Epidermal growth factor and the onset of epithelial epidermal wound healing.

At 10 days in ovo the embryonic chick epidermis acquires the ability to spread as a cohesive epithelial sheet when wounded. A tissue culture system has been constructed that supports epidermal cell outgrowth consistent with epidermal behaviour in vivo and permits experimental manipulation of the isolated tissue with growth factors and other hormones. This culture system consists of embryonic chick epidermis isolated at days 8, 10, and 12 of development, serum-free, chemically-defined culture medium, and the inner surface of the vitelline membrane of the hen's egg as the culture substratum. At 8 days the cellular outgrowth is mesenchymal in the absence of exogenous EGF. The 8 day tissues responds to added EGF by exhibiting precocious epithelial outgrowth. The results suggest that sensitivity to EGF or EGF-like growth factors is part of the mechanism underlying the developmental onset of epidermal wound healing in skin. The epidermal origin of the outgrowth is determined by antibody staining for specific cytokeratins. The epithelial character of the outgrowth is determined by visualizing actin microfilament distribution. The normal epithelial outgrowth shows apical/basal polarization of the sheet except at the edge. From 10 days on, the isolated epidermis exhibits epithelial outgrowth from explants in culture in the absence of exogenous EGF, suggesting endogenous production of an EGF-like factor. Glucocorticoid and mineralocorticoid hormones both produce a reduced amount of epithelial outgrowth. This retardation of the early outgrowth by glucocorticoids and mineralocorticoids could result from a reduced ability of the cut edge of the epidermis to 'disorganize' and assume the unpolarized migratory form required for rapid epidermal wound healing.     

Hamster tracheal organ culture in serum-free media: A quantitative comparison of in vitro epithelial morphology with that of in vivo controls

Summary The epithelial morphology of the hamster trachea in serum-free organ culture was compared with that of age-matched in vivo control tissues by collecting and statistically analyzing several quantifiable parameters. By this technique it was possible to detect both subtle and dramatic epithelial alterations. Midtracheal tissues from 6-wk-old male Syrian golden hamsters were used as the explants. Explants were placed on Gelfoam sponges and cultured for 1, 2, and 3 wk in CMRL 1066 alone and in CMRL 1066 to which seven factors were added: insulin and transferrin (5 μg/ml); hydrocortisone (5×10⁻⁷ M); epidermal growth facotr (5 ng/ml); bovine pituitary extract (0.5%); and phosphoethanolamine and ethanolamine (5×10⁻⁵ M). The following data were collected and statistically analyzed for each tracheal ring: number of epithelial cells; proportion and number of each cell type; basement membrane length; linear density of epithelial cells; epithelial height; and mitotic index. Compared to controls, ciliated cells decreased by 52% during washes in Leibovitz (L15) medium and tissue manipulation performed before culture and this loss persisted after cutlure for 1 wk. Explants culturedwithout the factors showed marked changes after 2 and 3 wk including epithelial thickening and folding, which was associated with increased linear density. Many cells in these specimens could not be categorized by type (22% were unidentifiable after 3 wk). Epithelial migration onto the outside of the explant was inhibited. In contrast, explants culturedwith the factors maintained a morphology similar to controls at 2 and 3 wk and epithelial migration onto the outside of the explant was supported. This study shows that explants in CMRL 1066 with the seven factors provide a useful biological model for the in vitro study of the mucociliary respiratory epithelium. This work was supported by grant HL24722 from the National Institutes of Health, Bethesda, Maryland.     

Ultrastructural observations on effects of different concentrations of calcium and thyroxine in vitro on larval epidermal cells of Rana catesbeiana tadpoles

Summary During anuran metamorphosis dramatic changes in morphogenesis and differentiation of epidermis occur under the influence of thyroid hormones. Modification of ionic calcium concentration also markedly alters the pattern of proliferation and differentiation in amphibian epidermal cells in vitro. The present study was designed to determine the direct effect of low (0.05 mM) and high (0.5mM) calcium (Ca²⁺) in the absence or presence of thyroxine (10⁻⁷ M) on epidermal cells of the body and tail tissue in vitro. When tail fin and body skin explants were maintained in low (0.05 mM) calcium for 48 h, normal ultrastructural morphology and integrity of the cells was observed in both the tissue types. When tissues were exposed to high levels of calcium (0.5mM) in culture medium, tail epidermis showed stratification, and skein cells exhibited apoptosis, both in the presence or absence of thyroid hormones. Under high calcium conditions, the body epidermis showed keratinization of apical cells, apoptosis of skein cells, and increased desmosome formation. These results suggest that (1) optimal Ca²⁺ concentration for larval epidermal cells is quite low (0.05 mM), (2) high Ca²⁺ leads to keratinization only in body epidermis, and (3) apoptosis occurred in skein cells of both the tissues at high Ca²⁺ concentrations (0.5mM). The present study therefore suggests that the extracellular calcium concentration regulates the process of cell death and differentiation inRana catesbeiana larval epidermis, and this effect may be similar to the effect of calcium on mammalian epidermal cells.     

