Short term retinol treatment in vitro induces stable transdifferentiation of chick epidermal cells into mucus-secreting cells

Summary Epidermal mucous metaplasia of cultured skin can be induced by treatment with excess retinol for several days (Fell 1957). In the induction of mucous metaplasia, retinol primarily affects the dermal cells and retinol-pretreated dermis can alter epidermal differentiation towards secretory epithelium (Obinata et al. 1987). In this work, we found that mucous metaplasia could be induced by culturing 13-day-old chick embryonic tarsometatarsal skin in medium containing retinol (20 M) for only 8–24 h, followed by culture in a chemically defined medium (BGJb) without retinol or serum for 6 days. The application of cycloheximide together with retinol during the first 8 h of culture inhibited epidermal mucous metaplasia during subsequent culture for 6 days in BGJb, indicating that induction of a signal(s) in the dermis by excess retinol requires protein synthesis. However, the presence of 20 nM hydrocortisone (Takata et al. 1981) throughout the culture period did not inhibit retinol-induced epidermal mucous metaplasia of the epidermis. This indicates that a brief treatment of the skin with excess retinol determines the direction of epithelial differentiation toward secretory epithelium; this is a simpler in vitro system for the induction of epidermal mucous metaplasia than those established before. Offprint requests to: A. Obinata     

Differentiation in vitro of chick-embryo skin in presence of histones. Analysis of protein synthesis in toto and of keratin in particular, with the use of labeled precursors]

Six-day limb skin from a chick embryo was cultured in vitro for seven days in a complete medium either supplemented or not with histones. At the end of the incubation period, the chick embryo skin cultured in the absence of histones was found to undergo keratinization, the converse being true for the limbs cultured in the presence of histones. In the latter, when H3-leucine and C14-cystine were added to the medium, a sharp decrease in the labeled amino acid incorporation was found.     

Temporal and spatial sequence expression of cytokeratin K19 in cultured human keratinocyte

The unique cytokeratin K19 specifically expresses in simple epithelial cells, basal cells of non-keratinized stratified squamous epithelium, epidermal cells during the embryonic stage and squamous carcinoma cells, but it is not expressed in adult epidermis. Interestingly, when epidermal cells are cultured in vitro, K19 is re-expressed in the supra-basal layer. K19 expression was used as a marker for epidermal cell growth and differentiation. In order to clarify the temporal and spatial sequential expression in cultured keratinocyte, two-stage human keratinocyte culture systems were used to examine K19 expression in keratinocytes in a proliferation and differentiation stages through immunoblotting and immunohistochemistry assay. According to our results, K19 was not expressed in cultured human keratinocytes in the proliferation stage but was re-expressed in keratinocytes three days after the cultured medium was changed to a differentiation medium. Immunohistochemical observation revealed that K19 was persistently expressed in the supra-basal layer of cultured keratinocytes during first three weeks of culturing, but none was detectable in the basal cell layer. When keratinocytes were cultured with an "inserted cultured dish," K19 was persistently expressed in all layers of keratinocytes nourished by medium both from an inner chamber and an outer chamber. The different expression of K19 in these two different culture systems seemed to indicate that down regulation of K19 expression in keratinocyte was related to the direction of medium supply.     

