A new in vitro wound model based on the co-culture of human dermal microvascular endothelial cells and human dermal fibroblasts

BACKGROUND INFORMATION: Different in vitro models, based on co-culturing techniques, can be used to investigate the behaviour of cell types, which are relevant for human wound and soft-tissue healing. Currently, no model exists to describe the behaviour of fibroblasts and microvascular endothelial cells under wound-specific conditions. In order to develop a suitable in vitro model, we characterized co-cultures comprising NHDFs (normal human dermal fibroblasts) and HDMECs (human dermal microvascular endothelial cells). The CCSWMA (co-culture scratch wound migration assay) developed was supported by direct visualization techniques in order to investigate a broad spectrum of cellular parameters, such as migration and proliferation activity, the differentiation of NHDFs into MFs (myofibroblasts) and the expression of endothelin-1 and ED-A-fibronectin (extra domain A fibronectin). The cellular response to hypoxia treatment, as one of the crucial conditions in wound healing, was monitored. RESULTS: The comparison of the HDMEC-NHDF co-culture with the respective mono-cultures revealed that HDMECs showed a lower proliferation activity when co-cultured, but their number was stable throughout a period of 48 h. NHDFs in co-culture were slightly slower at proliferating than in the mono-culture. The MF population was stable for 48 h in the co-culture, as well as in NHDF mono-culture. Co-cultures and HDMEC mono-cultures were characterized by a slower migration rate than NHDF mono-cultures. Hypoxia decreased both cell proliferation and migration in the mono-cultures, as well as in the co-cultures, indicating the general suitability of the assay. Exclusively, in co-cultures well-defined cell clusters comprising HDMECs and MFs formed at the edges of the in vitro wounds. CONCLUSIONS: On the basis of these results, the CCSWMA developed using co-cultures, including HDMECs, NHDFs and MFs, proved to be an effective tool to directly visualize cellular interaction. Therefore, it will serve in the future to evaluate the influence of wound-healing-related factors in vitro, as shown for hypoxia in the present study.     

Apoptotic cell death increases with senescence in normal human dermal fibroblast cultures

Normal human dermal fibroblasts have a limited life-span in vitro and stop proliferation after a fixed number of cell divisions. This process by which cells stop proliferation is called senescence. Senescence is also characterized by a decrease in the total cell number. In this study, we characterized an increase in cell death in normal human dermal fibroblasts in vitro as a function of increasing cell passage. With increasing passage, human fibroblasts showed an increase in the number of dead cells and increased DNA fragmentation as determined by flow cytometry. Serial passage of human fibroblasts also resulted in mitochondrial dysfunction, represented by a loss of mitochondrial membrane potential. The apoptotic markers caspase-3 and cytochrome c were both found to increase in senescent cells. These results suggest the activation of an apoptotic pathway within a population of human fibroblasts as a function of cell passage.     

In vitro wounding: effects of hypoxia and transforming growth factor β<Subscript>1</Subscript> on proliferation,migration and myofibroblastic differentiation in an endothelial cell-fibroblast co-culture model

The adequate reconstitution of human soft tissue wounds requires the coordinated interaction of endothelial cells and fibroblasts during the proliferation phase of healing. Endothelial cells assure neoangiogenesis, fibroblasts fill the defect and provide extracellular matrix proteins, and myofibroblasts are believed to support the reconstitution of microvessels. In the present study, we combined in vitro-wound size measurement and multicolour immunocytochemical staining of co-cultured human dermal microvascular endothelial cells and normal human dermal fibroblasts, recently introduced as co-culture scratch-wound migration assay. Applying antibodies for α-smooth-muscle actin, von Willebrand factor, extra domain A fibronectin and endothelin-1, we were able to monitor proliferation, migration and the differentiation process from fibroblasts to myofibroblasts as a response to hypoxia. Furthermore, we verified, whether transforming growth factor β₁ (TGFβ₁) and endothelin-1 are able to mediate this response. We show, that proliferation and migration of endothelial cells and fibroblasts decreased under hypoxia. The additional administration of TGFβ₁ did not significantly attenuate this decrease. Solely the myofibroblast population in co-culture adapted well to hypoxia, when cultures were supplemented with TGFβ₁. Considerating the data concerning TGFβ₁ and endothelin-1, we propose a model explaining the cellular interaction during early and late proliferation phase of human wound healing.     

Granulocyte macrophage colony stimulating factor (GM-CSF) biological actions on human dermal fibroblasts.

