Epidermal Cultures Research Articles

Modified epidermal lipid composition in air-exposed culture of non-bullous congenital ichthyotic erythroderma (NBCIE) keratinocytes.

Emerged epidermal cultures on dead de-epidermized dermis (DED) constitute an excellent model for in vitro reproduction of dermatoses linked to a keratinocyte defect. We used such cultures for studies of non-bullous congenital ichthyotic erythroderma (NBCIE). Keratinocytes of normal and pathological origin were expanded in submerged cell cultures and frozen keratinocytes from the resulting cell bank were subsequently used for seeding on DED. Lipid extracts from 14 day emerged cell cultures were assayed qualitatively and quantitatively using thin layer chromatography and compared with the neutral and non-polar lipid profiles obtained from normal epidermis extracts and with those from the plantar stratum corneum of healthy donors and untreated NBCIE patients. The ichthyotic cultures were found to contain significantly elevated levels of n-alkanes, as were the lipid extracts from the patients' plantar horny layer. Our results demonstrate that a major marker of the NBCIE epidermis can be reproduced under the emerged culture conditions. They also indicate that the characteristic n-alkane increase in NBCIE is indeed endogenous and not merely related to possible contamination from topical treatments.

Retinoid-induced beta-adrenergic inhibitory effect associated with increased thymidine incorporation of fetal rat keratinizing epidermal cells (FRSK cells)

Using fetal rat keratinizing epidermal cells (FRSK cells) we investigated the effects of retinoids on the cyclic AMP system. The beta-adrenergic adenylate cyclase response was significantly decreased by the treatment of cells with 1 × 10 −6 M Ro 10-1670. The effect was observed by 12h and remained for at least 48 h. The decreased beta-adrenergic response was accompanied by increased adenosine-and forskolin-induced cyclic AMP accumulations. Thymidine incorporation of FRSK cells was also increased by the retinoid treatment. Northern blot hybridization showed that none of the messages (the beta 2-adrenergic receptor, the stimulatory guanine nucleotide binding protein (Gs-α), or the inhibitory guanine nucleotide binding proteins (Gi-2α, Gi-3α)) were significantly altered by the retinoid treatment. We have already reported that the beta-adrenergic response as well as the beta 2-adrenergic receptor mRNA is increased by the dexamethasone treatment of FRSK cells. The addition of both Ro 10-1670 and dexamethasone in the incubation medium resulted in the loss of the beta-adrenergic augmentation effect by dexamethasone. These results indicate that retinoids decrease the beta-adrenergic response and increase the thymidine incorporation of FRSK cells. This is contrary to the previous results using pig epidermal organ culture system.

Effects of the Selective Protein Kinase C Inhibitor, Ro 31-7549, on the Proliferation of Cultured Mouse Epidermal Keratinocytes

We have investigated the effects of Ro 31-7549, a selective protein kinase C (PKC) inhibitor, on DNA synthesis and proliferation in two primary mouse epidermal keratinocyte culture systems. In differentiating keratinocytes incubated in medium containing 10% serum and high calcium (approximately 0.5 mM), Ro 31-7549 blocked the inhibitory effect of the phorbol ester 12-0-tetradecanoyl-13-acetate (TPA) (a PKC activator) on keratinocyte DNA synthesis at 24 h [50% maximal response concentration (EC50) = 1 microM], consistent with inhibition of PKC-mediated differentiation. Continuous treatment of the differentiative culture system with the PKC inhibitor resulted in a marked (fourfold) stimulation of [3H]thymidine incorporation at day 7 of exposure, with an EC50 of 0.25 microM. The potencies of these effects of Ro 31-7549 are comparable to that reported for inhibition of TPA-induced platelet 47-kD protein phosphorylation [50% inhibitory concentration (IC50) = 4.4 microM]. The time course of [3H]thymidine incorporation indicated that Ro 31-7549 did not directly stimulate DNA synthesis but instead prevented the loss of proliferative capacity associated with continued culture in this medium. Maximal stimulation (2.6 times) of DNA synthesis was observed on day 4, whereas DNA synthesis at day 1 was unaffected. In a highly proliferative culture system using serum-free medium containing 25 microM calcium, TPA dose-dependently inhibited proliferation with an IC50 of approximately 0.3 mM. This antiproliferative effect of TPA was largely reversed by 0.1 microM Ro 31-7549. In the proliferative culture system, 0.75 microM Ro 31-7549 also essentially reversed the inhibition of proliferation caused by switching to high (1.0 mM) calcium. These results suggest that the loss of proliferative capacity in differentiating epidermal keratinocyte cultures may be mediated, at least in part, by PKC.