Epithelial-Directed Mesenchyme Differentiation in vitro

To assess the requirement for specific or possibly non-specific epithelial instructions for mesenchymal cell differentiation, we designed studies to evaluate and compare homotypic with heterotypic tissue recombinations across vertebrate species. These studies further tested the hypothesis that determined dental papilla mesenchyme requires epithelial-derived instructions to differentiate into functional odontoblast cells using a serumless, chemically-defined medium. Theiler stage 25 C57BL/6 or Swiss Webster cap stage mandibular first molar tooth organs or trypsin-dissociated, homotypic epithelial-mesenchymal tissue recombinants resulted in the differentiation of odontoblasts within 3 days. Epithelial differentiation into functional ameloblasts was observed within 7 days. Trypsin-dissociated and isolated mesenchyme did not differentiate into odontoblasts under these experimental conditions. Heterotypic recombinants between quail Hamburger-Hamilton stages 22–26 mandibular epithelium and Theiler stage 25 dental papilla mesenchyme routinely resulted in odontoblast differentiation within 3 days in vitro. Odontoblast differentiation and the production of dentine extracellular matrix continued throughout the 10 days in organ culture. Ultrastructural observations of the interface between quail and mouse tissues indicated the reconstitution of the basal lamina as well as the maintenance of an intact basal lamina during 10 days in vitro. Quail epithelial cells did not differentiate into ameloblasts and no enamel extracellular matrix was observed. These results show that quail mandibular epithelium can provide the required developmental instructions for odontoblast differentiation in the absence of serum or other exogenous humoral factors in a chemically-defined medium. They also suggest the importance of reciprocal epithelial-mesenchymal interactions during epidermal organogenesis.     

Reconstituted skin in culture:a simple method with optimal differentiation

Human skin is a unique organ, which can be reconstituted in vitro and represents an interesting system for studying cell proliferation and differentiation. A simple technique for producing reconstituted skin with optimal epidermal differentiation is described and characterized. A 4-mm punch biopsy of normal human skin is deposited on the epidermal side of mortified de-epidermized human dermis maintained at the air-liquid interface with a metallic support. The culture medium contains insulin, epidermal growth factor (EGF), cholera toxin, hydrocortisone, penicillin/streptomycin and fungizone. A well-differentiated epidermis develops within 15 days. Morphological and ultrastructural studies show a neoepidermis resembling normal skin. Differentiation markers such as involucrin, filaggrin, and various cytokeratins detected with pancytokeratin antibody are present and confirm this resemblance. The keratin profile is comparable to that observed in other skin culture models. A basement-membrane-like structure is reconstituted with hemidesmosomes and anchoring-filament formation. Bullous pemphigoid (BP) antigen is observed at the dermo-epidermal junction after 21 days of culture. Moreover, both dermal substrates and punch biopsies can be kept frozen for long-term storage, with little or no loss of epidermal growth kinetics and morphology. This skin culture technique is rapid, simple, economical and reproducible. Characterization has here shown high-quality epidermal differentiation. Scientists interested in epidermal in vitro studies should take interest in all these advantages.     

Role of leukemia inhibitory factor in the regulation of the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture

Mouse epidermal melanoblasts/melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanoblast/melanocyte-proliferation medium supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Leukemia inhibitory factor (LIF) supplemented to the medium from initiation of primary culture increased the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes. Pure cultured primary melanoblasts or melanocytes were further cultured with the medium supplemented with LIF from 14 days (keratinocyte depletion). LIF stimulated the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes in the absence of keratinocytes. Moreover, anti-LIF antibody supplemented to the medium from initiation of primary culture inhibited the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes. These results suggest that LIF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture in cooperation with cAMP elevator and bFGF.     

Inhibited morphological terminal differentiation and enhanced proliferation of cultured mouse epidermal cells at different concentrations of dimethyl sulphoxide

Dimethyl sulphoxide (DMSO), at concentrations of 1-2%, induces terminal differentiation in several different cell types in vitro and enhances the growth of newborn mouse epidermal cells in primary culture under conditions that also permit terminal differentiation. We have found that DMSO concentrations approaching 4% reversibly inhibited (with little overt toxicity) terminal differentiation of normal epidermal cells from newborn SENCAR mice. Cells cultured in medium containing 4% DMSO and calcium in excess of 1 mM did not stratify extensively or slough large amounts of keratinized debris into the medium as occurred in control cultures, nor did they form large numbers of squamous cells or keratin bundles, as revealed by light and electron microscopy. The number of detergent-insoluble cornified envelopes was similarly reduced. Long-term growth of epidermal colonies in secondary culture was optimum in 1% DMSO, this concentration also permitting normal terminal differentiation of these cells. Since DMSO had these effects on epidermal cells in vitro, it may also affect epidermal cell proliferation and terminal differentiation in vivo, an important consideration should DMSO ever be approved for topical use in the US.     