Short-term cultivation of murine thymic epithelial cells in a serum-free medium

Summary Thymic epithelial cells were grown in defined medium without unknown serum factors and without concurrent growth of other cell types. Thymic tissue was obtained from 1- to 4-wk-old mice, disaggregated, and incubated in a mixture of collagenase-dispase-DNAse. The resulting organoids were seeded on collagen-coated flasks. The culture medium consisted of DME-F12 with low or high concentration of Ca²⁺ supplemented with insulin, epidermal growth factor, cholera toxin, hydrocortisone, and transferrin. Under these conditions, explants attached to the substrate within 2 d, and expanding epithelioid monolayer islets emerged from the organoids during the following days. [³H]Thymidine incorporation revealed a growth fraction of the cells close to 5%. By omitting either epidermal growth factor, insulin, or cholera toxin from the medium, pronounced reduction in sizes of islets and in [³H]thymidine incorporation was found. Throughout the culture period, the islets appeared as continuous sheets of polygonal cells. The epithelial nature of the expanding cell islets was confirmed by demonstration of cytokeratins and of desmosomes. Ultrastructural evaluation of early cultures revealed clusters of epithelial cells intermixed with lymphocytes, and late cultures showed a typical pattern of stratified keratinizing epithelium. However, squamous metaplasia was avoided by the use of low Ca²⁺ medium, which also proved essential for cell transfer. MHC class II antigen was detected on the majority of the cultured cells, and culture supernatants contained co-mitogenic activity for thymocytes and GM-colony stimulating activity. This work supported by The Danish Research Council, grant 12-8148.     

Dietary selenium levels determine epidermal langerhans cell numbers in mice

Selenium (Se) is a dietary trace element that is essential for effective immunity and protection from oxidative damage induced by ultraviolet radiation (UVR). Langerhans cells (LC) represent the major antigen-presenting cells resident in the epidermis; a proportion migrate from the skin to the draining lymph nodes in response to UVR. Because it is known that Se deficiency impairs immune function, we determined what effect this has on LC numbers. CH3/HeN mice were weaned at 3 wk and placed on diets containing <0.005 ppm of Se (Se deficient) or 0.1 ppm of Se (Se adequate, control mice). After 5 wk on the diet, the epidermal LC numbers in the Se-adequate group were 966±51 cells/mm² and LC counts in the epidermis of the Se-deficient mice were 49% lower (p<0.05). Glutathione peroxidase-I (GPx) activity was measured in the epidermis, lymph nodes, and liver. In the epidermis, the activity of GPx in the Se-deficient mice was only 39% (p<0.01) of that seen in epidermis from Se-adequate mice (1.732 U/mg protein). The mice were then irradiated with one dose of 1440 J/m² of broadband UVB or mock irradiated. After 24 h, the decrease in LC number after UVB was greater in the Se-adequate mice, (40% decrease) compared to the Se-deficient group (10%). Thus, Se deficiency reduces epidermal LC numbers, an effect that might compromise cutaneous immunity.     

Establishment of an outgrowth culture system to study growth regulation of normal human epithelium

Summary To study the growth regulation of epithelial cells as a sheet, I developed an outgrowth culture system for normal human ectocervical epithelial (NHCE) cells, whereby outgrowths from tissue explants increase their radius in a constant rate over time. Cinematographic observation revealed that throughout the outgrowths the cells coordinately migrate and proliferate. To date, all 59 specimens examined have shown similar growth characteristics, with explant size not causing any difference in the growth rate; 10⁸ cells/specimen can easily be obtained in 3 wk. Cell densities of outgrowths also remain constant. Moreover, there is no fibroblast contamination, and removal of explants does not affect growth rate. Therefore, pure epithelial outgrowth in uniform growth condition can be prepared for further experiments. The results demonstrate that the outgrowth culture system is an attractive model for analysis of growth control mechanisms in normal human epithelium in vitro.     

