Characterisation of human fibroblasts as keratinocyte feeder layer using p63 isoforms status.
ABSTRACT Large-scale culture of primary keratinocytes allows the production of large epidermal sheet surfaces for the treatment of extensive skin burns. This method is dependent upon the capacity to establish cultures of proliferating keratinocytes in conditions compatible with their clonal expansion while maintaining their capacity to differentiate into the typical squamous pattern of human epidermis. Feeder layers are critical in this process because the fibroblasts that compose this layer serve as a source of adhesion, growth and differentiation factors. In this report, we have characterise the expression patterns of p63 isoforms in primary keratinocytes cultured on two different feeder layer systems, murine 3T3 and human fibroblasts. We show that with the latter, keratinocytes express a higher ratio of Delta N to TAp63 isoform, in relation with higher clonogenic potential. These results indicate that human fibroblasts represent an adequate feeder layer system to support the culture of primary human keratinocytes.
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ABSTRACT: Tissue-engineered autologous skin is a potential alternative to autograft for burn coverage, but produces poor clinical responses such as unsatisfactory graft intake due to insufficient vascularization. Endothelialized skin equivalents comprising human umbilical vein endothelial cells (HUVECs) survive significantly longer due to inosculation with the capillaries of the host, but these cells are allogeneic by definition. The aim of this study was to reconstruct an autologous endothelialized skin equivalent by incorporating progenitor or pre-differentiated endothelial cells derived from adipose tissue, easily accessible source for autologous transplantation. Human adipose tissue-derived stem cells were isolated from lipoaspirates and amplified to obtain endothelial progenitor cells, which were subsequently differentiated into endothelial cells. These cells were then seeded along with human fibroblasts into a porous collagen-glycosaminoglycan-chitosan scaffold to obtain an endothelialized dermal equivalent. Then, human keratinocytes give rise to a endothelialized skin equivalent. Immunohistochemistry and transmission electron microscopy results demonstrate the presence of capillary-like tubular structures in skin equivalents comprising pre-differentiated endothelial cells, but not endothelial progenitor cells. The former expressed both EN4 and von Willebrand factor, and Weibel-Palade bodies were detected in their cytoplasm. This study demonstrates that adipose tissue is an excellent source of autologous endothelial cells to reconstruct endothelialized tissue equivalents, and that pre-differentiation of stem cells is necessary to obtain vasculature in such models.Journal of Tissue Engineering and Regenerative Medicine 07/2011; 6(7):512-8. · 2.83 Impact Factor
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ABSTRACT: To adequately and permanently restore organ function after grafting, human tissue-engineered skin substitutes (TES) must ultimately contain and preserve functional epithelial stem cells. It is therefore essential that a maximum of stem cells be preserved during each in vitro step leading to the production of TES such as the culture process and the elaboration of a skin cell bank by cryopreservation. To investigate the presence and functionality of epithelial stem cells within the human TES made by the self-assembly approach, slow-cycling cells were identified using 5-bromo-2'-deoxyuridine (BrdU) in the 3D construct. A subset of basal epithelial cells retained the BrdU label and was positive for the stem cell-associated marker keratin 19 within TES after a chase of 21 days in culture post BrdU-labeling. Moreover, keratinocytes harvested from TES gave rise to stem cell-like colonies in secondary monolayer subcultures indicating that stem cells were preserved within TES. To evaluate the effect of cryopreservation with dimethyl sulfoxide and storage in liquid nitrogen on stem cells, human epithelial cells were extracted from skin samples, amplified in culture and used to produce TES, before cryopreservation as well as after thawing. We found that the proportion and the growth potential of epithelial stem cells in monolayer culture and in TES remained constant before and after cryopreservation. Furthermore, the functionality of these substitutes was demonstrated by successfully grafting human TES on athymic mice for 6 months. We conclude that human epithelial skin stem cells are adequately preserved upon human tissue reconstruction. Thus, these TES produced by the self-assembly approach are suitable for clinical applications.Tissue Engineering Part A 11/2012; · 4.64 Impact Factor
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ABSTRACT: 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.International Journal of Molecular Sciences 01/2013; 14(3):4684-704. · 2.46 Impact Factor