Basic fibroblast growth factor stimulates the sustained proliferation of mouse epidermal melanoblasts in a serum-free medium in the presence of dibutyryl cyclic AMP and keratinocytes.

Basic fibroblast growth factor (bFGF) stimulated the sustained proliferation of mouse epidermal melanoblasts derived from epidermal cell suspensions in a serum-free medium supplemented with dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP). The melanoblasts could be subcultured in the serum-free medium supplemented with the two factors in the presence of keratinocytes, but not in the absence of keratinocytes. In these conditions, some melanoblasts proliferated without differentiating for more than 20 days including a subculture. This is the first report of a successful culture of melanoblasts from mammalian skin. This culture system is expected to clarify further markers for melanoblasts and requirements for their proliferation and differentiation.     

Using paracrine effects of Ad-MSCs on keratinocyte cultivation and fabrication of epidermal sheets for improving clinical applications

Recent advances in wound healing have made cell therapy a potential approach for the treatment of various types of skin defects such as trauma, burns, scars and diabetic leg ulcers. Cultured keratinocytes have been applied to burn patients since 1981. Patients with acute and chronic wounds can be treated with autologous/allograft cultured keratinocytes. There are various methods for cultivation of epidermal keratinocytes used in cell therapy. One of the important properties of an efficient cell therapy is the preservation of epidermal stem cells. Mesenchymal Stem Cells (MSCs) are major regulatory cells involved in the acceleration of wound healing via induction of cell proliferation, angiogenesis and stimulating the release of paracrine signaling molecules. Considering the beneficial effects of MSCs on wound healing, the main aim of the present study is investigating paracrine effects of Adipose-derived Mesenchymal Stem Cell (Ad-MSCs) on cultivation of keratinocytes with focusing on preservation of stem cells and their differentiation process. We further introduced a new approach for culturing isolated keratinocytes in vitro in order to generate epidermal keratinocyte sheets without using a feeder layer. To do so, Ad-MSC conditioned medium was applied as an alternative to commercial media for keratinocyte cultivation. In this study, the expression of several stem/progenitor cell (P63, K19 and K14) and differentition (K10, IVL and FLG) markers was examined using real time PCR on days 7, 14 and 21 of culture in keratinocytes in Ad-MSC conditioned medium. P63 and α6 integrin expression was also evaluated via flow cytometry. The results were compared with control group including keratinocytes cultured in EpiLife medium and our data indicated that this Ad-MSC conditioned medium is a good alternative for keratinocyte cultivation and producing epidermal sheets for therapeutic and clinical purposes. The reasons are the expression of stem cell and differentiation markers and overcoming the requirement for feeder layer which leads to a xenograft-free transplantation. Besides, this approach has low cost and is easier to perform. However, more in vitro and in vivo experiments as well as safety evaluation required before clinical applications.     

Attenuation of neurotoxicity following anoxia or glutamate receptor activation in EGF- and hippocampal extract-treated neuronal cultures

Neurotoxicity following anoxia or glutamate receptor activation was studied in primary neuronal cultures grown in serum-free, chemically defined CDM R12 medium. Exposure to 1 mM KCN, 0.5 mM kainic acid and 0.5 mM N-methyl-D-aspartate led to progressive neuronal degeneration. This damage was quantified by measuring lactate dehydrogenase released in the culture medium. The toxic effects were observed early during the development of the neuronal culture (from 4 days in vitro on) and seemed to be neuron-specific since astrocyte cultures were not affected. Chronic treatment of the neuronal cultures with epidermal growth factor at 10 ng/ml and hippocampal extract at dil. 1/833 (w/v) induced morphological alterations, increased beta-adrenergic receptor coupled adenylate cyclase activity, increased level of total lactate dehydrogenase activity in the case of epidermal growth factor-treated cultures, and attenuation of lactate dehydrogenase release following exposure to KCN or glutamate receptor agonists. The alterations observed are probably due to the proliferation and differentiation of glial cells in these treated cultures. This suggests that glial cells protect neurons in vitro from degeneration induced by anoxia or glutamate receptor activation.     

