In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.     

In vitro cultures of Cinchona species

The uptake of l-[methylene-¹⁴C]-tryptophan from culture medium into root organs of Cinchona ledgeriana and the subsequent incorporation of the radiolabel into quinine and quinidine is reported. In addition, feeding unlabelled l-tryptophan at levels of 500mg/l to the cultures results in a 5-fold increase in the yields of both quinoline alkaloids.     

In vitro genotoxic effects of four Helichrysum species in human lymphocytes cultures

Helichrysum sanguineum, Helichrysum pamphylicum, Helichrysum orientale, Helichrysum noeanum (Asteraceae) are medicinal plants. For centuries, they have been used as tea in Turkey because of their medicinal properties. So far no scientific evidence has been found in a literature survey regarding the genotoxic effects of these plants. This work evaluated the genotoxic effects on human lymphocyte cultures induced by methanol extracts of these plants, assayed in different concentrations (0.01, 0.05, 0.1, 0.5 and 1 mg/mL). According to the results, Helichrysum noeanum, Helichrysum pamphylicum and Helichrysum sanguineum induced the formation of micronuclei and decreased the mitotic and replication indexes. Helichrysum orientale did not affect these parameters, whereas Helichrysum noeanum, Helichrysum pamphylicum and Helichrysum sanguineum were clearly genotoxic. They should therefore not be used freely in alternative medicine, although their antiproliferative activity may suggest antimitotic and anticarcinogenic properties. Helichrysum orientale could be used in alternative medicine.     

In vitro human keratinocyte migration rates are associated with SNPs in the KRT1 interval

Efforts to develop effective therapeutic treatments for promoting fast wound healing after injury to the epidermis are hindered by a lack of understanding of the factors involved. Re-epithelialization is an essential step of wound healing involving the migration of epidermal keratinocytes over the wound site. Here, we examine genetic variants in the keratin-1 (KRT1) locus for association with migration rates of human epidermal keratinocytes (HEK) isolated from different individuals. Although the role of intermediate filament genes, including KRT1, in wound activated keratinocytes is well established, this is the first study to examine if genetic variants in humans contribute to differences in the migration rates of these cells. Using an in vitro scratch wound assay we observe quantifiable variation in HEK migration rates in two independent sets of samples; 24 samples in the first set and 17 samples in the second set. We analyze genetic variants in the KRT1 interval and identify SNPs significantly associated with HEK migration rates in both samples sets. Additionally, we show in the first set of samples that the average migration rate of HEK cells homozygous for one common haplotype pattern in the KRT1 interval is significantly faster than that of HEK cells homozygous for a second common haplotype pattern. Our study demonstrates that genetic variants in the KRT1 interval contribute to quantifiable differences in the migration rates of keratinocytes isolated from different individuals. Furthermore we show that in vitro cell assays can successfully be used to deconstruct complex traits into simple biological model systems for genetic association studies.     

In vitro simulation of cytoplasmic membrane senescence in cotyledons

The loss of microsomal NADH-cytochrome c reductase activity (EC 1.6.99.3) in cotyledons, known to accompany germination of Phaseolus vulgaris and thought to reflect the progress of cytoplasmic membrane senescence, can be simulated in an in vitro system in which isolated microsomes from 2-day-old tissue are treated with cytosol fractions (microsomal supernatants). Inactivation of the enzyme is comparatively low when the microsomes are treated for 4 hours with cytosol fractions from 1- and 2-day-old tissue, but increases to about 68% upon treatment with a corresponding fraction from 3-day-old cotyledons. This temporal pattern is consistent with the pronounced in situ decline in NADH-cytochrome c reductase detectable between the 2nd and 4th days of germination. Extensive in vitro inactivation was also effected by cytosol fractions prepared from older tissue, including that harvested after 9 days of germination by which time the cotyledons were beginning to abscise.     

