Human hair follicle dermal sheath and papilla cells support keratinocyte growth in monolayer coculture.

Traditional skin grafting techniques are effective but limited methods of skin replacement. Autologous transplantation of rapidly cultured keratinocytes is successful for epidermal regeneration, but the current gold-standard technique requires mouse fibroblast feeders and serum-rich media, with serum-free systems and dermal fibroblast (DF) feeders performing relatively poorly. Here, we investigated the capacity of human hair follicle dermal cells to act as alternative supports for keratinocyte growth. Dermal papilla (DP) dermal sheath (DS), DF and 3T3 cells were used as inactivated feeder cells for human keratinocyte coculture. Under conditions favouring dermal cells, proliferation of keratinocytes in the presence of either DS or DP cells was significantly enhanced compared with DF cells, at levels comparable to keratinocytes cultured under gold-standard conditions. Secreted protein acidic and rich in cysteine (SPARC) expression increased DS and DP cells relative to DFs; however, further experiments did not demonstrate a role in keratinocyte support.

Human hair follicle dermal cells and skin fibroblasts show differential activation of NF-κB in response to pro-inflammatory challenge.

The underlying mechanism of immune privilege in hair follicle cell dermal papilla (DP) and sheath (DS) populations is not well understood, and the responsiveness of hair follicle dermal cells to pro-inflammatory challenge presently remains unknown. In this work, we describe acute NF-κB activation in human DS, DP and dermal fibroblast (DF) cells challenged with TNF-alpha and IL1-beta. In contrast, the DS and DP cells revealed an unexpected tolerance to bacterial LPS challenge relative to DF cells. Understanding follicle cell responses to typical pro-inflammatory stimuli is critical for diseases where collapse of hair follicle immune privilege is observed, and to further applications in autologous stem cell/wound healing therapeutics.

Sodium salicylate inhibits TNF-alpha-induced NF-kappaB activation, cell migration, invasion and ICAM-1 expression in human melanoma cells.

We have previously shown that tumour necrosis factor-alpha (TNF-alpha) upregulates human melanoma cell integrin expression, migration and invasion in vitro. The aim of this study was to investigate the effect of the non-steroidal anti-inflammatory agent sodium salicylate on TNF-alpha-induced activation of the transcription factor nuclear factor-kappaB (NF-kappaB) and upregulation of intercellular adhesion molecule-1 (ICAM-1), and TNF-alpha-stimulated cell migration and invasion through fibronectin. HBL human melanoma cells were pre-incubated with sodium salicylate prior to stimulation with TNF-alpha for 24 h. NF-kappaB activation was measured using an assay that detects changes in the expression of a luciferase reporter gene under the direct control of NF-kappaB transcriptional activity. The effect of sodium salicylate and TNF-alpha on HBL cell invasion over 20 h and migration over 24 h was studied using fibronectin invasion and 'scratch wound' migration models in vitro, as described previously. Sodium salicylate inhibited TNF-alpha-stimulated NF-kappaB activation in melanoma cells in a concentration-dependent manner, and this was achieved with pre-incubation times as short as 15 min. TNF-alpha-stimulated ICAM-1 expression in HBL cells was also downregulated by sodium salicylate, although in a manner inversely related to the concentration of this agent. In functional assays, TNF-alpha stimulated migration and invasion, and sodium salicylate significantly reduced the extent of melanoma invasion and migration in both the presence and absence of TNF-alpha. In conclusion, sodium salicylate effectively inhibited TNF-alpha-induced upregulation of NF-kappaB, ICAM-1 expression, in-vitro migration and invasion in human melanoma cells, indicating that non-steroidal anti-inflammatory drugs may be a useful therapeutic approach to oppose inflammation-induced melanoma invasion and metastasis in vivo.

Alpha-melanocyte stimulating hormone cytoprotective biology in human dermal fibroblast cells.

Alpha-melanocyte stimulating hormone (alpha-MSH) has been identified as a potent anti-inflammatory peptide effective in various tissues including skin. It acts by inhibiting the production and action of several pro-inflammatory stimuli including TNF-alpha, IL-1beta and LPS in a number of cell types. The role of such stimuli in inducing cellular apoptosis is also well described; however the precise role of alpha-MSH in apoptosis is presently unclear, with studies reporting both anti- and pro-apoptotic activity. The present study demonstrates that cultured human dermal fibroblasts respond to serum depletion and TNF-alpha, IL-1beta and LPS with an increase in membrane permeability, a decrease in viability and an increase in phosphatidylserine externalization (indicative of apoptosis) over 48-96 h. alpha-MSH (at 10(-6) M, but not 10(-9) M) was found to inhibit the serum free and pro-inflammatory mediated reduction in membrane permeability and cellular viability and also inhibited increases in apoptosis. In conclusion, data support a cytoprotective and anti-apoptotic role of the alpha-MSH peptide in human dermal fibroblast cells.

Generation and characterization of multipotent stem cells from established dermal cultures.

Human multipotent skin derived precursor cells (SKPs) are traditionally sourced from dissociated dermal tissues; therefore, donor availability may become limiting. Here we demonstrate that both normal and diseased adult human dermal fibroblasts (DF) pre-cultured in conventional monolayers are capable of forming SKPs (termed m-SKPs). Moreover, we show that these m-SKPs can be passaged and that cryopreservation of original fibroblast monolayer cultures does not reduce m-SKP yield; however, extensive monolayer passaging does. Like SKPs generated from dissociated dermis, these m-SKPs expressed nestin, fibronectin and versican at the protein level. At the transcriptional level, m-SKPs derived from normal adult human DF, expressed neural crest stem cell markers such as p75NTR, embryonic stem cell markers such as Nanog and the mesenchymal stem cell marker Dermo-1. Furthermore, appropriate stimuli induced m-SKPs to differentiate down either mesenchymal or neural lineages resulting in lipid accumulation, calcification and S100ß or ß-III tubulin expression (with multiple processes). m-SKP yield was greater from neonatal foreskin cultures compared to those from adult DF cultures; however, the former showed a greater decrease in m-SKP forming capacity after extensive monolayer passaging. m-SKP yield was greater from adult DF cultures expressing more alpha-smooth muscle actin (αSMA). In turn, elevated αSMA expression correlated with cells originating from specimens isolated from biopsies containing more terminal hair follicles; however, αSMA expression was lost upon m-SKP formation. Others have shown that dissociated human hair follicle dermal papilla (DP) are a highly enriched source of SKPs. However, conversely and unexpectedly, monolayer cultured human hair follicle DP cells failed to form m-SKPs whereas those from the murine vibrissae follicles did. Collectively, these findings reveal the potential for using expanded DF cultures to produce SKPs, the heterogeneity of SKP forming potential of skin from distinct anatomical locations and ages, and question the progenitor status of human hair follicle DP cells.