Human epidermis reconstructed in vitro: A model to study keratinocyte differentiation and its modulation by retinoic acid

Summary It was possible to reconstruct epidermis in vitro by seeding dissociated keratinocytes on de-epidermized dermis and growing such recombined cultures for 1 wk, exposed to air, at the surface of the culture medium. These conditions were chosen to mimic the transdermal feeding and the exposure to the atmosphere that occur in vivo. Contrary to classical cultures performed on plastic dishes covered with culture medium, which show rudimentary differentiation and organization, the architecture of the stratified epithelium obtained in reconstructed cultures and the distribution of differentiation markers such as suprabasal keratins, involucrin, and membrane-bound transglutaminase were similar to those of the epidermis of skin biopsies; moreover, biochemical studies showed that the synthesis of the various keratins and the production of cornified envelopes was similar to what is found with skin specimens. The reconstructed epidermis model was found to be very useful to study in vitro the effect of retinoic acid on keratinocyte differentiation and epidermal morphogenesis.     

Human oral cavity as a model for the study of genome-genome interactions

The enormous diversity of culturable bacteria within the oral microbial community coupled with experimental accessibility renders the human oral cavity a valuable model to investigate genome-genome interactions. The complex interactions of oral bacteria result in the formation of biofilms on the surfaces of the oral cavity. One mechanism thought to be important in biofilm formation is the coaggregation of bacterial partners. In this paper, we examine the role of coaggregation in oral biofilms and develop protocols to elucidate the spatial organization of bacterial species retained within oral biofilms. To explore these issues, we have employed two experimental systems: the saliva-coated flowcell and the retrievable enamel chip. From flowcell studies, we have determined that coaggregation can greatly influence the ability of an oral bacterial species to grow and be retained within the developing biofilm. To examine the spatial architecture of oral biofilms, fluorescent in situ hybridization protocols were developed that successfully target specific members of the oral microbial community. Together, these approaches provide insight into the development of oral biofilms and expand our understanding of genome-genome interactions.     

Lineus as a model for studying developmental processes in animals reconstructed from adult pieces

The difficulties that prevent reconstruction of animals by piecing together body fragments from several adults are overcome by using nemerteans of the genus Lineus. For 25 years I have managed to make viable composite worms by grafting body parts cut out of Lineus specimens either from the same clonal strain (syngeneically reconstructed worms) or from the same species (allogeneically reconstructed worms) or else from different species (xenogeneically reconstructed worms). Body reconstruction has usually been carried out orthotopically, i.e., the components of composite animals have been selected so as to be anatomically complementary. However, reconstruction has been made heterotopically when it was essential to obtain morphogenetic events in the adults. Here, I shall review some of the developmental processes that took place in such reconstructed animals. First, I shall report immune responses in composite worms derived from various combinations of body pieces grafted together. Second, I shall study sex differentiation during gonad development in growing or regenerating chimeric worms made by the grafting of male and female components. I shall refer also to gonadogenesis in the asexual progeny of bipartite chimeras derived from lateral body halves of both sexes fused together (clones of bilaterally allophenic worms). Third, I shall analyze regulative processes (regeneration, transgeneration) during localized morphogenesis occurring in heterotopically reconstructed worms. The data show how reconstructed Lineus may be exploited to increase our knowledge of developmental mechanisms, especially in the misunderstood field of organismal pattern homeostasis.     

Microbial diversity, producer-decomposer interactions and ecosystem processes: a theoretical model

Interactions between the diversity of primary producers and that of decomposers--the two key functional groups that form the basis of all ecosystems--might have major consequences on the functioning of depauperate ecosystems. I present a simple ecosystem model in which primary producers (plants) and decomposers (microbes) are linked through material cycling. The model considers a diversity of plant organic compounds and a diversity of microbial species. Nutrient recycling efficiency from organic compounds to decomposers is then the key parameter that controls ecosystem processes (primary productivity, secondary productivity, producer biomass and decomposer biomass). The model predicts that microbial diversity has a positive effect on nutrient recycling efficiency and ecosystem processes through either greater intensity of microbial exploitation of organic compounds or functional niche complementarity, much like in plants. Microbial niche breadth and overlap should not affect ecosystem processes unless they increase the number of organic compounds that are decomposed. In contrast, the model predicts that plant organic compound diversity can only have a negative effect or, at best, no effect on ecosystem processes, at least in a constant environment. This creates a tension between the effects of plant diversity and microbial diversity on ecosystem functioning, which may explain some recent experimental results.     