In vitro fermentation of chitosan derivatives by mixed cultures of human faecal bacteria

Stirred, pH controlled batch cultures were carried out with faecal inocula and various chitosans to investigate the fermentation of chitosan derivatives by the human gut flora. Changes in bacterial levels and short chain fatty acids were measured over time. Low, medium and high molecular weight chitosan caused a decrease in bacteroides, bifidobacteria, clostridia and lactobacilli. A similar pattern was seen with chitosan oligosaccharide (COS). Butyrate levels also decreased. A three-stage fermentation model of the human colon was used for investigation of the metabolism of COS. In a region representing the proximal colon, clostridia decreased while lactobacilli increased. In the region representing the transverse colon, bacteroides and clostridia increased. Distally a small increase in bacteroides occurred. Butyrate levels increased. Under the highly competitive conditions of the human colon, many members of the microflora are unable to compete for chitosans of low, medium or high molecular weight. COS were more easily utilised and when added to an in vitro colonic model led to increased production of butyrate, but some populations of potentially detrimental bacteria also increased.     

In vitro modulation of human keratinocyte pro- and anti-inflammatory cytokine production by the capsule of Malassezia species

Malassezia spp. are commensal, cutaneous fungi that are implicated in seborrhoeic dermatitis. We hypothesize that the lipid-rich capsule of Malassezia spp. masks the organism from host detection, and depletion of this layer elicits an inflammatory response. To test this, preparations of capsulated or acapsular [10% (v/v) Triton X-100 treated], viable and nonviable, exponential or stationary phase Malassezia furfur, Malassezia globosa, Malassezia obtusa, Malassezia restricta, Malassezia slooffiae and Malassezia sympodialis, were incubated with normal human keratinocytes. Proinflammatory (IL-6, IL-8, IL-1alpha and tumour necrosis factor-alpha) and anti-inflammatory cytokine (IL-10) release and intracellular IL-10 concentrations were quantified using enzyme-linked immunosorbent assays. Capsulated Malassezia yeasts stimulated limited or no production of inflammatory cytokines, and increased intracellular IL-10 (P < 0.05). Removal of the capsule of many Malassezia preparations caused a significantly increased production of IL-6, IL-8 and IL-1alpha, and a decrease in intracellular IL-10. Notably, acapsular viable, stationary phase M. globosa caused a 66-fold increase in IL-8 production (P < 0.001) and acapsular nonviable, stationary phase M. furfur caused a 38-fold increase in IL-6 production (P < 0.001) and a 12-fold decrease in intracellular IL-10 (P < 0.001). These results support the hypothesis that the lipid layer of Malassezia spp. modulates cytokine production by keratinocytes. This has implications in the pathogenesis of seborrhoeic dermatitis.     

Epithelial-specific gene expression during differentiation of stratified primary human keratinocyte cultures.

Cultured epithelial cells are used to generate extensive patches of autologous skin equivalent for patients with burns or wounds and to investigate the growth and differentiation of epithelia in vitro. We have undertaken a comprehensive study of the morphological and molecular events that occur during culturing of human foreskin keratinocytes at the liquid-air interface on a dermal equivalent consisting of a collagen matrix containing fibroblasts. Using radioactively labeled RNA probes for mRNAs and monoclonal antibodies for proteins, we found that the expression of a comprehensive set of differentiation stage-specific genes was affected by the type of fibroblasts included in the matrix as well as by the age of the culture. The expression of these genes was not always coordinated and could not be predicted from the histological appearance of the stratified epithelium. Surprisingly, the mouse fibroblasts promoted epithelial differentiation much more closely resembling foreskin than did the homologous primary foreskin fibroblasts.     

In vitro male germ cell cultures of zebrafish

Transgenic modification of sperm before fertilization has the advantages of a much shorter timeline for the production of transgenic animals. A culture system using primary cultures of zebrafish male germ cells, in which the differentiation of spermatogonia to functional sperm can occur in vitro, allows us to introduce foreign DNA into the cultured sperm and to produce transgenics from the sperm. This chapter describes methods for the co-culture of male germ cells and a Sertoli cell feeder layer and the introduction of foreign DNA with retroviruses. This male germ cell culture system should prove useful not only in producing genetically modified sperm, but also in analyzing the regulatory function of Sertoli cells for spermatogenesis in vertebrates.     