Restricted growth of rat kidney proximal tubule cells cultured in serum-supplemented and defined media

Proximal tubules suitable for in vitro culture were prepared from rat kidney cortex by a Ficoll-gradient centrifugation technique which yielded greater than 94% purity. The tubules were seeded into culture dishes, and cell growth was monitored in both Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and in a defined medium consisting of 50:50 Ham's F12 and Dulbecco's supplemented with insulin, transferrin, and hydrocortisone. Growth in serum-containing medium was continuous; however, the specific activity of the brush border enzyme alkaline phosphatase decreased rapidly with time, and the culture morphology became fibroblastic by 6 days. Neither collagen-coating of the dishes nor addition of the differentiation inducer hexamethylene-bisacetamide had any significant effect on growth or enzyme activity of the cultured cells. Theophylline, another inducer of differentiation, proved cytotoxic. Growth of proximal tubule cells in defined medium proceeded for 4 days before irreversible growth arrest occurred. Alkaline phosphatase activity and epithelial morphology remained relatively constant throughout the culture period. Additions of the growth factors triiodothyronine, prostaglandin E2, and epidermal growth factor were unable to unblock the growth arrest. If cells cultured in defined medium for 3 days were switched to serum-supplemented medium, continuous growth occurred, but both alkaline phosphatase activity and epithelial morphology were rapidly lost. As a test of the culture method, rabbit proximal tubule cells were cultured under similar conditions in defined medium. Growth was prolific and continuous for up to, but not exceeding, 30 days, and differentiated properties were retained. It was concluded that both rat and rabbit proximal tubule cells have a limited proliferative capacity in vitro but that the capacity of the rat cell to divide is much reduced relative to the rabbit cell.     

Morphological differentiation and changes in polypeptide synthesis pattern during regeneration of human epidermal tissue developed in vitro

By incubating multilayered primary cultures of human keratinocytes in low-calcium medium the suprabasal cell layers can be stripped off leaving a basal cell monolayer. When this monolayer is re-fed normal calcium medium a reproducible series of cell kinetic, morphological, and biochemical changes takes place resulting in the reestablishment of a multilayered tissue. Analysis of cell-cycle-specific proteins indicated that, during regeneration, a large cohort of cells became synchronized undergoing DNA replication after 3 days. Examination of culture morphology at the ultrastructural level confirmed the capacity of the basal cell monolayer to gradually reestablish a multilayered, differentiated epithelium. The ultrastructural appearance at 7 days poststripping was similar to that of unstripped cultures and was indicative of a tissue in steady state. Quantitation of cornified envelope formation at different times during regeneration showed that an increasing proportion of the cells were able to undergo terminal differentiation. In general, the pattern of keratin synthesis in the original epidermal explant labelled in vitro was similar to the pattern observed in human epidermis in vivo; however, in contrast to epidermis in vivo the explant also synthesized the hyperproliferative keratins 6 and 16. The in vitro differentiated keratinocytes showed underexpression of several proteins identified as differentiation markers, whereas several basal cell markers were overexpressed compared to the original explant. In addition, the in vitro differentiated keratinocytes synthesized some new proteins, notably keratins 7, 15 and 19. The basal layer remaining after stripping mainly expressed basal cell markers; however, during recovery, some of the differentiation-specific markers (e.g. keratin 10 and 15) were again expressed together with keratin no. 19, which is also expressed during wound healing in vivo. It is suggested that the present system of regenerating epidermal tissue cultures may serve as an experimental model to investigate certain aspects of the regulation of epidermal tissue homeostasis.     