A Fully Differentiated Epidermis Developed in Vitro

The study of cell lines in tissue culture often allows a more complete understanding of their biochemistry, cell biology, physiology, and role in normal tissue function. As the science of tissue culture has progressed, it has become possible to recreate parts of normal, functioning whole tissues. For example, epidermal culture …

Potassium Channel Openers Stimulate DNA Synthesis in Mouse Epidermal Keratinocyte and Whole Hair Follicle Cultures

We have conducted studies using primary mouse epidermal keratinocyte and whole hair follicle cultures to investigate the mechanism of the hypertrichotic activity of potassium channel openers. In a time course study, the extent of stimulation of epidermal keratinocyte DNA synthesis by minoxidil increased as the rate of DNA synthesis in control cultures declined. Minoxidil stimulation of DNA synthesis in 7-day cultures required prolonged (> 1 day) exposure to the agent. Pinacidil and diazoxide also stimulated DNA synthesis in mouse epidermal keratinocyte cultures. In addition, minoxidil, pinacidil, diazoxide, and cromakalim stimulated DNA synthesis in whole-organ cultures of mouse hair follicles. These results suggest that potassium channel openers retard the loss of proliferative activity of differentiating keratinocytes and support the hypothesis that these agents stimulate hair growth through a direct effect on hair follicles.

Lateral growth and terminal differentiation during repeated epidermal regeneration in vitro. Age dependence and modulation by cholera toxin.

By incubating multilayered primary cultures of human epidermal keratinocytes in a low calcium medium, the suprabasal layers can be stripped off leaving a basal cell monolayer. When this monolayer is refed normal calcium medium a reproducible series of cell kinetic, morphological and biochemical changes take place resulting in the regeneration of a multilayered tissue. The stripping procedure seems to induce the selective proliferation of a cohort of basal cells that is committed to vertical migration and rapid terminal differentiation. In contrast, when the basal cells are allowed to regenerate in the presence of the strong mitogen, cholera toxin, lateral growth and continued proliferation are favoured at the expense of the capacity of the cells to differentiate. Repeated stripping of the same cultures disclosed a considerable heterogeneity in the capacity of the basal cells to regenerate the suprabasal layers. The number of times the basal cells could restore the suprabasal layers after repeated stripping varied from four to nine times. A negative correlation between donor age and regenerative capacity was observed. The experiments with repeated stripping of the same cultures also showed that the capacity to proliferate and to restore the multilayering was fully retained for at least four cycles of stripping-regeneration, whereas the capacity to terminally differentiate was rapidly lost. It is suggested that the present system of regenerating epidermal tissue cultures may serve as an experimental model for the study of epidermal tissue homeostasis and cellular aging.