Fibroblasts are involved in all pathologies characterized by increased ExtraCellularMatrix synthesis, from wound healing to fibrosis. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) is a cytokine isolated as an hemopoietic growth factor but recently indicated as a differentiative agent on endothelial cells. In this work we demonstrated the expression of the receptor for GM-CSF (GM-CSFR) on human normal skin fibroblasts from healthy subjects (NFPC) and on a human normal fibroblast cell line (NHDF) and we try to investigate the biological effects of this cytokine. Human normal fibroblasts were cultured with different doses of GM-CSF to study the effects of this factor on GM-CSFR expression, on cell proliferation and adhesion structures. In addition we studied the production of some Extra-Cellular Matrix (ECM) components such as Fibronectin, Tenascin and Collagen I. The growth rate of fibroblasts from healthy donors (NFPC) is not augmented by GM-CSF stimulation in spite of increased expression of the GM-CSFR. On the contrary, the proliferation of normal human dermal fibroblasts (NHDF) cell line seems more influenced by high concentration of GM-CSF in the culture medium. The adhesion structures and the ECM components appear variously influenced by GM-CSF treatment as compared to fibroblasts cultured in basal condition, but newly only NHDF cells are really induced to increase their synthesis activity. We suggest that the in vitro treatment with GM-CSF can shift human normal fibroblasts towards a more differentiated state, due or accompanied by an increased expression of GM-CSFR and that such "differentiation" is an important event induced by such cytokine.     

The cytotoxic effects of a novel IH636 grape seed proanthocyanidin extract on cultured human cancer cells

This study was conducted to investigate the effects of aging on collagen and collagenase expression by human dermal fibroblasts. To evaluate this effect, the expression of these ECM was determined and compared between either fetal and adult fibroblasts or dermal fibroblasts at various passages. A total of 13 cell strains, 8 fetal foreskin and 5 adult dermal fibroblasts, were grown to 80-90% confluency and their rates of cell proliferation and expression of mRNA for collagenase (MMP-1) and pro 1(I) chain of type I collagen was determined and compared. Fetal cells had a significantly higher rate of proliferation relative to adult fibroblasts evaluated within 10 days of culture. Northern analysis was used to evaluate the steady state levels of mRNA in these cells. The result of these experiments revealed a significantly greater expression of mRNA for collagenase (58.6 ± 7.7 vs. 9.9 ± 1.5, p < 0.05) in strains of adult fibroblasts. This was consistent with collagenase activity of conditioned medium derived from adult cells relative to fetal fibroblasts. However the expression of pro 1(I) chain of type I collagen mRNA was not significantly (56.2 ± 5.2 vs. 58.5 ± 3.5) different between adult and fetal fibroblasts. This finding was confirmed by measuring total collagen production present in conditioned medium of these cells using hydroxyproline as an index for collagen production. The cellular response to IGF-1 and IFN-2b as representatives of fibrogenic and anti-fibrogenic factors were also evaluated. When expression of collagenase was used as an indication for cellular response, the degree of this response to IGF-1 but not IFN-2b was significantly greater in fetal relative to adult cells. Serial passage was also used as an in vitro model for aging fibroblasts and found a gradual reduction in pro 1(I) chain of type I collagen mRNA and hydroxyproline formation due to passaging. In conclusion, a slower rate of proliferation, a greater collagenase activity and expression of collagenase mRNA by aging fibroblasts could be some of the main reasons for attenuation of wound healing in elderly patients.     

Ellagitannins from Phyllanthus muellerianus (Kuntze) Exell.: Geraniin and furosin stimulate cellular activity, differentiation and collagen synthesis of human skin keratinocytes and dermal fibroblasts