The dynamic properties of neuronal chromatin are modulated by triiodothyronine

The effect of triiodothyronine (T3) on the rate of synthesis of nuclear proteins was studied during terminal differentiation of rat cortical neurons cultured in a serum-free medium. To this aim total and acid soluble nuclear proteins were analyzed by different electrophoretic techniques. Our results show that: 1) during maturation in vitro, neuronal nuclei undergo a dramatic change in the rate at which different classes of histones and high mobility group (HMG) proteins are synthesized; the synthetic activity, measured as incorporation of radioactive precursors into nuclear proteins, slows indeed down with age: especially evident is the decrease in core histones synthesis; at day 15, on the other hand, HMG 14 and 17 and ubiquitinated H2A (A24) are synthesized at a high rate, especially in T3-treated neurons; 2) neurons treated with T3 show, at any age tested, a higher level of lysine incorporation into nuclear proteins; 3) even if during the first days of culture neurons synthesize core histones more actively in the presence of T3, there is no accumulation of these proteins at later stages, as compared with untreated cells. Possible implications of these data and relationship with the chromatin rearrangement which accompanies neuronal terminal differentiation are discussed.     

Magnesium and phosphate enrichment of culture medium stimulates the proliferation of epidermal cells from newborn and adult mice

The proliferation and differentiation of mouse epidermal cells can be sequentially analyzed by modification of extracellular calcium. Newborn cells cultured in low calcium medium (less than 0.1 mM) proliferate as a monolayer and maintain a typical basal cell phenotype in culture but have a limited proliferative capacity and short lifespan. Elevation of the magnesium content of the culture medium from 1 to 5 mM stimulated the proliferation of newborn mouse (1-3 days old) keratinocytes. Maximal DNA synthesis rates, as determined on day 5 of culture, were up to 2-3-fold higher in the magnesium-enriched cultures. Exposure to high magnesium caused 3-4-fold increases in the DNA content of newborn keratinocyte cultures, and extended the confluent phase of epidermal cell growth to over 10 days. Other divalent cations (strontium, copper, zinc, nickel, beryllium, and barium) did not improve keratinocyte growth in culture. Keratinocytes from the tail skin of adult (3 months old) mice displayed an absolute requirement for high phosphate in the culture medium. The medium containing an optimal (10 mM) phosphate concentration prevented the cell detachment caused by the standard low (1 mM) phosphate medium, and in combination with an elevated magnesium content (10-15 mM) it markedly increased both DNA synthesis rates and DNA content of the adult cell cultures. Optimally growing, newborn or adult cultures contained less cells in the G1 phase of the cell cycle and more cells in S and G2 +M. The addition of phosphate and magnesium per se did not induce keratinocyte differentiation and did not interfere with the high calcium (1 mM)-induced differentiation.     

ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in serum-free primary culture

It is well known that alpha-melanocyte stimulating hormone (MSH) induces the differentiation of mouse epidermal melanocytes in vivo and in vitro. Although adrenocorticotropic hormone (ACTH) possesses the same amino acid sequence as MSH does, it is not clear whether the peptide and its fragments induce the differentiation of mouse epidermal melanocytes. In this study, the differentiation-inducing potencies of human ACTH and its fragments were investigated by adding them into a culture medium (0.001-1,000 nM) from the initiation of primary culture of epidermal cell suspensions. Their potencies were compared with the potency of alpha-MSH. After 2-4 days of primary cultures with ACTH(1-13), ACTH(1-17), ACTH(1-24), ACTH(1-39), ACTH(4-12), ACTH(4-13), and alpha-MSH, pigment granules appeared in the cytoplasms and dendrites of melanoblasts that were in contact with the adjacent keratinocyte colonies. By 14 days, cultures contained mostly pigmented melanocytes. The order of potencies of ACTH fragments and alpha-MSH shown by the ED(50) value was as follows: alpha-MSH = ACTH(1-13) = ACTH(1-17) = ACTH(4-12) = ACTH(4-13) > ACTH(1-24) > ACTH(1-39). The length of their peptide chains was inversely proportional to the potency. On the contrary, ACTH(1-4), ACTH(11-24), and ACTH(18-39) failed to induce the differentiation of melanocytes. In contrast, ACTH(1-10), ACTH(4-10), ACTH(4-11), and ACTH(5-12) possessed a weak potency at high doses only (100 and 1,000 nM). These results suggest that ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in culture completely. The amino acids of Met(4) and Pro(12) are suggested to be important for its potency.     