An organ culture of adult mouse skin: An in vitro model for studying the molecular mechanism of skin tumor promotion

A simple method to culture explants of adult mouse skin in a modified Eagle's HeLa cell medium was developed in order to further study the biochemical responses to the tumor promoting phorbol esters. The skin explants remained viable for at least 48 hr, as determined by their ability to incorporate ³H-thymidine into DNA as well as to induce epidermal ornithine decarboxylase (EC 4.1.1.17) activity following 12-0-tetradecanoylphorbol-13-acetate addition. The time course of induction of ornithine decarboxylase activity by the tumor promoter was similar to that observed with intact mice. Furthermore, the addition of retinoic acid and indomethacin, the agents that are known to inhibit the induction of ornithine decarboxylase activity by topically applied TPA, also inhibited the induction of ornithine decarboxylase activity by TPA in skin explants.     

Formation of desmosomes and other contact specializations in cultured skin of the frog (Rana esculenta)

Summary Small trypsinized explants from ventral skin of frogs (Rana esculenta) were maintained in culture for 4 days during which a newly formed epithelium differentiated along the cut edges of the dermis. During the first 6 h adjacent cells produced numerous interdigitating lamellipodia. After 2 days, epithelial polarity was restored by the formation of zonulae occludentes and the epithelial cells were joined by a few small newly formed desmosomes and by numerous interdigitations. Bipartite junctional complexes consisting of a zonula occludens, followed by a series of typical desmosomes, and characteristic of adult frog epidermis were formed only after 4 days. When cultured in the presence of an inhibitor of protein synthesis (cycloheximide) the trypsinized epidermis no longer formed desmosomes. Therefore pools of one or more crucial desmosomal proteins must be very low or non-existent. However, cycloheximide did not prevent the formation of cell contact specializations, consisting of a highly developed system of complex lamellar interdigitations, between adjacent cells.     

Differentiation of pure chick embryo epidermis grown in primary serum-free culture

The differentiation of precocious embryonic epidermis in serum-free primary culture was analyzed by light and electron microscopic methods. Explants of 7-day chick embryo epidermis were grown on collagen or poly-L-lysine substrates in the absence of dermal mesenchyme. The serum substitute consisted of a mixture of insulin, transferrin, putrescine and seleneous acid together with (or without) Nerve Growth Factor. These culture conditions were shown to support proliferation, growth and development (evaluated using morphological criteria) of the epidermal explants up to 4-5 days; during this period, the epidermis underwent stratification; well-developed desmosomes as well as tonofilaments were formed and the epidermis achieved a morphology close to that of 10-11 day epidermis in ovo. However long-term survival of the explants was not obtained as cellular death, starting on day 5, progressively led to the necrosis of most parts of the explant. This morphological study demonstrates that the early phases of epidermal growth and maturation can occur to some extent in the virtual absence of dermal elements and serum factors. Chick embryo epidermal cells may thus possess the intrinsic ability to go through, at least for short periods in vitro, their differentiation programme. Then, at the onset of epidermal keratinization (12 days in ovo), they require specific exogenous factors to fully differentiate in vitro.     

Histidase function in human epidermal cells

The activity of histidase was studied in (1) epidermal tissue scraped from human infant foreskin, (2) fibroblast-like cells in monolayer serial culture from human foreskin, and (3) epithelial-like (epidermal) outgrowth from foreskin primary explants. Foreskin epidermal tissue without in vitro culture and epidermal outgrowth in primary culture from explants of foreskin showed equivalent mean levels of histidase activity, 5.22 × 10^?3 and 5.01 × 10^?3 μMoles urocanic acid produced per milligram protein per minute. Under the same assay conditions, there was no measurable histidase activity in cultured fibroblast-like cells from foreskin at various times after subculture. The K_m for enzyme from human foreskin epidermal tissue ranged between 2 and 5 × 10^?3 M histidine. Ability to demonstrate the presence or absence of this tissue-specific enzyme function in cultured cells suggests a useful means for studying differentiation, as well as a more precise way to identify epidermal origin of cultured cell types than morphological characteristics alone would permit.     