Human skin cell stress response to GSM-900 mobile phone signals. In vitro study on isolated primary cells and reconstructed epidermis

In recent years, possible health hazards due to radiofrequency radiation (RFR) emitted by mobile phones have been investigated. Because several publications have suggested that RFR is stressful, we explored the potential biological effects of Global System for Mobile phone communication at 900 MHz (GSM-900) exposure on cultures of isolated human skin cells and human reconstructed epidermis (hRE) using human keratinocytes. As cell stress markers, we studied Hsc70, Hsp27 and Hsp70 heat shock protein (HSP) expression and epidermis thickness, as well as cell proliferation and apoptosis. Cells were exposed to GSM-900 under optimal culture conditions, for 48 h, using a specific absorption rate (SAR) of 2 W x kg(-1). This SAR level represents the recommended limit for local exposure to a mobile phone. The various biological parameters were analysed immediately after exposure. Apoptosis was not induced in isolated cells and there was no alteration in hRE thickness or proliferation. No change in HSP expression was observed in isolated keratinocytes. By contrast, a slight but significant increase in Hsp70 expression was observed in hREs after 3 and 5 weeks of culture. Moreover, fibroblasts showed a significant decrease in Hsc70, depending on the culture conditions. These results suggest that adaptive cell behaviour in response to RFR exposure, depending on the cell type and culture conditions, is unlikely to have deleterious effects at the skin level.     

Development of water buffalo (Bubalus bubalis) embryos from in vitro matured oocytes reconstructed with fetal skin fibroblast cells as donor nuclei

The present study was carried out to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to find out the developmental competence of embryos following transfer of these nuclei to in vitro matured enucleated buffalo oocytes. Skin cells were isolated from 1 to 2-month-old fetuses obtained from slaughterhouse, by enzymatic digestion (0.5% w/v trypsin +0.05% w/v collagenase in Dulbecco's PBS) for 15-20 min. The cells were washed 4 times with Dulbecco's PBS and then once with RPMI-1640+10% FBS by centrifugation at 600 x g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 2-3 days. Cumulus-oocyte complexes (COCs) collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 5 microg/ml FSH-P + 10 microg/ml LH+10% FBS) for 20-22 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body with 10-15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electro fused and incubated in the activation medium (TCM-199 + 8 microg/ml cytochalasin-B+10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199+10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 48 h. The cleaved embryos were then co-cultured with buffalo oviduct cells in embryo development media (EDM). Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electro fused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to 2-cell stage, 3 (3.8%) reached to 4-cell stage and 3 (3.8%) reached to 8-cell stage. In the synchronized group, out of 62 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40 (65%) were electrofused, activated and cultured, out of which 4 (10%) developed to 2-cell stage, 3 (7.50%) to 4-cell stage, 2 (5.0%) to early morula stage and 1 (2.50%) to blastocysts stage. These results suggest that buffalo fetal skin fibroblasts could be used as donor nuclei for the production of buffalo embryos after nuclear transfer to enucleated in vitro matured buffalo oocytes.     

Characterization of a multiple particle delivery complex and determination of cellular photodamage in skin fibroblast and breast cancer cell lines

Zinc metallized Phthalocyanine (ZnPcS_mix), a potent photosensitizer, is conjugated to gold dendrimer encapsulated nanoparticles (AuDENPs) in order to improve the efficacy of photodynamic therapy (PDT) using MCF‐7 breast cancer cells and WS1 fibroblast cells as a control. Both ZnPcS_mix and AuDENPs are mixed in a nitrogen atmosphere for 48 hours and characterization analysis conducted using ultraviolet‐visible (UV‐vis) spectrometry for spectral properties, transmission electron microscopy (TEM) for morphological features and zeta potential measurement for surface stability and size distribution of the compound obtained or of the multiple particles delivery complex (MPDC). Cell viability, proliferation and membrane damage following PDT are assessed by the trypan blue exclusion test, adenosine triphosphate luminescence and lactate dehydrogenase cytotoxicity assays, respectively. Stable MPDCs are spherical shaped with a diameter lesser than 5 nm, and have a maximum absorption peak at 676 nm. The MPDC‐mediated PDT induces a decrease in cell viability and proliferation, and increased membrane damage or cytotoxicity. The conjugation enhances the therapeutic efficiency of PDT by improving drug delivery and targeting of MCF‐7 cancer cells.      

Human blood platelets, PMN leukocytes and their interactions in vitro. Responses to selective and non-selective stimuli.