Leaves from Phyllanthus muellerianus (Kuntze) Exell. are traditionally used for wound healing in Western Africa. Aqueous extracts of dried leaves recently have been shown to stimulate proliferation of human keratinocytes and dermal fibroblasts. Within bioassay-guided fractionation the ellagitannins geraniin (1), corilagin (2), furosin (3), the flavonoids quercetin-3-O-β-d-glucoside (isoquercitrin), kaempferol-3-O-β-d-glucoside (astragalin), quercetin-3-O-d-rutinoside (rutin), gallic acid, methyl gallate, caffeic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid and caffeoylmalic acid (phaselic acid) have been identified in P. muellerianus for the first time. Geraniin was shown to be the dominant component of an aqueous extract.Suitable analytical methods for quality control of geraniin in P. muellerianus extract (methanol/water, 70/30) have been developed and validated based on ICH guidelines (ICH-compliant protocol).Geraniin and furosin increased the cellular energy status of human skin cells (dermal fibroblasts NHDF, HaCaT keratinocytes), triggering the cells towards higher proliferation rates, with fibroblasts being more sensitive than keratinocytes. Highest stimulation of NHDF by geraniin was found at 5 μM, and of keratinocytes at 50-100 μM. Furosin stimulated NHDF at about 50 μM, keratinocytes at about 150-200 μM. Necrotic cytotoxicity of geraniin, as measured by LDH release, was observed at 20 μM for NHDF and 150 μM for keratinocytes. Toxicity of furosin - less than that of geraniin - was observed at >400 μM.Furosin and geraniin stimulated the biosynthesis of collagen from NHDF at 50 μM and 5-10 μM respectively. Geraniin at 105 μM significantly stimulated the differentiation in NHEK while furosin had a minor influence on the expression of involucrin and cytokeratins K1 and K10. The study proves clearly that hydrophilic extracts from P. muellerianus and especially the lead compound geraniin exhibit stimulating activity on dermal fibroblasts and keratinocytes, leading to increased cell proliferation, barrier formation and formation of extracellular matrix proteins. From these findings the traditional clinical use of such extracts for wound healing seems to be justified.     

Salmon nasal cartilage proteoglycan enhances growth of normal human dermal fibroblast through Erk1/2 phosphorylation

Proteoglycan (PG) is a heavily glycosylated protein, localized to cell surface and extracellular matrix, and has various functions. Recently, it has been gradually revealed that PG interacts with various growth factors and morphogens and regulates cellular functions. Although salmon nasal cartilage PG (Salmon-PG) increases proliferation of immortalized cells, its mechanism remains unclear. In this study, we confirmed the effect of Salmon-PG on normal human dermal fibroblast (NHDF) and investigated the mechanism of PG action on NHDF. Salmon-PG dose- and time-dependently increased NHDF proliferation. Receptor tyrosine kinase array revealed that Salmon-PG increased only Erk1/2 signaling. Erk1/2 phosphorylation was significantly increased by Salmon-PG in a time-(10 min) and dose-(400 or 800 μg/mL) dependent manner. MEK inhibitor suppressed the enhancement of NHDF proliferation by Salmon-PG. The overall findings indicate that Salmon-PG plays a role as a growth factor in NHDF via Erk1/2 activation, suggesting that Salmon-PG contributes to the maintenance of skin homeostasis.     

A Novel Technique for Culture of Human Dermal Microvascular Endothelial Cells under either Serum-Free or Serum-Supplemented Conditions: Isolation by Panning and Stimulation with Vascular Endothelial Growth Factor

Several physiological and pathophysiological events involving vascular endothelium occur at the microvascular level. Studies on human microvasculature require homogenous primary cultures of microvascular endothelial cells. However, procedures available for isolating and culturing human dermal microvascular cells (HDMEC) result in significant contamination with fibroblasts. To eliminate contamination with fibroblasts or other cells, we developed a procedure to isolate HDMEC from neonatal human foreskin by panning the cells using EN4, an anti-endothelial cell monoclonal antibody. Panned cells uniformly expressed von Willebrand factor and CD36, confirming their microvascular endothelial characteristics, whereas cells cultured without panning showed a significant degree of contamination with fibroblasts. In the presence of vascular endothelial growth factor (VEGF), HDMEC could be cultured under serum-free conditions. VEGF stimulated the growth of HDMEC in a dose-dependent manner in serum-free medium or in media supplemented with either human serum or newborn calf serum. Since differences exist between large vessel endothelial cells and microvascular endothelial cells, we compared the response to VEGF stimulation of HDMEC with human umbilical vein endothelial cells (HUVEC). The dose response of the two cell types to VEGF was different. This effect of VEGF on endothelial cells may be mediated by the VEGF receptorkdr,since mRNA forkdrwas detected using RT–PCR in both HDMEC and HUVEC. The procedure described in this study will make possible the culture of highly enriched HDMEC without contamination with fibroblasts and facilitate studies with these cells under defined assay conditions in a serum-free environment.     