The tumor promoter 12-0-tetradecanoylphorbol-13-acetate induces ornithine decarboxylase in proliferating basal cells but not in differentiating cells from mouse epidermis

The cultivation of mouse epidermal cells in medium of reduced calcium concentration (0.02–0.1 mM) selects for basal cell growth. Elevation of medium calcium levels above 0.1 mM results in rapid and well defined differentiative changes. This model was utilized to determine which cell type in mouse epidermis responds to the phorbol ester tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), by an induction of the enzyme ornithine decarboxylase (ODC). Previous data had shown that TPA induces ODC in primary mouse epidermal cells only during the first 36 hr after plating in medium containing 1.44 mM Ca²⁺. In contrast, the induction in cells grown in low calcium medium was 2–10-fold greater, and inducibility persisted for at least 4 weeks. The greater inducibility of ODC in low calcium cells is not paralleled by increased thymidine incorporation after TPA treatment, probably because these cells are already proliferating at a maximum rate. When low calcium cells grown in 0.07 mM Ca²⁺ medium were switched to 1.2 mM Ca²⁺, there was a rapid loss of ODC inducibility. These results strongly suggest that the basal cells of the epidermis constitute the major target cells for the induction of ODC by TPA. The induction of ODC by ultraviolet light was not enhanced by growth of cells in low calcium medium, indicating that extracellular calcium concentration per se does not determine ODC inducibility. When epidermal cells grown in 1.2 mM or 0.07 mM Ca²⁺ medium were exposed to both UV light and TPA, there was a significant synergistic effect of combined treatment over the sum of each individual response, suggesting that factors in addition to differentiation determine the extent of ODC induction.     

Elimination of epiplakin by gene targeting results in acceleration of keratinocyte migration in mice

Epiplakin (EPPK) was originally identified as a human epidermal autoantigen. To identify the function of epiplakin, we generated epiplakin "knockout" mice. These mice developed normally, with apparently normal epidermis and hair. Electron microscopy after immunostaining revealed the presence of EPPK adjacent to keratin filaments in wild-type mice, suggesting that epiplakin might associate with keratin. The appearance and localization of keratin bundles in intact epidermal keratinocytes of EPPK-/- mice were similar to those in wild-type mice. Wounds on the backs of EPPK-/- mice closed more rapidly than those on the backs of wild-type and heterozygous mice. The outgrowth of keratinocytes from skin explants from knockout mice was enhanced compared to outgrowth from explants from wild-type mice, even in the presence of mitomycin C, suggesting that the difference in keratinocyte outgrowth might be due to a difference in the speed of migration of keratinocytes. At wound edges in wild-type mice, EPPK was expressed in proliferating keratinocytes in conjunction with keratin 6. In EPPK-/- mice, no similar proliferating keratinocytes were observed, but migrating keratinocytes weakly expressed keratin 6. EPPK was coexpressed with keratin 6 in some keratinocytes in explant cultures from wild mice. We propose that EPPK might be linked functionally with keratin 6.     

Growth, adipose, brain, and skin alterations resulting from targeted disruption of the mouse peroxisome proliferator-activated receptor beta(delta)

To determine the physiological roles of peroxisome proliferator-activated receptor beta (PPARbeta), null mice were constructed by targeted disruption of the ligand binding domain of the murine PPARbeta gene. Homozygous PPARbeta-null term fetuses were smaller than controls, and this phenotype persisted postnatally. Gonadal adipose stores were smaller, and constitutive mRNA levels of CD36 were higher, in PPARbeta-null mice than in controls. In the brain, myelination of the corpus callosum was altered in PPARbeta-null mice. PPARbeta was not required for induction of mRNAs involved in epidermal differentiation induced by O-tetradecanoylphorbol-13-acetate (TPA). The hyperplastic response observed in the epidermis after TPA application was significantly greater in the PPARbeta-null mice than in controls. Inflammation induced by TPA in the skin was lower in wild-type mice fed sulindac than in similarly treated PPARbeta-null mice. These results are the first to provide in vivo evidence of significant roles for PPARbeta in development, myelination of the corpus callosum, lipid metabolism, and epidermal cell proliferation.     