The effect of 2,2′-dichlorodiethyl sulfide on DNA synthesis of a murine stratified keratinocyte culture system

A primary stratified keratinocyte culture resembling the epidermis in situ was used as a model for studying the effects of exposure to 2,2′-dichlorodiethyl sulfide, or sulfur mustard (SM), on DNA synthesis. A method that distinguishes between semi-conservative (s.c.) DNA synthesis and repair synthesis was used to determine if the former was inhibited following treatment with SM. In this method the density of the newly synthesized DNA was increased by incorporation of 5-bromo-2-deoxyuridine. Density gradient centrifugation was then used to isolate the heavy DNA for quantification. It was demonstrated that topically applied SM in the dose range of 1–10 nmole/cm 2 inhibited s.c. DNA synthesis (replication) in a dose and time related manner. Inhibition of DNA replication by SM would result in inhibition of cell division which must be preceded by s.c. DNA synthesis. This failure to replace damaged germinative cells may lead to the destruction of the basal layer which is observed in vivo and in our epidermal culture following exposure to SM. This may also be related to development of vesication observed in exposed intact human skin.

Epidermal growth factor affects both glia and cholinergic neurons in septal cell cultures

The effects of epidermal growth factor on high density primary cultures of fetal (embryonic day 17) rat septal cells were examined. Under serum-free conditions, the continuous exposure of these cultures to epidermal growth factor for seven days significantly decreased choline acetyltransferase (EC 2.3.1.6) activity in a dose-dependent manner. Maximal decreases were observed from 1 to 10 ng/ml epidermal growth factor. This effect was completely abolished by the addition of anti-epidermal growth factor antibodies. The epidermal growth factor-mediated decrease in choline acetyltransferase activity was culture-time dependent, being first detectable after five days of factor application and may likely represent an inhibition of the spontaneous increase in enzyme activity that occurs with time in culture. Concomitant with changes in enzyme activity, epidermal growth factor produced a significant and proportional decrease in the number of acetylcholinesterase-positive neurons. This decrease in acetylcholinesterase-positive cells did not reflect a decrease in cholinergic cell survival as nerve growth factor could restore the number of acetylcholinesterase-positive neurons in epidermal growth factor-treated cultures to control levels. Furthermore, in these high-density cultures, epidermal growth factor did not affect general neuronal survival, while it did produce an increase in the number and intensity of glial fibrillary acidic protein-immunoreactive astroglia as well as in the number of macrophage-like cells. The proliferative response of these non-neuronal cells to epidermal growth factor, as assessed by [ 3H]thymidine incorporation, was evident after three days of epidermal growth factor application, persisted thereafter, and could be antagonized by the inclusion of the antimitotic 5-fluorodeoxyuridine. Furthermore, 5-fluorodeoxyuridine completely blocked the epidermal growth factor-mediated decrease in choline acetyltransferase activity. However, when epidermal growth factor was tested in pure glial cultures, it only directly induced proliferation of astrocytes. These results suggest that the proliferative response of either one or both of these glial cell types in the mixed cultures may be indirectly affecting cholinergic cell expression.

Serial cultivation of normal human keratinocytes: a defined system for studying the regulation of growth and differentiation.

We have developed a defined method for human epidermal keratinocyte culture. The minimally supplemented basal medium supported establishment of primary cultures from neonatal foreskin in a defined environment. It also supported serial cultivation and rapid expansion of cell number. Casein replaced serum for defined cryopreservation. Cells were serially cultivated in medium containing 0.08 mM calcium. The rate of cell division however remained high after addition of 1.8 mM calcium. The particulate transglutaminase activity of the cultures was low at confluence, even in the presence of 1.88 mM calcium, indicating an enrichment of the basal cell population. Culture with small amounts (0.3%) of chelated serum increased particulate transglutaminase activity approximately 2.2-fold in low calcium cultures and approximately 3.5-fold in high calcium cultures. A gradual reduction in growth rate of serum-treated cultures upon serial cultivation also indicated a depletion of cells with basal cell character. Bovine hypothalamic extract and cholera toxin were able to avert, in part, the differentiation-promoting effects of serum. Keratinocytes serially cultivated in the defined medium maintained the ability to develop normally into a morphologically differentiated epidermis.

