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Reconstructed Epidermis (reconstructed + epidermis)
Selected AbstractsExploration of the functional hierarchy of the basal layer of human epidermis at the single-cell level using parallel clonal microcultures of keratinocytesEXPERIMENTAL DERMATOLOGY, Issue 4 2010Nicolas O. Fortunel Please cite this paper as: Exploration of the functional hierarchy of the basal layer of human epidermis at the single-cell level using parallel clonal microcultures of keratinocytes. Experimental Dermatology 2010. Abstract:, The basal layer of human epidermis contains both stem cells and keratinocyte progenitors. Because of this cellular heterogeneity, the development of methods suitable for investigations at a clonal level is dramatically needed. Here, we describe a new method that allows multi-parallel clonal cultures of basal keratinocytes. Immediately after extraction from tissue samples, cells are sorted by flow cytometry based on their high integrin-,6 expression and plated individually in microculture wells. This automated cell deposition process enables large-scale characterization of primary clonogenic capacities. The resulting clonal growth profile provided a precise assessment of basal keratinocyte hierarchy, as the size distribution of 14-day-old clones ranged from abortive to highly proliferative clones containing 1.7 × 105 keratinocytes (17.4 cell doublings). Importantly, these 14-day-old primary clones could be used to generate three-dimensional reconstructed epidermis with the progeny of a single cell. In long-term cultures, a fraction of highly proliferative clones could sustain extensive expansion of >100 population doublings over 14 weeks and exhibited long-term epidermis reconstruction potency, thus fulfilling candidate stem cell functional criteria. In summary, parallel clonal microcultures provide a relevant model for single-cell studies on interfollicular keratinocytes, which could be also used in other epithelial models, including hair follicle and cornea. The data obtained using this system support the hierarchical model of basal keratinocyte organization in human interfollicular epidermis. [source] Carbohydrate expression and modification during keratinocyte differentiation in normal human and reconstructed epidermisEXPERIMENTAL DERMATOLOGY, Issue 5 2003Bruno Méhul Abstract:, Using fluorescein isothiocyanate (FITC)-labeled lectins we were able to demonstrate the presence of specific carbohydrate moieties in normal human and reconstructed epidermis. Evidence is provided that in both cases the strongly reduced lectin staining at the level of the stratum corneum is the result of a hindered accessibility of the lectins in this lipid-rich hydrophobic environment. Isolated corneocytes and purified cornified envelopes (CEs) exhibited clearly glycosylated structures reacting with distinct lectins. The presence of glycosidase activity, particularly in the upper layers of the epidermis characterized by an acidic environment (pH 5.5), indicates that modifications of the sugar residues might be important in epidermal homeostasis, barrier behavior and desquamation. Absent or strongly reduced glycosidase activity in the stratum corneum of reconstructed epidermis with an impaired pH gradient could be in part responsible for the reduced barrier function and the lack of desquamation in this model. [source] Lack of desquamation , the Achilles heel of the reconstructed epidermisINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 5 2002M. Ponec Synopsis The use of human skin equivalents for screening tests aiming to assess repetitive application of various test agents is hampered by the lack of desquamation in vitro. The present study was undertaken to examine whether the desquamation can be induced by various treatments including mechanical stress, application of various agents that should decrease the surface pH and calcium level, activate the enzymes involved in desquamation process or UV irradiation. In addition, the effect of ,-hydroxyacids, known to enhance desquamation and to improve the stratum corneum barrier function in vivo, was examined as well. Human epidermis reconstructed on de-epidermized dermis or on fibroblast-populated collagen matrices during a 2-week culture at the air,liquid interface underwent various treatments during an additional 3-week period. The effects of treatments were evaluated on the basis of tissue morphology and lipid composition. The results of the present study revealed that cell shedding could only be induced by a mild repetitive mechanical treatment. The lack of desquamation, under most in vitro conditions, has a practical consequence, since it may hamper the use of reconstructed epidermis for various screening studies aiming to examine the repetitive exposure to topical agents or UV irradiation. The gradual thickening of the stratum corneum will lead to its higher resistance to the environmental stimuli and in this way affect the outcome of the tests. Furthermore, from the results obtained in the present study, it became evident that one should be careful in selecting endpoints when, for example, the effects of agents known to modulate melanogenesis are examined. Résumé L'utilization d'équivalents cutanés humains dans les procédures de criblage, afin d'estimer l'action répétée de divers agents, est entravée par l'absence de desquamation in vitro. La présente étude a été entreprise afin de déterminer dans quelle mesure la desquamation peut être induite par différents traitements tels que stress mécanique, application d'agents divers qui conduiraient à une chute du pH de surface et du taux de Calcium, activeraient les enzymes impliquées dans le processus de desquamation, ou l'irradiation UV. De plus, l'effet des , hydroxy-acides, connus pour favouriser la desquamation et d'améliorer la fonction barrière du Stratum-Corneum in vivo, a étéétudié. L'épiderme humain reconstruit sur un derme dé-épidermisé ou sur des matrices de collagène colonisées par des fibroblastes pendant 2 semaines de culture, en interface air × liquide, a subi divers traitements pendant une période additionnelle de 3 semaines. Les effets de ces traitements étaient évalués sur des critères morphologiques du tissu ainsi que la composition en lipides. Les résultats de cette étude montrent que l'élimination cellulaire ne peut être induite que par un léger traitement mécanique répété. L'absence de desquamation dans la plupart des conditions in vitro a une conséquence pratique puisqu'elle peut entraver l'utilization de l'épiderme reconstruit à des fins diverses de criblage en vue d'appréhender les expositions répétées à des agents topiques, ou l'irradiation UV. L'épaississement progressif du Stratum-Corneum lui confèrera une résistance accrue aux stimuli environnementaux qui, en retour, modifiera les résultats des tests. De plus, les résultats de cette présente étude impliquent à l'évidence une précaution dans la sélection des cinétiques de mesures lorsque, par exemple, les effets des agents connus pour moduler la mélanogénèse sont étudiés. [source] Keratinocytes Control the Pheo/Eumelanin Ratio in Cultured Normal Human MelanocytesPIGMENT CELL & MELANOMA RESEARCH, Issue 6 2002Christine Duval The pheo/eumelanin ratio of cultured normal human melanocytes is distinct from the ratio observed for the same cells in vivo where they are in close contact with keratinocytes. To study the possible involvement of keratinocytes in the control of melanogenesis, we compared quantitatively and qualitatively the melanin production in melanocyte mono-cultures, in melanocyte,keratinocyte co-cultures and in pigmented reconstructed epidermis. Pheomelanin and eumelanin contents were assessed by high-performance liquid chromatography with electrochemical and fluorometric detection of their specific degradation products and revealed striking differences in the presence of keratinocytes. In the absence of keratinocytes (melanocyte mono-cultures), we observed a very limited eumelanin production and a very high pheomelanin synthesis. The pheo/eumelanin ratio in mono-cultures could be slightly influenced by changing the composition of the culture medium, however, the very strong imbalance in favor of pheomelanin remained unchanged. An induction of eumelanin synthesis accompanied by an important reduction of pheomelanin formation was only observed in the presence of keratinocytes. The pheo/eumelanin ratio in melanocyte mono-culture dropped from 1043 down to about 25 in the presence of keratinocytes (co-cultures). The same observations were made when the melanocytes were integrated into a reconstructed human epidermis. Interestingly, under co-culture conditions resulting in only a partial contact between melanocytes and keratinocytes, the reduction of the pheo/eumelanin ratio were less pronounced. From these results we conclude that keratinocytes play an important role in the melanin production, affecting the melanogenic pathways. [source] | ||||||||||