Differential Reactivity in Epidermal Cells of Begonia rex Excised and Grown in vitro

Thin explants composed of the epidermis and underlying collenchyma excised from leaf veins of Begonia rex and cultured in vitro are capable of neoformation of unicellular hairs, roots and buds. Unicellular hairs were formed over the entire surface of the explant when 10⁻⁶M indole acetic acid or 10⁻⁷M naphthaleneacetic acid (NAA) was added to the basal medium; each epidermal cell was potentially involved. The epidermis was most sensitive to a NAA treatment during the first few days of culture but 30% of the explants could still react after 4 days of culture without NAA. When NAA (5 × 10⁻⁷M) and a cytokinin, zeatin (10⁻⁷M), were added together, roots were formed from epidermal tissue after numerous divisions in the original cells. Their initiation was not related to particular cells. Buds were formed when a cytokinin (10⁻⁶M) was added to the basal medium; bud meristems were formed from small groups of cells surrounding basal cells of glandular hairs. Hair formation was inhibited by either high (32–27°C) or low (12°C) temperatures applied continuously. 32–27°C seemed to inhibit elongation of the hairs specifically, whereas 12°C inhibited earlier phases in hair formation. This hypothesis was supported by short temperature treatments applied at different times during hair formation.     

Growth-promoting effect of haemocytes on insect epidermis in vitro

The epidermis of 21-day-old leg regenerates of cockroaches (Leucophaea maderae) was cultivated in vitro. Outgrowth of the epidermis only occurred in connexion with haemocytes.Haemocytes contaminating the epidermal explants show strong adherence to epidermal cells. The epidermal cells adhering to moving haemocytes are stretched out to long projections or completely pulled out of the epithelium. When more haemocytes are present, they can form an uninterrupted line at the margin of the epidermis. By the adhesion of marginal cells of the epidermis to the moving haemocytes, the epithelium is apparently pulled out into broad tongues. In these tongues the epidermal cells become highly flattened, especially at the front, and soon begin to divide. Outgrowth in the tongues continues only as long as there are haemocytes at the front. When they have disappeared, outgrowth stops, the flattened epidermal cells detach from the glass surface, round up, and the outgrown tissue may withdraw again.For further analysis of the interactions of haemocytes and epidermal cells the epidermis is placed on a monolayer of haemocytes. The epidermis rapidly grows out on such a monolayer. The epidermal cells either move over or under the haemocytes indicating that there are substances on both sides of the haemocytes which are attractive to the epidermal cells and cause their flattening and outgrowth. Similar outgrowth occurs on fixed monolayers of haemocytes. There is no outgrowth on areas where the monolayer has been scraped away. No principal differences can be found between monolayers consisting almost exclusively of either plasmatocytes or granular haemocytes.The similarities of the observed interactions of haemocytes and epidermal cells to encapsulation and wound healing are pointed out. A hypothesis is presented which assumes that the haemocytes during wound healing not only serve as a mechanical support but also as a chemical guide by which the closure of the wound by epidermal cells is enhanced.     

Optimization of in vitro bud induction and plantlet formation from mature embryos of Aleppo pine (Pinus halepensis Mill.)

Summary Studies were undertaken to optimize tissue culture conditions for micropropagation of Aleppo pine (Pinus halepensis Mill.) from mature embryos and various explants of the embryo. Over 90% of the embryo explants gave rise to adventitious buds within 4 wk. Intact embryos were the most suitable explants for shoot bud induction. Both isolated cotyledons and hypocotyls produced adventitious buds, but these developed slowly and failed to elongate. N⁶-Benzyladenine (BA) alone at 5.0μM was the most effective cytokinin when added to gelled to gelled von Arnold and Eriksson’s (AE) medium containing 3% sucrose. Adventitious bud development was achieved on hormone-free AE medium, and shoot elongation was optimum on three quarter-strength Bornman’s MCM medium, with 0.1% conifer-derived activated charcoal. Shoots were multiplied on three-quarter strength MCM medium, containing 5μM BA. To induce adventitious roots on the elongated shoots, pulse treatment with 1 mM IBA for 6 h, followed by the transfer of the shoots to sterile peat:vermiculite (1:1) mixture, was beneficial. After acclimatization for 3 to 4 wk under mist, almost all the rooted shoots could be transplanted successfully to the greenhouse, where the plants exhibited normal growth habit. Histologic studies on the ontogeny of adventitious shoot formation from mature embryo explants revealed temporal structural changes in different parts of the explant. Induction of mitotic divisions on the shoot-forming medium resulted in the formation of meristemoids in the epidermal and subepidermal layers of the explant, located initially at both the tips of the cotyledons and the axils of adjacent cotyledons. Shoot buds arising in the axils of adjacent cotyledons were due to new cell division and not to any preexisting meristem.     