Using simultaneous recording of aggregation and chemiluminescence, responses of human polymorphonuclear leukocytes, blood platelets and their mixture were investigated after stimulation by specific as well as non-specific stimuli for each cell. In our experimental settings, aggregation of platelets and PMN leukocytes was increased in the following order of stimuli: PMA相似文献   

Human embryonic stem cell-derived cardiomyocytes as an in vitro model to study cardiac insulin resistance

Patients with type 2 diabetes (T2D) and/or insulin resistance (IR) have an increased risk for the development of heart failure (HF). Evidence indicates that this increased risk is linked to an altered cardiac substrate preference of the insulin resistant heart, which shifts from a balanced utilization of glucose and long-chain fatty acids (FAs) towards an almost complete reliance on FAs as main fuel source. This shift leads to a loss of endosomal proton pump activity and increased cardiac fat accumulation, which eventually triggers cardiac dysfunction. In this review, we describe the advantages and disadvantages of currently used in vitro models to study the underlying mechanism of IR-induced HF and provide insight into a human in vitro model: human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Using functional metabolic assays we demonstrate that, similar to rodent studies, hESC-CMs subjected to 16 h of high palmitate (HP) treatment develop the main features of IR, i.e., decreased insulin-stimulated glucose and FA uptake, as well as loss of endosomal acidification and insulin signaling. Taken together, these data propose that HP-treated hESC-CMs are a promising in vitro model of lipid overload-induced IR for further research into the underlying mechanism of cardiac IR and for identifying new pharmacological agents and therapeutic strategies. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

UVA exposure of human skin reconstructed in vitro induces apoptosis of dermal fibroblasts: subsequent connective tissue repair and implications in photoaging

The skin reconstructed in vitro has been previously shown to be a useful model to investigate the effects of UVB exposure (Bernerd and Asselineau, 1997). The present study describes the response to UVA irradiation. Major alterations were observed within the dermal compartment. Apoptosis of fibroblasts located in the superficial area of the dermal equivalent was observed as soon as 6 h after irradiation, leading to their disappearance after 48 h. This effect was obtained without major alterations of epidermal keratinocytes suggesting a differential cell type sensitivity to UVA radiations. In addition, collagenase I was secreted by dermal fibroblasts. The UVA dermal effects could be observed even after removal of the epidermis during the post irradiation period, demonstrating that they were independent of the keratinocyte response. The analysis of the tissue regeneration during the following 2 weeks revealed a connective tissue repair via fibroblasts proliferation, migration and active synthesis of extracellular matrix proteins such as fibronectin and procollagen I. This cellular recolonization of the superficial part of the dermal equivalent was due to activation of surviving fibroblasts located deeply in the dermal equivalent. The direct damage in the dermis and the subsequent connective tissue repair may contribute to the formation of UVA-induced dermal alterations.     

Human liver slices as an in vitro model to study toxicity-induced hepatic stellate cell activation in a multicellular milieu

INTRODUCTION: Hepatic stellate cell (HSC) activation is a key event in wound healing as well as in fibrosis development in the liver. Previously we developed a technique to induce HSC activation in slices from rat liver. Although this model provides a physiologic, multicellular milieu that is not present in current in vitro models it might still be of limited predictive value for the human situation due to species-differences. Therefore, we now aimed to evaluate the applicability of human liver slices for the study of HSC activation. METHOD: Liver slices (8 mm diameter, 250 microm thickness) were generated from human liver tissue and incubated for 3 or 16 h with 0-15 microl of carbon tetrachloride (CCl4) after which ATP-content and expression levels of HSC (activation) markers was determined. RESULTS: Human liver slices remained viable during incubation as shown by constant ATP levels. Incubation with CCl(4) caused a dose-dependent decrease in viability and an increase in mRNA expression of the early HSC activation markers HSP47 and alphaB-crystallin, but not the late markers for HSC activation, alphaSMA and pro-collagen 1a1. Synaptophysin mRNA expression remained constant during incubation with or without CCl4, indicating a constant number of HSC in the liver slices. CONCLUSION: We developed a technique to induce early toxicity-induced HSC activation in human liver slices. This in vitro model provides a multicellular, physiologic milieu to study mechanisms underlying toxicity-induced HSC activation in human liver tissue.     

Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-β1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.     

The canola microspore-derived embryo as a model system to study developmental processes in plants

The ability of microspores to undergo embryo development after a successful induction treatment provides a unique experimental system to study a variety of developmental processes in plants. Recent published results focus on the cellular and molecular aspects of the early induction process. In this review, besides summarizing the current findings, the advantages of using the MDE system to study other aspects of embryo development are emphasized. The continual improvement of culturing procedures, media components, and molecular methods guarantees exciting new findings in the near future.     