Bisphosphonates induce the osteogenic gene expression in co‐cultured human endothelial and mesenchymal stem cells

Bisphosphonates (BPs) are known to affect bone homeostasis and also to have anti-angiogenic properties. Because of the intimate relationship between angiogenesis and osteogenesis, this study analysed the effects of Alendronate (AL) and Zoledronate (ZL) in the expression of endothelial and osteogenic genes on interacting endothelial and mesenchymal stem cells, an issue that was not previously addressed. Alendronate and ZL, 10⁻¹²–10⁻⁶ M, were evaluated in a direct co-culture system of human dermal microvascular endothelial cells (HDMEC) and human bone marrow mesenchymal stem cells (HMSC), over a period of 14 days. Experiments with the respective monocultures were run in parallel. Alendronate and ZL caused an initial dose-dependent stimulation in the cell proliferation in the monocultures and co-cultures, and did not interfere with their cellular organization. In HDMEC monocultures, the expression of the endothelial genes CD31, VE-cadherin and VEGFR2 was down-regulated by AL and ZL. In HMSC monocultures, the BPs inhibited VEGF expression, but up-regulated the expression of the osteogenic genes alkaline phosphatase (ALP), bone morphogenic protein-2 (BMP-2) and osteocalcin (OC) and, to a greater extent, osteoprotegerin (OPG), a negative regulator of the osteoclastic differentiation, and increased ALP activity. In co-cultured HDMEC/HMSC, AL and ZL decreased the expression of endothelial genes but elicited an earlier and sustained overexpression of ALP, BMP-2, OC and OPG, compared with the monocultured cells; they also induced ALP activity. This study showed for the first time that AL and ZL greatly induced the osteogenic gene expression on interacting endothelial and mesenchymal stem cells.     

HIF1A/miR-20a-5p/TGFβ1 axis modulates adipose-derived stem cells in a paracrine manner to affect the angiogenesis of human dermal microvascular endothelial cells

Angiogenic cytokines secreted by the adipose-derived stem cells (ADSCs) might promote the angiogenesis of endothelial cells. In the present study, we hypothesize that miR-20a targets TGFB1 to modulate the transforming growth factor β1 (TGFβ1) secretion by ADSCs, therefore affecting the angiogenesis. We found that hypoxia-inducible factor 1A (HIF1A) and TGFβ1 expressions were increased by hypoxia, accompanied with promoted ADSC cell viability. Incubation with conditioned medium from ADSCs treated with hypoxia significantly enhanced the angiogenesis capacity of human dermal microvascular endothelial cells (HDMECs), while TGFB1-silenced ADSCs medium significantly reverses HDMECs angiogenesis. miR-20a suppresses the expression of TGFB1 and secretion of TGFβ1 by ADSCs via binding to its 3′untranslated region, therefore modulating the HDMEC angiogenesis via affecting the paracrine from ADSCs; the effects of miR-20a-overexpressed conditioned medium on HDMEC angiogenesis were significantly reversed by TGFB1-overexpressed conditioned medium. Finally, HIF1A suppressed the expression of miR-20a via targeting its promoter region, subsequently promoting the paracrine from ADSCs and HDMEC angiogenesis.     

Restimulation of cell cycle progression by hypoxic tumour cells with deoxynucleosides requires ppm oxygen tension

Continuous exposure of Ehrlich ascites tumour cells to argon-CO2 under in vitro conditions caused rapid cessation of cell proliferation. On fixing the O2 level at 10 ppm in the protective atmosphere (0.001% in comparison with about 20% in normoxic atmosphere), G1 and early S cells remained stationary while G2 cells continued to pass from G2 into mitosis, to remain in a non-growing state in G1 of the subsequent cycle. Re-aeration of cells following 12 h hypoxia induced up to 25% of the population to continue DNA synthesis without a preceding cell division, as revealed by flow-cytometric analysis. Supplementation of cells cultured under hypoxia with a combination of deoxynucleosides (100 microM deoxycytidine, 10 microM deoxyadenosine, 10 microM deoxyguanosine) was found to stimulate reprogression through the cycle, provided the residual oxygen tension in the protective atmosphere exceeded 40 ppm. The increase in the number of cells with a DNA content of more than 4 C and in the number of binucleate cells observed after re-aeration of hypoxic cells was not prevented by deoxynucleosides or by uridine, which were present in the medium either during hypoxia of from the beginning of reoxygenation. These results indicate that the development of polyploidy as a result of oxygen deficiency cannot be influenced by improvement of RNA and DNA synthetic precursors.     