Distribution and changes of reserve materials in cotyledon cells of Panax ginseng related to direct somatic embryogenesis and germination

Cotyledon explants from zygotic embryos of Panax ginseng produced somatic embryos on Murashige and Skoog basal medium without growth regulators. Somatic embryos developed directly from epidermal cells at the cotyledon base. Somatic embryos were always formed from the side of the cotyledon opposite to the one attached to the medium surface regardless of cotyledon orientation. The frequency of somatic embryo formation from the abaxial epidermis (66%) was much higher than that from the adaxial epidermis (12%). Differences in embryogenic response were likely related to cell structure. Abaxial epidermal cells were filled with reserve materials (lipid bodies), while adaxial epidermal cells were devoid of any prominent reserves. During germination, the reserve materials in the cells of the cotyledons disappeared rapidly. At the same time, the competency of somatic embryo formation from cotyledon explants declined rapidly to zero. Upon culture of the cotyledon explants (for somatic embryo induction), lipid bodies slowly disappeared, but starch grains accumulated prominently. Reserve materials disappeared after commencement of embryogenic cell division. During germination, lipid bodies rapidly disappeared, and chloroplasts developed instead of starch grains. Received: 29 January 1997 / Revised version received: 16 April 1997 / Accepted: 9 May 1997     

Enhancement of Neurite Outgrowth and Aspartate-Glutamate Uptake Systems in Retinal Explants Cultured with Chick Gizzard Extract

Chicken gizzard extract promoted a long and radially directed neurite outgrowth from retinal explants of 8-day-old chick embryo in cultures of 2–3 days. The neurite outgrowth from retinal explants cultured in the absence of gizzard extract was short and restricted to the explant perimeter. The neurite outgrowth promoted by gizzard extract depended strictly on several factors. (a) Fetal calf serum and polycationic substratum were required in this culture system, (b) Pretreatment of the polyornithine-coated substratum with gizzard extract allowed the retinal explants to extend neurites even in the absence of gizzard extract in the medium. (c) Maximal neurite outgrowth was observed in retinal explants dissected from 8-day embryos, but thereafter the explants’response to gizzard extract rapidly declined and was almost lost at the 12th day. As a biochemical parameter of differentiation of cultured neuroretina, uptake systems for neurotransmitter candidates were examined in homogenates of retinal explants cultured in the absence or presence of gizzard extract. After 3 days in culture with gizzard extract, the uptake increased for aspartate and glutamate 1.6- to 1.8-fold and for γ-aminobutyric acid to a lesser degree when examined at a concentration for high-affinity uptake (10^-6M). In contrast, the uptake capacity for glycine, choline, and dopamine was not altered in explants cultured with or without gizzard extract. Kinetic analysis showed that the enhanced capacity to accumulate aspartate was not due to an alteration of K^m, but to an increase of V^max. The results suggest that one or several factors in chick gizzard muscle promote not only neurite outgrowth but also the aspartate-glutamate uptake systems in the developing neuroretina, probably related to ganglion cells.     

Progress in outgrowth culture from rabbit tracheal explants: Balance between proliferation and maintenance of differentiated state in epithelial cells

Summary Primary cultures of rabbit tracheal cells were obtained as outgrowths from explants of tracheal mucosa. A 30% collagen substratum containing serum and minimal essential medium was required for obtaining an outgrowth of epithelial cells keeping their differentiated characteristics. The tracheal epithelial cells obtained near the explant in the first days of culture presented morphologic similarities with normal tracheal epithelium. Cultures contained basal cells and epithelial polarized cells that exhibited apical tight junctions and desmosomes. Ciliated cells stayed functional during all time culture. Their number slightly increased at the beginning of the culture and then stayed constant when the total number of cells increased. Development of the outgrowth was rapid and significant inasmuch as the outgrowth surface reached 30 times that of the explant after less than 8 days. This was linked to cellular proliferation, as demonstrated by the incorporation of bromodeoxyuridine (BrdU) in phase-S nuclei and the revelation of BrdU using an immunofluorescence technique. The epithelial nature of the outgrowth cells and the absence of contamination with fibroblasts were established by positive staining with anti-keratin antibody and by negative staining with anti-vimentin antibody, respectively. This work was supported by DRET and by CIFRE grant awarded to S. R.