (DLK), an upstream activator of the MAP kinase pathways, is sufficient by itself to induce Hsp27 phosphorylation, cell periphery localization, and redistribution to the insoluble protein fraction (cytoskeleton) in poorly differentiated keratinocytes. This redistribution correlates with the insolubilization of cornified envelope-associated proteins such as involucrin. Interestingly, the effects of DLK on Hsp27 were blocked by PD98059, a selective inhibitor of the extracellular signal-regulated protein kinase (ERK) pathway. Moreover, downregulation of Hsp27 by small interfering RNA in epithelial cells expressing DLK was accompanied by attenuated expression of involucrin in the cytoskeleton. Thus, these observations suggest that the DLK-ERK signaling pathway may act as a regulator of the interaction that occurs between Hsp27 and the cytoskeleton during the formation of the cornified cell envelope, a process conferring to the skin its crucial barrier function.

Dual leucine zipper-bearing kinase (DLK) is an inducer of keratinocyte differentiation, a complex process also involving microtubule reorganization to the cell periphery. However, signaling mechanisms involved in this process remain to be elucidated. Here, we demonstrate that DLK enhances and is required for microtubule reorganization to the cell periphery in human cell culture models and in Dlk knockout mouse embryos. In tissue-engineered skins with reduced DLK expression, cortical distribution of two microtubule regulators, LIS1 and HSP27, is impaired as well as desmosomal and tight junction integrity. Altered cortical distribution of desmosomal and tight junction proteins was also confirmed in Dlk knockout mouse embryos. Finally, desmosomal and tight junction defects were also observed after microtubule disruption in nocodazole-treated tissue-engineered skins, thus confirming a role for microtubules in the maintenance of these types of cell junctions. Globally, this study demonstrates that DLK is a key regulator of microtubule reorganization to the cell periphery during keratinocyte differentiation and that this process is required for the maintenance of desmosomal and tight junction integrity.

Progress in the identification of skin stem cells and the improvement of culture methods open the possibility to use stem cells in regenerative medicine. Based on their quiescent nature, the development of label retention assays allowed the localization of skin stem cells in the bulge region of the pilosebaceous units and in the bottom of rete ridges in glabrous skin. The development of markers such as keratin 19 also permits their study in human tissues. In this chapter, protocols to identify skin stem cells based on their slow-cycling property and their expression of keratin 19 will be described in detail. The methods include the labeling of skin stem cells within mouse or rat tissues in vivo, the labeling of proliferative human cells in vitro using 5-bromo-2-deoxyuridine (BrdU), and the detection of keratin 19 and BrdU by immunofluorescence or immunoperoxidase staining.

Human beings are greatly preoccupied with the unavoidable nature of aging. While the biological processes of senescence and aging are the subjects of intense investigations, the molecular mechanisms linking aging with disease and death are yet to be elucidated. Tissue engineering offers new models to study the various processes associated with aging. Using keratin 19 as a stem cell marker, our studies have revealed that stem cells are preserved in human skin reconstructed by tissue engineering and that the number of epithelial stem cells varies according to the donor's age. As with skin, human corneas can also be engineered in vitro. Among the epithelial cells used for reconstructing skin and corneas, significant age-dependent variations in the expression of the transcription factor Sp1 were observed. Culturing skin epithelial cells with a feeder layer extended their life span in culture, likely by preventing Sp1 degradation in epithelial cells, therefore demonstrating the pivotal role played by this transcription factor in cell proliferation. Finally, using the human tissue-engineered skin as a model, we linked Hsp27 activation with skin differentiation.