Aflatoxin toxicity in cultured human epidermal cells: stimulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Aflatoxin B1 (AFB1) at 1 microgram/ml was markedly toxic to human epidermal cells grown in the Rheinwald-Green 3T3 feeder layer system. At 0.1 microgram/ml, the toxicity was barely evident, as assessed by colony expansion during a 2 week exposure, but it was dramatically stimulated by 5 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which was non-toxic alone. Neither AFB1-dihydrodiol nor AFB2 were toxic in the presence or absence of TCDD, indicating that metabolism to the 8,9-epoxide was responsible for the AFB1 toxicity. Stimulation of AFB1 epoxidation by TCDD was also indicated by the > 20-fold increase in DNA adduct formation in cultures exposed to [14C]AFB1 and TCDD for 4 days as compared to AFB1 alone. Analysis of free metabolites in culture medium by reverse-phase HPLC revealed that confluent epidermal cultures metabolized AFB1 to AFM1, AFB2a and aflatoxicol. In the presence of TCDD, the levels of AFM1 were higher (14 versus 3% of dose) as were those of AFB2a (3 versus 0.5% of dose), while aflatoxicol levels were lower (0.8 versus 2% of dose). In the absence of irradiated 3T3, the toxicities of AFB1, AFB2, AFM1 and aflatoxicol to cells in serum-free medium (0.15 mM Ca2+) were similar to those in the feeder layer system. Although this moderately low calcium concentration appeared quite satisfactory for observing toxicity, the response was attenuated at a lower calcium concentration (0.09 mM Ca2+).

Changes of lysosomal proteinase activities and their expression in rat cultured keratinocytes during differentiation

The cathepsins B, H and L, lysosomal cysteine proteinases, play a major role in intracellular protein degradation. These proteinase activities and expressions were examined in a Ca 2+ regulated epidermal culture system which consists of two morphological cell types: undifferentiated cells grown in low Ca 2+ (0.1 mM concentration) and differentiated cells grown in high Ca 2+ (1.8 mM concentration), respectively. Cathepsin B and L activities of the differentiated cells showed a several-fold increase compared to that of the undifferentiated cells. In addition, by using CM-cellulose column chromatography, cathepsin B and L were separated and the level of cathepsin L activity increased significantly. Cathepsin B, L and H were also detected by using an immunoblotting procedure in which their bands were expressed after differentiation was induced by the increasing calcium concentration. Cathepsin L activity and immunostaining intensity reached a maximum at 1 or 2 days of differentiation. In contrast, cystatin α (an endogenous inhibitor of cysteine-dependent cathepsins) appeared in the final stage of differentiation. These results indicate that the expression of epidermal cathepsins and their endogenous inhibitor are involved in part of the program of cell differentiation and the terminal differentiation process in cultured rat keratinocytes.

Transforming growth factor alpha induces collagen degradation and cell migration in differentiating human epidermal raft cultures.

When cultured on plastic and treated with transforming growth factor alpha (TGF alpha), human keratinocytes exhibit an increase in proliferation at the colony periphery, apparently as a consequence of enhanced cell migration (Barrandon and Green, 1987). To investigate the effects of TGF alpha on a differentiating stratified squamous epithelium and to begin to examine the molecular basis mediating this influence, we cultured human epidermal cells on a gelled lattice of collagen and fibroblasts, floating on the air-liquid interface. Under these conditions, raft cultures differentiate and exhibit morphological and biochemical features of human skin in vivo (Asselineau et al., 1986; Kopan et al., 1987). When 3-wk-old raft cultures were treated with TGF alpha, basal cells showed a marked increase in cell proliferation. At elevated concentrations of TGF alpha, the organization of cells within the artificial tissue changed and islands of basal cells entered the collagen matrix. Biochemical analysis of the response revealed that type I collagenase and gelatinase were induced by keratinocytes within 12 h after TGF alpha treatment. In contrast, invasion of basal cells into the collagen matrix was not significant until 48-72 h post-treatment, suggesting that collagenase and gelatinase production may be a prerequisite to this phenomenon. These results have important implications for the possible role of TGF alpha in squamous cell carcinoma and tumor invasion.

