Neonatal Dermal Cells Research Articles

Dominant-negative Sox18 function inhibits dermal papilla maturation and differentiation in all murine hair types

SOX family proteins SOX2 and SOX18 have been reported as being essential in determining hair follicle type; however, the role they play during development remains unclear. Here, we demonstrate that Sox18 regulates the normal differentiation of the dermal papilla of all hair types. In guard (primary) hair dermal condensate (DC) cells, we identified transient Sox18 in addition to SOX2 expression at E14.5, which allowed fate tracing of primary DC cells until birth. Similarly, expression of Sox18 was detected in the DC cells of secondary hairs at E16.5 and in tertiary hair at E18.5. Dominant-negative Sox18 mutation (opposum) did not prevent DC formation in any hair type. However, it affected dermal papilla differentiation, restricting hair formation especially in secondary and tertiary hairs. This Sox18 mutation also prevented neonatal dermal cells or dermal papilla spheres from inducing hair in regeneration assays. Microarray expression studies identified WNT5A and TNC as potential downstream effectors of SOX18 that are important for epidermal WNT signalling. In conclusion, SOX18 acts as a mesenchymal molecular switch necessary for the formation and function of the dermal papilla in all hair types.

Triiodothyronine (T3) inhibits hyaluronate synthesis in a human dermal equivalent by downregulation of HAS2

Triiodothyronine (T3) is a thyroid hormone that can have varying effects on skin. In order to assess the effects of T3 on the human dermis, we prepared dermal equivalents using neonatal dermal cells via the process of self-assembly in the presence of differing concentrations of T3. These dermal equivalents were prepared in the absence of serum and a three dimensional matrix allowing for the direct assessment of different concentrations of T3 on dermal extracellular matrix formation. Three different concentrations of T3 were chosen, 20pM, which is part of the base medium, 0.2nM T3 and 2nM T3. We find that self-assembled dermal equivalents formed under these conditions show a progressive "thinning" with increasing T3 concentrations. While we observed no change in total collagen content, inhibition of hyaluronate (HA) synthesis was observed in the 0.2- and 2-nM T3 constructs as compared to the 20-pM construct. Other glycosaminoglycan synthesis was not affected by increasing T3 concentrations. In order to identify the gene(s) responsible for inhibition of HA synthesis in the 2-nM T3 dermal equivalent, we conducted a differential gene array analysis. The results of these experiments demonstrate the differential expression of 40 genes, of these, 34 were upregulated and 6 genes were downregulated. The results from these experiments suggest that downregulation of HAS2 may be responsible for inhibition of hyaluronate synthesis in the self-assembled 2-nM T3 human dermal matrix.

Tissue-engineered fetal dermal matrices

In the early to mid-gestation fetus, skin wounds heal with no scar formation and perfect restoration of dermal architecture. This phenomenon is intrinsic to fetal skin. The intrinsic phenotypic properties of the fetal fibroblast are believed to be "the effector of scarless repair". We sought to prepare dermal matrices with high similarity to the mid-gestation fetal dermis using the technology of "self-assembly" with fetal dermal cells of 18, 20, and 22wk gestation. Comparison of these dermal constructs to those prepared with neonatal dermal cells, adult skin, neonatal foreskin, and mid-gestation fetal skin demonstrates that these fetal dermal matrices bear marked morphological and biochemical resemblance to the mid-gestation fetal dermis. In order to shed further light on the genes involved in scarless wound healing, we conducted a differential gene array analysis of the neonatal and fetal dermal matrices. Using a gene chip (GLYCOv4 gene chip) of approximately 1,260 human genes, we observed differential expression of 67 genes. A number of fibrotic genes were observed to be downregulated and anti-fibrotic genes upregulated.

Clonal Growth of Dermal Papilla Cells in Hydrogels Reveals Intrinsic Differences between Sox2-Positive and -Negative Cells In Vitro and In Vivo

In neonatal mouse skin, two types of dermal papilla (DP) are distinguished by Sox2 expression: CD133+Sox2+ DP are associated with guard/awl/auchene hairs, whereas CD133+Sox2− DP are associated with zigzag (ZZ) hairs. We describe a three-dimensional hydrogel culture system that supports clonal growth of CD133+Sox2+, CD133+Sox2−, and CD133−Sox2− (non-DP) neonatal dermal cells. All three cell populations formed spheres that expressed the DP markers alkaline phosphatase, α8 integrin, and CD133. Nevertheless, spheres formed by CD133− cells did not efficiently support hair follicle formation in skin reconstitution assays. In the presence of freshly isolated P2 dermal cells, CD133+Sox2+ and CD133+Sox2− spheres contributed to the DP of both AA and ZZ hairs. Hair type did not correlate with sphere size. Sox2 expression was maintained in culture, but not induced significantly in Sox2− cells in vitro or in vivo, suggesting that Sox2+ cells are a distinct cellular lineage. Although Sox2+ cells were least efficient at forming spheres, they had the greatest ability to contribute to DP and non-DP dermis in reconstituted skin. As the culture system supports clonal growth of DP cells and maintenance of distinct DP cell types, it will be useful for further analysis of intrinsic and extrinsic signals controlling DP function.

Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis

In this protocol, we describe how to isolate keratinocytes from adult mouse epidermis, fractionate them into different sub-populations on the basis of cell surface markers and examine their function in an in vivo skin reconstitution assay with disaggregated neonatal dermal cells. We also describe how the isolated keratinocytes can be subjected to clonal analysis in vitro and in vivo and how to enrich for hair follicle-inducing dermal papilla cells in the dermal preparation. Using these approaches, it is possible to compare the capacity of different populations of adult epidermal stem cells to proliferate and to generate progeny that differentiate along the different epidermal lineages. Isolating, fractionating and grafting cells for the skin reconstitution assay is normally spread over 2 d. Clonal growth in culture is assessed after 14 d, while evaluation of the grafts is carried out after 4-5 weeks.

Epithelial Stem Cells: A Folliculocentric View

Putative epithelial stem cells were identified in the hair follicle bulge as quiescent "label retaining cells". The study of these cells was hindered until the identification of bulge cell molecular markers, such as CD34 expression and K15 promoter activity. This allowed for the isolation and characterization of bulge cells from mouse follicles. Bulge cells possess stem cell characteristics, including multipotency, high proliferative potential, and their cardinal feature of quiescence. Lineage analysis demonstrated that all epithelial layers within the adult follicle and hair originated from bulge cells. Bulge cells only contribute to the epidermis during wound healing, but after isolation, when combined with neonatal dermal cells, they regenerate new hair follicles, epidermis, and sebaceous glands. Bulge cells maintain their stem cell characteristics after propagation in vitro, thus ultimately they may be useful for tissue engineering applications. Understanding the signals important for directing movement and differentiation of bulge cells into different lineages will be important for developing treatments based on stem cells as well as clarifying their role in skin disease.

Clothing the Nude: A New Model for Trichogenesis

Considering how hard evolution has worked to denude humans of our fur covering it seems paradoxical how much time and effort we spend on enhancing and preserving the little that remains. This effort is mirrored in the huge (and continually growing) scientific literature on hair. What all this study has revealed is that the hair follicle, and related structures, is an excellent, accessible and fruitful model for many fields such as developmental biology, epithelial– mesenchymal interactions, regeneration and stem cell biology. Indeed a great deal of extremely interesting and useful data has been obtained from studies of hair development and cycling, particularly with the advent of transgenic tools. These studies are, however, currently limited by two important factors. One is the relative inaccessibility of mammalian embryos to manipulation during development. Thus researchers using the chick system to study skin and feather development have benefited from the ability to manipulate and follow the developmental embryos in ovo. In the chick system it has been possible to observe and intervene at all stages of skin development, even before dermis development. Moreover, by using transfection, via electroporation or viral infection, experiments, the critical importance of different pathways in initiation and morphogenesis of epidermal appendages has been highlighted (Yu et al, 2004). In mammalian systems, on the other hand, until the advent of targeted transgenic methods, our understanding of hair induction and development was restricted to the results obtained via dermal–epidermal recombination experiments. The other major impediment is the lack of a dermal specific promoter for targeted transgenics. Since the introduction of epidermal specific promoters, the cre-lox system and other mechanisms of controlling transgene expression, numerous, very elegant studies have been carried out where specific genes can be expressed or knocked out in an epidermal specific manner. The lack of a dermal specific promoter, however, means that genes of the mesenchymal component can, for the most part, only be studied indirectly, for example by expression of a diffusible inhibitor in the epidermis (Andl et al, 2002). This is except in the rare cases when a general knockout affecting hair formation can be shown not to have an effect in the epidermis (Botchkarev et al, 2002). The lack of an in vitro or easily manipulated in vivo system is thus a great impediment to experimental study of hair development and induction. There is hence a great need for a simple and repeatable in vitro or in vivo assay for hair reconstitution and growth and methods to test for the inductiveness of dermal cells. Lacking these, using cells manipulated ex vivo in order to study hair induction and development has been difficult. Previous models for trichogenesis in vivo have been developed, including the implantation of dermal papillae (Cohen, 1961), and subsequently cultured dermal papilla cells (Jahoda et al, 1984) in rat ear or grafts under the kidney capsule of nude mice (Inamatsu et al, 1998; Takeda et al, 1998). More recently models that allows grafting of defined mesenchymal and epidermal cell populations, for example, using silicone chambers and nude mice (Lichti et al, 1993; Weinberg et al, 1993) have been developed and have proved extremely useful. These assays are, however, quite time and materials intensive, particularly in the large number of (often precious) cells used and the time required for a positive result. It is for this reason that the method presented in this issue by the group of Kurt Stenn (Zheng et al, 2005) and similar methods recently developed (Morris et al, 2004; Pouliot et al, 2005) is of interest. In their paper they present a detailed and elegant system that they call the ‘‘hair patch’’ assay, for studying trichogenesis, using dissociated cells transplanted in the dermis of nude mice. The chief advances and advantages of this system over earlier methods are its simplicity, relative rapidity in terms of seeing results, and the small numbers of cells required. The method involves injecting a mixture of isolated neonatal dermal cells with epidermal aggregates into the dermis of nude mice. These are then able to interact and undergo relatively normal hair morphogenesis to give rise to cycling hair follicles within 8–12 d. Because of the simplicity of the method (a simple injection) and the fact that the ‘‘grafts’’ are small and spatially restricted, a number, six or more, can be performed on the same mouse, which represents large potential savings in terms of animals and their upkeep. In addition relatively small numbers of cells, i.e., 10 dermal and 5 10 epidermal cells, are used as compared with other methods, for example the chamber graft assay requires 10-fold more cells. A number of interesting experiments can be envisaged with this system as is in terms of testing the interaction of mesenchymal and epithelial cells from different sources or genetic backgrounds. It will, moreover, be extremely inter-