Ionizing radiation induces early, sustained increases in collagen biosynthesis: a 48-week study in mouse skin and skin fibroblast cultures

Groups of 10 CF1 female mice, irradiated to the thorax with a dual-head 137Cs gamma-RAY source, received single doses of 0, 5, 10, 15, or 25 Gy. One to forty-eight weeks later collagen synthesis was measured in minced skin specimens incubated in medium containing [3H]proline and then assayed for radioactive hydroxyproline. A progressive, generally dose-dependent increase in collagen biosynthesis, up to 50% above control sites, was found 1, 4, and 12 weeks after radiation exposure. These changes showed further small fluctuations at 12-36 weeks, increasing again at the 48-week interval. At the same times throughout the study fibroblasts were cultured from skin explants. Following the second subculture, these cells were also incubated in medium containing [3H]proline, and collagen synthesis was again determined by [3H]hydroxyproline assay. At all radiation dose levels studied, collagen production increased threefold by 12 weeks postradiation and remained elevated for the 48-week duration of the study. In vitro radiation dose response differences were not observed.     

Human skin mast cells: their dispersion, purification, and secretory characterization

Digestion of human foreskin with collagenase and hyaluronidase disperses approximately 3.4 X 10(7) nucleated cells per gram of tissue, of which mast cells constitute 4.7%. These may be purified to 80% by use of density gradient centrifugation. The majority of mast cells (79%) measured between 9 and 13 micron in diameter, and the mean histamine content was 4.6 pg/cell. Viability was demonstrated by trypan blue exclusion by 93% of the cells and the low spontaneous histamine secretion of less than 7% in functional studies. Anti-IgE released up to 17.5% of cell-associated histamine within 5 to 7 min. Calcium ionophore-induced release was optimal with 0.3 microM A23187 when 28.6% histamine was released. Unlike human lung mast cells, skin mast cells released histamine in response to compound 48/80 and poly-L-lysine. This release, which was complete within 20 sec, was totally dependent on intact glycolysis and oxidative phosphorylation and partially dependent on extracellular calcium. The same characteristics were observed with secretion induced by substance P and morphine. The weak activity of eledoisin and physalaemin suggests that the substance P receptor, like that of the rat mast cell, is not of the classical types described for smooth muscle. Morphine-induced secretion was partially blocked by naloxone in a manner not compatible with competitive antagonism at a classical opioid receptor. The sensitivity of skin mast cells to nonimmunologic stimulation clearly distinguishes them from mast cells of the lung and lymphoid tissues and provides evidence of functional heterogeneity within human mast cells.     

Polar angle as a determinant of amphipathic alpha-helix-lipid interactions: a model peptide study

Various physicochemical properties play important roles in the membrane activities of amphipathic antimicrobial peptides. To examine the effects of the polar angle, two model peptides, thetap100 and thetap180, with polar angles of 100 degrees and 180 degrees, respectively, were designed, and their interactions with membranes were investigated in detail. These peptides have almost identical physicochemical properties except for polar angle. Like naturally occurring peptides, these peptides selectively bind to acidic membranes, assuming amphipathic alpha-helices, and formed peptide-lipid supramolecular complex pores accompanied by lipid flip-flop and peptide translocation. Despite its somewhat lower membrane affinity, thetap100 exhibited higher membrane permeabilization activity, a greater flip-flop rate, as well as more antimicrobial activity due to a higher pore formation rate compared with thetap180. Consistent with these results, the peptide translocation rate of thetap100 was higher. Furthermore, the number of peptides constituting thetap100 pores was less than that of thetap180, and thetap100 pores involved more lipid molecules, as reflected by its cation selectivity. The polar angle was found to be an important parameter determining peptide-lipid interactions.     

Compromising human skin in vivo and ex vivo to study skin barrier repair

Ex vivo regenerated stratum corneum (SC) after tape-stripping can be used as a model to study the barrier function of compromised skin. Yet, details about how close the regenerated SC model mimics the lipid properties (e.g. lipid composition and lipid ordering) of the in vivo situation are not known. Here, we examined using a comprehensive ceramide analysis whether human ex vivo regenerated SC showed similar lipid properties as human in vivo regenerated SC. Both in vivo and ex vivo regenerated SC had an altered ceramide subclass composition, with increased percentages of sphingosine-based subclass and decreased percentages of phytosphingosine-based subclass ceramides, a reduced mean ceramide chain length, and a higher percentage of unsaturated ceramides. Overall, regenerated SC ex vivo showed more pronounced but similar changes compared to the in vivo response. One of the purposes of these models is to use them to mimic compromised skin of inflammatory skin diseases. The altered lipid properties in regenerated SC were comparable to those observed in several inflammatory skin diseases, which makes them a valuable model for the barrier properties in inflammatory skin diseases.     

Escherichia coli as a model system to study DNA repair genes of eukaryotic organisms

The bacteria Escherichia coli has been widely employed in studies of eukaryotic DNA repair genes. Several eukaryotic genes have been cloned by functional complementation of mutant lineages of E. coli. We examined the similarities and differences among bacterial and eukaryotic DNA repair systems. Based on these data, we examined tools used for gene cloning and functional studies of DNA repair in eukaryotes, using this bacterial system as a model.