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

Abstract 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 post-stripping 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 overex-pressed 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.

Barrier Function of Human Keratinocyte Cultures Grown at the Air-Liquid Interface

Stratum corneum (SC), the outermost and least permeable layer of skin, is the major barrier to passive transepidermal water loss. In the research described in this paper, we have used human keratinocyte cultures, grown at the air-liquid (A/L) interface, to examine the relationship between epidermal differentiation (including SC formation) and barrier function. Histologically, the A/L culture showed several markers of complete differentiation, including the presence of well-organized and defined epidermal cell layers, keratohyalin granules, and a multilayered SC. The permeability of tritiated water through epidermal cultures, which had grown for 3 weeks at the A/L interface, was measured with a microdiffusion apparatus. The results of these experiments demonstrated that: a) the human keratinocyte cultures developed a substantial barrier (i.e., a multilayered SC) to water diffusion across the entire surface. If the relative humidity of the culturing environment was lowered from 100% to around 75%, the barrier was significantly improved; b) the differentiation promoter, 1.25-dihydroxy-vitamin-D3, increased the number of SC layers and reduced water permeation through the culture; c) the nature of the keratinocyte support matrix could be altered to improve the morphology as well as the barrier function of the epidermal cultures. Overall, the observations are consistent with the relationship that is believed to exist between SC intercellular lipid content and percutaneous penetration. Confirmation of this hypothesis will further the considerable potential of human keratinocyte A/L cultures as a valuable and relevant model in which to study drug absorption and metabolism.

Characterization of 230-kD Bullous Pemphigoid Antigen Associated with Detergent-Insoluble Fraction of Cultured Keratinocytes

The 230-kD protein identified by antibodies from patients with bullous pemphigoid (BP) has a dual location in cultured normal human epidermal keratinocytes: part in a high-speed supernatant of homogenized cells and part in a particulate fraction, where it is resistant to extraction by non-ionic detergent or mild base. Antibodies were affinity purified from the particulate 230-kD BP antigen, which can be extracted in the presence of urea. The affinity-purified antibodies bind not only the cytosolic 230-kD protein, showing that it is related, if not identical, to the particulate form, but also produce a discontinuous granular pattern by indirect immunofluorescence in the basement membrane zone of rabbit esophagus. In stratifying epidermal cultures, expression of the 230-kD BP antigen is limited to basal cells. These data are consistent with 230-kD BP antigen involvement in keratinocyte basal cell interaction with extracellular matrix and indicate that the cultured cell may provide a useful model for analysis of 230-kD BP antigen function.

Two classes of continuous cell lines established from Syrian hamster 9 day gestation embryos: Preneoplastic cells and progenitor cells

Primary cultures of 9-d-gestation Syrian hamster embryo (E9) cells are distinct from primary cultures of later gestational age in terms of their growth and differentiation. First, primary E9 cell cultures express multiple mesenchymal differentiation lineages (e.g., adipocyte, myoblast) only rarely seen in cultures of 13-d-gestation fetal (F13) cells. Second, although most primary E9 cultures have a limited in vitro proliferative life span and exhibit cellular senescence similar to primary cultures of F13 cells, E9 cultures seem to have higher frequency of escape from senescence and conversion to continuous cell lines compared to F13 cells. Moreover, this frequency can be further increased 4- to 5-fold by continuous exposure of the E9 cells to tumor promoters or epidermal growth factor. Eleven continuous cell lines have been isolated from untreated, promoter-treated, or epidermal growth factor-treated primary E9 cultures. Seven of these are neoplastic or preneoplastic. However, the remaining four do not show any evidence of being in neoplastic progression and three of these continue to express the same differentiated phenotype observed in ther parental primary cell cultures.