Influence of age and medium on formation of epithelial cords in the rat fetal ovary in vitro

Fetal ovaries of 14.5-day-old rats were cultured for periods of up to 19 days in control medium or in medium conditioned by the preliminary culture of testes from fetal or young rats. In all ovaries, after 12 days of culture in either medium, epithelial cords were noted having an aspect identical to that of seminiferous cords present in fetal testes explanted at 14.5 days and also cultured for 12 days, i.e. the epithelial cords appeared in ovaries when there was no 'male' or testicular influence. The appearance of histological preparations suggested that the disappearance of the germ cells might bring about a reorganization of the follicular cells in epithelial cords during the differentiation period of the first follicles. With ovaries cultured in conditioned medium, degeneration of the germ cells was more marked, follicles were rare and intra-ovarian cords were greater in number than in ovaries cultured in control medium. The ovaries thus transformed produced the anti-Müllerian hormone (AMH) although they lacked the "germinostatic activity" normally developed by testes of fetal or young rats. This germinostatic activity prevents the multiplication of oogonia when the testes and ovaries are co-cultured in vitro. The transformed ovaries therefore do not have all the functional capacities of fetal testes.     

Effect of epidermal growth factor on the sheet formation of the human keratinocyte in cell culture

Epidermal growth factor is an important element in maintaining keratinocyte proliferation and maturation. To evaluate its effect on keratinocyte growth in vitro, human foreskins were cultured. The epidermal keratinocyte growth in culture was separated into two stages by a conditional medium: the proliferation stage, in which the cells were maintained in a monolayer; and the differentiation stage, in which the cells grew to stratification and keratinization. The keratinocytes were cultured in various concentrations of epidermal growth factor, and their morphology and growth behavior were closely observed. Our results demonstrated that cultured keratinocytes grew in a confluent layer under the influence of epidermal growth factor. In contrast, in a culture without epidermal growth factor, the proliferation rate of cultured keratinocytes slowed down and eventually the cells stopped growing. During serum stimulation, with or without additional exogenous epidermal growth factor, the cultured keratinocytes grew continuously to the normal terminal differentiation. Under this two-stage culture model, the cultured keratinocytes could grow into an intact sheet of graftable epidermis.     

Granulocyte-macrophage colony-stimulating factor is a keratinocyte-derived factor involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture

Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and a melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with granulocyte-macrophage colony-stimulating factor (GMCSF) from 14 days (keratinocyte depletion). GMCSF stimulated the number of melanoblasts/melanocytes as well as the percentage of differentiated melanocytes in keratinocyte-depleted cultures. Flow cytometry analysis showed that melanoblasts and melanocytes in the S and G(2)/M phases of the cell cycle were increased by the treatment with GMCSF. Moreover, anti-GMCSF antibody added to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts/melanocytes as well as the differentiation of melanocytes. Enzyme-linked immunosorbent assay of culture media revealed that GMCSF was secreted from keratinocytes, but not from melanocytes. These results suggest that GMCSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanoblasts/melanocytes in culture in cooperation with cAMP elevator and bFGF.