Low molecular weight hyaluronan mediated CD44 dependent induction of IL-6 and chemokines in human dermal fibroblasts potentiates innate immune response

Complex regulation of the wound healing process involves multiple interactions among stromal tissue cells, inflammatory cells, and the extracellular matrix. Low molecular weight hyaluronan (LMW HA) derived from the degradation of high molecular weight hyaluronan (HMW HA) is suggested to activate cells involved in wound healing through interaction with HA receptors. In particular, receptor CD44 is suggested to mediate cell response to HA of different MW, being the main cell surface HA receptor in stromal tissue and immune cells. However, the response of dermal fibroblasts, the key players in granulation tissue formation within the wound healing process, to LMW HA and their importance for the activation of immune cells is unclear. In this study we show that LMW HA (4.3 kDa) induced pro-inflammatory cytokine IL-6 and chemokines IL-8, CXCL1, CXCL2, CXCL6 and CCL8 gene expression in normal human dermal fibroblasts (NHDF) that was further confirmed by increased levels of IL-6 and IL-8 in cell culture supernatants. Conversely, NHDF treated by HMW HA revealed a tendency to decrease the gene expression of these cytokine and chemokines when compared to untreated control. The blockage of CD44 expression by siRNA resulted in the attenuation of IL-6 and chemokines expression in LMW HA treated NHDF suggesting the involvement of CD44 in LMW HA mediated NHDF activation. The importance of pro-inflammatory mediators produced by LMW HA triggered NHDF was evaluated by significant activation of blood leukocytes exhibited as increased production of IL-6 and TNF-α. Conclusively, we demonstrated a pro-inflammatory response of dermal fibroblasts to LMW HA that was transferred to leukocytes indicating the significance of LMW HA in the inflammatory process development during the wound healing process.     

Cellular senescence induced by cathepsin X downregulation

Cellular senescence represents a powerful tumor suppressor mechanism to prevent proliferation and invasion of malignant cells. Since tumor cells as well as primary fibroblasts lacking the lysosomal cysteine-type carboxypeptidase cathepsin X exhibit a reduced invasive capacity, we hypothesized that the underlying reason may be the induction of cellular senescence. To investigate the cellular and molecular mechanisms leading to diminished migration/invasion of cathepsin X-deficient cells, we have analyzed murine embryonic fibroblasts (MEF) derived from cathepsin X-deficient mice and neonatal human dermal fibroblasts (NHDF) transfected with siRNAs targeting cathepsin X. Remarkably, both cell types exhibited a flattened and enlarged cell body, a characteristic phenotype of senescent cells. Additional evidence for accelerated senescence was obtained by detection of the common senescence marker β-galactosidase. Further examination revealed increased expression levels of senescence-associated genes such as p16, p21, p53, and caveolin in these cells along with a reduced proliferation rate. The accelerated cellular senescence induced by cathepsin X deficiency was rescued by simultaneous expression of exogenous cathepsin X. Finally, cell cycle analysis confirmed a marked reduction of the synthesis rate and prolongation of the S-phase, while susceptibility to apoptosis of cathepsin X-deficient cells remained unchanged. In conclusion, cathepsin X deficiency leads to accelerated cellular senescence and consequently to diminished cellular proliferation and migration/invasion implying a potential role of cathepsin X in bypassing cellular senescence.     

Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro.

CD36 is an 88-kDa glycoprotein that has been identified on platelets, monocytes, and some endothelial cells. Experimental evidence suggests that CD36 mediates the binding of Plasmodium falciparum-infected RBC to a variety of cells, and therefore may play a role in the vascular complications associated with malaria. Additionally, CD36 may also bind the extracellular matrix proteins thrombospondin and collagen. Human umbilical vein endothelial cells have been used in in vitro models examining the binding of P. falciparum RBC to endothelial cells, but they do not consistently express cell surface CD36. Inasmuch as human dermal microvascular endothelial cells (HDMEC) differ in a variety of ways from large vessel endothelial cells, we have examined HDMEC for cell surface expression of CD36 in vivo and in vitro. Direct immunofluorescence of skin showed bright staining of HDMEC with antibody recognizing CD36 and flow cytometric analysis of cultured HDMEC revealed cell surface expression. In contrast, large vessel endothelial cells were not stained with antibody recognizing CD36 in vivo and cultured cells derived from umbilical vein failed to express cell surface CD36 in vitro. Western immunoblots of lysates of HDMEC but not human umbilical vein endothelial cells demonstrated an 88-kDa protein that comigrated with CD36 from platelets. Functional studies demonstrated that adherence of PRBC to HDMEC was inhibited up to 66% by mAb recognizing CD36. Furthermore, the expression of CD36 on HDMEC was increased in a dose- and time-dependent manner by IFN-gamma, and was decreased by protein kinase C agonists. These data demonstrate that HDMEC express functionally active CD36 and this expression can be positively and negatively regulated by soluble factors. This study demonstrates that HDMEC are useful in the study of CD36-mediated binding of PRBC to endothelial cells in vitro and provides further evidence of distinct phenotypic differences between HDMEC and large vessel endothelial cells.     