Cellular confluence determines injury-induced prostaglandin E2 synthesis by human keratinocyte cultures

When keratinocyte cultures become confluent, their prostaglandin E2 synthesis is suppressed. To determine whether the injury response is characterized by increased prostaglandin E2 synthesis, an in vitro injury model was developed. When confluent keratinocyte cultures were focally lethally irradiated using ultraviolet light B, a dose-dependent increase in prostaglandin E2 synthesis was induced by the injury. After irradiation, confluent cultures' prostaglandin E2 synthesis increased for 2 days to 8-fold more than controls, then decreased to control values by day 6. Increased prostaglandin E2 synthesis was first detected 8 h after injury. Focal irradiation of non-confluent cultures (killing isolated colonies) caused no change in prostaglandin E2 synthesis, indicating that culture continuity must be disrupted before synthesis increases. In addition, partial irradiations of petri dishes demonstrated that enhanced metabolism was confined to cells adjacent to the injury site and was not mediated by a soluble factor. When confluent and injured cultures were incubated with [14C]arachidonic acid, and the products formed analyzed by thin layer chromatography, 10-fold more prostaglandin E2 microgram protein was seen in irradiated cultures relative to confluent controls. The products formed by each group were the same, and no consistent increases in metabolites other than prostaglandin E2 were observed. The increased synthesis of prostaglandin E2 by injured cultures was apparently due to an increase in cyclooxygenase activity as determined by kinetic experiments. These data indicate that the pattern of metabolism of arachidonic acid seen in non-confluent cultures is similar to that seen in injury, and that cell-cell contact modulates enhanced prostaglandin E2 synthesis.     

Lipid content and metabolism of human keratinocyte cultures grown at the air-medium interface

The differentiation of human keratinocytes in most culture systems is incomplete; e.g., lamellar bodies, the characteristic lipid-delivery organelles of epidermis, are not present. Moreover, their lipid profile does not reflect the distinctive composition found in cornifying epidermis. In contrast, keratinocytes that grow at an air-medium interface exhibit more complete differentiation. In this study, we compared the elaboration of lamellar bodies, the lipid content, and the lipid metabolism of human keratinocytes, cultured both under standard immersed conditions and after lifting to an air-medium interface. Whereas submerged cultures neither elaborated lamellar bodies nor displayed a lipid distribution characteristic of cornifying epidermis, lifted cultures displayed advanced cornification, elaborated lamellar bodies which were deposited in intercellular domains, and a lipid profile more typical of cornifying epidermis. Moreover, lipid biosynthesis was 5-10-fold more active in lifted than in immersed cultures, and was not inhibited by exogenous lipoproteins. These findings are consistent with recent studies that demonstrate both high rates of lipogenesis in differentiating layers of the epidermis as well as autonomy of lipogenesis from the influence of circulating lipoproteins. Thus, the lipid content and metabolism of human keratinocyte cultures, grown at an air-medium interface, demonstrate features that simulate the epidermis.     

Volatile profiles of human skin cell cultures in different degrees of senescence

It is known that skin releases volatile organic compounds to the environment, and also that its emission pattern changes with aging of the skin. It could be considered, that these compounds are intermediaries in cell metabolism, since many intermediaries of metabolic pathways have a volatile potential. In this work, a simple and non-destructive method consisting of SPME sampling and GC/MS analysis was developed to identify volatile organic emanations from cell cultures. This technique, applied to skin cells culture, indicates that the cells or cell metabolism produce several skin emissions. Chemometric analysis was performed in order to explore the relationship between a volatile profile and the senescence of cell cultures. Volatile profiles were different for cell cultures in different degrees of senescence, indicating that volatile compound patterns could be used to provide information about the age of skin cells.     