Effects of extra- and intracellular calcium concentration on DNA replication, lateral growth, and differentiation of human epidermal cells in culture

Variation in the extra- and intra-cellular concentration of calcium ([Ca]e and [Ca]i) affected the 3H-thymidine labeling pattern of sorted S-phase cells in human epidermal cultures. A lowering of [Ca]e resulted in retarded lateral growth but, unless [Ca]e was extremely low, caused an increase in the proportion of strongly labelled (rapidly cycling) S-phase cells. An increased desquamation of superficial cells due to a reduced cellular cohesiveness was also observed in low calcium medium. Thus, a lowering of [Ca]e might stimulate the proliferation of a pool of cycling cells destined for rapid terminal differentiation and tissue regeneration, whereas proliferation destined for lateral growth is inhibited. Attempts to decrease the [Ca]i with the calcium chelator quin-2 at low [Ca]e seemed to elevate the proportion of strongly labelled S-phase cells, whereas an increased [Ca]i obtained with the ionophore A23187 caused a dramatic decrease in the proportion of S-phase cells that showed strong 3H-thymidine incorporation. This implies that variation in both [Ca]i and [Ca]e may play a role in the regulation of proliferation and differentiation, in keratinocytes.

Synthetic epidermal pentapeptide and related growth regulatory peptides inhibit proliferation and enhance differentiation in primary and regenerating cultures of human epidermal kératinocytes

A pentapeptide that inhibits proliferation of mouse epidermal keratinocytes in vivo and in vitro has been purified from mouse skin extracts. In the present study the effect of a synthetic analog of the epidermal pentapeptide on proliferation and differentiation of cultured human epidermal keratinocytes was investigated. In young, rapidly growing primary cultures the pentapeptide caused a dramatic decrease in mitotic activity and also induced pronounced changes in the balance between kinetically defined subpopulations of proliferating cells. A dipeptide derived from the pentapeptide was found to be at least as potent. A serine derivative of a hemoregulatory peptide also seemed to be active. When tested in epidermal cultures regenerating after removal of the suprabasal cell layers, both the pentapeptide and the dipeptide were shown to cause a delay in the proliferative response. Both peptides were also able to stimulate early (increase in cell size) and late (cornified envelope formation) events in the differentiation pathway of the keratinocyte. The apparent stimulatory effect on differentiation was most clearly seen in regenerating cultures, whereas the effect on primary cultures varied with the experimental set-up. It is suggested that homologous epidermal peptide(s) may play a major role in the regulation of human epidermal homeostasis.

Growth of Melanocytes in Human Epidermal Cell Cultures

Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin. Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages. Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli. When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient.

Proliferation and differentiation of cultured epidermal cells from patients with X-linked ichthyosis and ichthyosis vulgaris.

The growth, differentiation, and regeneration of epidermal cultures from patients with X-linked and autosomal dominant ichthyosis and normal individuals were compared. Cell proliferation was studied by combining the technique of fluorescence-activated cell sorting with [3H]thymidine labelling and autoradiography. As in normal epidermal cultures, a marked heterogeneity in the labelling intensity of S-phase cells was observed in the ichthyotic cultures with totally unlabelled as well as very strongly labelled cells. However, in contrast to normal cultures, by far the largest proportion of S-phase cells in the ichthyotic cultures were very strongly labelled with a corresponding, severe reduction in the proportion of un- and weakly labelled cells. The increased labelling intensity of S-phase cells was observed in primary as well as in regenerating cultures, although it was most pronounced in the latter case. There was no difference between cultures from the two types of ichthyotic skin. The morphologic differentiation in the cultures was assessed by measurement of mean diameter of sorted S-phase cells and by quantitation of cornified envelop formation. Both parameters were reduced in the ichthyotic cultures, compared with normal ones. Taken together, these findings are indicative of a hyperproliferative state in the ichthyotic cultures.