Apoptotic primary normal human dermal fibroblasts for in vitro models of fibrosis

Recent studies show that apoptosis affects surrounding tissue, playing a role in diseases such as fibrosis, a significant global disease burden. Elucidating the mechanisms by which the different apoptotic cells present during fibrotic wound healing affect their environment would enable development of new therapies. We describe here a simple, rapid, and cost-effective method for inducing apoptosis of primary normal human dermal fibroblasts without affecting the overall cell viability of the population. Such population could be used for in vitro models of fibrotic wound healing in co-culture with other cells involved in this process to study events such as apoptosis-induced proliferation.     

Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming

Induced pluripotent stem cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell‐like state. Notwithstanding their autologous origin and their potential to differentiate towards cells of all three germ layers, iPSC reprogramming is still affected by low efficiency. As dermal fibroblast is the most used human cell for reprogramming, we hypothesize that the variability in reprogramming is, at least partially, because of the skin fibroblasts used. Human dermal fibroblasts harvested from five different anatomical sites (neck, breast, arm, abdomen and thigh) were cultured and their morphology, proliferation, apoptotic rate, ability to migrate, expression of mesenchymal or epithelial markers, differentiation potential and production of growth factors were evaluated in vitro. Additionally, gene expression analysis was performed by real‐time PCR including genes typically expressed by mesenchymal cells. Finally, fibroblasts isolated from different anatomic sites were reprogrammed to iPSCs by integration‐free method. Intriguingly, while the morphology of fibroblasts derived from different anatomic sites differed only slightly, other features, known to affect cell reprogramming, varied greatly and in accordance with anatomic site of origin. Accordingly, difference also emerged in fibroblasts readiness to respond to reprogramming and ability to form colonies. Therefore, as fibroblasts derived from different anatomic sites preserve positional memory, it is of great importance to accurately evaluate and select dermal fibroblast population prior to induce reprogramming.     

Outer root sheath (ORS) cells organize into epidermoid cyst-like spheroids when cultured inside Matrigel: a light-microscopic and immunohistological comparison between human ORS cells and interfollicular keratinocytes

In organotypic cultures, outer root sheath (ORS) cells of the human hair follicle develop into a stratified epithelium largely reminiscent of the epidermis; this apparently reflects their importance during wound healing. In the present study, ORS cells were grown inside a three-dimensional network of extracellular matrix proteins (Matrigel), together with different mesenchymal cells, in an attempt to mimic their follicular environment. Thus, inside Matrigel, ORS cells formed spheroids differentiating toward the center and showing all the markers of epidermal keratinization. Under identical conditions, normal epidermal keratinocytes developed similar spheroids, but of a significantly smaller size. Human dermal fibroblasts and dermal papilla cells, cocultured in the matrix, had a positive influence on both the proliferation and differentiation within both types of spheroids. Epidermal differentiation markers, such as suprabasal keratins, involucrin, filaggrin, gp80 and pemphigoid antigen, were readily expressed in ORS spheroids, whereas hard (hair) keratins were not detectable by immunostaining. Cells positive for an epithelial membrane antigen, strongly expressed in sebaceous glands, were seen in numerous spheroids. In contrast to organotypic surface epithelia, the expression and location of different integrin chains was normalized in ORS spheroids, indicating an enhanced mesenchymal influence in this in vitro system.     

Transfection of epithelioma papulosum cyprini (EPC) carp cells

The variables involved in the transfection of epithelioma papulosum cyprini (EPC) cells (a representative carp fish cell line) with the genes for -galactosidase from E. coli, for luciferases from firefly or renilla, for the G protein of viral haemorrhagic septicemia virus or for green fluorescent protein under the cytomegalovirus, the SV40 or the T7 polymerase promoters have been studied. Fugene was selected among 10 transfection different reagents because it is simpler to use and it induced maximum efficiences of transfection of 37% equivalent to 10–15 ng -galactosidase per 500000 EPC cells.