In vitro study on effect of bardoxolone methyl on cisplatin-induced cellular senescence in human proximal tubular cells

Molecular and Cellular Biochemistry - Bardoxolone methyl [methyl-2-cyano-3, 12-dioxooleana-1, 9(11)dien-28-oate (CDDO-Me)], an activator of the nuclear factor erythroid-derived 2-related factor2...     

Modifications of proteoglycans produced by human skin fibroblast cultures during replicative senescence

The properties of proteoglycans (PGs) produced by normal human skin fibroblast were investigated with increasing passage. The increase of subculture number was associated with a constant increase in PG molecular size, which was particularly evident in cell layer extracts. In the cell layer, the ratio of DS-PGs/HS-PGs was markedly higher in early passage cultures. Moreover, the cell layer from young cells contained lower amounts of radioactivity incorporated into the most hydrophobic PG populations, suggesting that the PG core protein might also undergo significant modification with increasing subcultures. There was no significant difference in energy charge value between early and late passage cultures, whereas the NAD/NADH ratio was found to decrease markedly in senescent cells.     

In vitro plant tissue cultures accumulate polyisoprenoid alcohols

In vitro cultivated plant cells and tissues were found to synthesize polyisoprenoids. Taxus baccata suspension cell cultures accumulated polyisoprenoids of the same pattern as the parental tissue; methyl jasmonate or chitosan treatment almost doubled their content. All the root cultures studied accumulated dolichols as predominant polyisoprenoids. Roots of Ocimum sanctum grown in vitro accumulated approx. 2.5-fold higher amount of dolichols than the roots of soil-grown plants. Dolichols dominated over polyprenols in all Triticum sp. tissues studied.     

In vitro toxicity evaluation of low doses of pesticides in individual and mixed condition on human keratinocyte cell line

The induced toxicity of three pesticides (alpha-Hexachlorocyclohexane: α-HCH; Parathion methyl:PM; Carbofuran: CN) in single and four possible combination on human keratinocyte cell line have been investigated. There was no significant change in toxicity (cyto and genotoxicity) on cell line exposed by individual pesticides except α-HCH. But, a synergistic effect was observed when we tested mixture of pesticides. The intracellular ROS and cytotoxicity assay revealed maximum reduction in cell viability (60%) was found in tri mixture of pesticides. All the possible combination of these pesticides demonstrated genotoxic activity in terms of olive tail moment and % tail DNA on cell line at low concentration. The order of toxicity was ranked as α-HCH+PM+CN>α- HCH+CN>PM+CN>α-HCH+PM. Our results call for more research to be undertaken in order to understand the mechanisms behind the synergy observed and quantify the extent of its environmental impacts.     

Stem cells (p63(+)) in keratinocyte cultures from human adult skin

Epidermal stem cells (ESC) are responsible for maintaining skin cellular homeostasis, as they give rise to fast-dividing transit amplifying cells committed to terminal differentiation, while retaining their self-renewal capacity. However, no pure ESC cultures are available and no highly specific cytochemical marker was identified. We report here the experimental conditions allowing the selective enrichment in ESC, using cultured adult human keratinocytes. The main step was the selection of cells able to rapidly adhere to human collagen type IV in vitro . Thus, an increased proportion of putative ESC of about 65% was obtained, as demonstrated by p63 expression.     

In vitro cultures of Cinchona species. Part II

Factors affecting the regeneration of shoots from Nicotiana plumbaginifolia colonies were studied. As opposed to results obtained with tobacco, phytohormone pretreatments of those colonies did not appreciably affect their subsequent aptitude to develop shoots. Hexoses generally inhibited shoot morphogenesis at concentrations higher than 20 mM, but ribose sustained efficient shoot induction at higher concentration. Shoot induction was promoted by a high nitrogen supply. Nitrate promoted greater shoot induction than ammonium succinate or glutamine. Among the micronutrients tested, zinc was stringently required for efficient shoot induction, concentrations ranging between 10 and 100 M being optimal. Under optimized conditions of regeneration, wild type as well as nitrate reductase deficient mutants could be efficiently regenerated.