3T3 Feeder Layer Research Articles

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+).

Effect of Retinoids on Hyperproliferation-Associated Keratins K6 and K16 in Cultured Human Keratinocytes: A Quantitative Analysis

The keratin patterns of human epidermal keratinocytes cultured on a 3T3-feeder layer in the presence of 10(-8) M non-aromatic (all-trans retinoic acid and 13-cis retinoic acid) and polyaromatic (arotinoid, arotinoid-sulfone, and free arotinoic-acid) retinoids were analyzed by high resolution one- and two-dimensional gel electrophoresis and immunoblotting. Laser densitometric evaluation of one-dimensional gels allowed to quantitate the changes within the keratin patterns and revealed an increase in the expression of keratins K13, K15, and K19 as induced by both non-aromatic and polyaromatic retinoids, except for the parent compound arotinoid. This would then indicate that such keratinocytes are pursuing a more embryonic type of differentiation. In evaluating the data for the hyperproliferation-associated keratins K6 and K16 we noticed an unexpected result: except for all-trans retinoic acid, these two keratins showed opposite responses. As compared to control cultures, the amount of K6 did generally increase, while K16 was reduced, with arotinoid acid being the most effective retinoid. The apparently uncoupled expression of K6 and K16 appeared also to be concentration dependent when 13-cis retinoic acid at concentrations of 10(-9), 10(-8), and 10(-7) M was analyzed. Considering the overall antiproliferative potency of retinoids, we therefore conclude that K16 alone, rather than the pair K6/K16, should be regarded as a proliferation-related keratin and as such may be used as a sensitive marker to evaluate keratinocyte proliferation.

Cytokeratins as molecular markers in the evaluation of the precise differentiation stage of human gingival epithelium reconstituted in vitro

Cytokeratins are considered to be molecular markers for different types of epithelial differentiation. They were used to investigate the precise differentiation stage of gingival epithelium, reconstituted in vitro, following two different culture procedures. Human trypsin-dissociated gingival keratinocytes were seeded either on a feeder layer of irradiated mouse 3T3 fibroblasts or on a connective tissue equivalent (lattice) made up of human fibroblasts in a collagen gel. The cytokeratins were extracted and analysed by two-dimensional gel electrophoresis. Although both methods showed on histological sections that cultured gingival keratinocytes formed a multilayered non-keratinizing epithelium, the cytokeratins patterns showed great differences. The gingival epithelium-like structure reconstituted on 3T3 feeder layer expressed some cytokeratins characteristic of the in situ gingival epithelium (K 5, 6, 14, 16, 17) and some which do not exist in the normal tissue (K 8, 18, 19, traces of K 13 and K 15) and are specific for embryonic, simple and tumour epithelia. However, the gingival epithelium reconstituted on connective tissue equivalent expressed all the cytokeratins present in the normal tissue (K 5, 6, 14, 16, 17), except those specific for terminal differentiation (K 1, 2, and 10/11). These findings suggest that the culture of gingival keratinocytes on connective tissue equivalents allows them to reproduce physiological stages of differentiation.

Relationship of sebaceous cell stage to growth in culture.

The maturational stage at which sebaceous epithelial cells are irreversibly committed to autolysis has not been determined in a biologic system. The purpose of these experiments was to determine the relationship of sebocyte maturation to their ability to grow in culture. Single cell suspensions from adult rat preputial glands were prepared, and sebocyte stages were classified using lipid staining as one criterion of maturation. Cells were subjected to one-step, isokinetic discontinuous density gradient centrifugation in Percoll. Cells were cultured on 3T3 feeder layers in an epithelial cell medium containing 20% fetal calf serum, growth factors, and antibiotics. Growth in culture occurred from sebocytes in all gradient fractions. The least growth was found from cells in the lightest density fraction (1.020), which contained sebocytes with the most lipid, i.e., the most mature. Over fivefold more growth occurred from cells in the most dense fraction (1.080), which contained undifferentiated and immature sebocytes. Cell growth in culture was then correlated with the type of sebocyte in each fraction plated. Although cell growth in culture correlated significantly with the number of undifferentiated cells (r2 = 0.460), epithelial colonies were found in the absence of discernible undifferentiated cells in most fractions. Growth in culture correlated much better with the number of early plus mid-differentiated sebocytes (r2 = 0.702). These data suggest that, whereas mature sebocytes are not capable of attachment/proliferation, early differentiation of sebocytes is compatible with retention of the capacity to proliferate. If this property is shared by human sebocytes, it would contribute to the intransigence and diversity of acne-form lesions.

Relationship of sebaceous cell stage to growth in culture

The maturational stage at which sebaceous epithelial cells are irreversibly committed to autolysis has not been determined in a biologic system. The purpose of these experiments was to determine the relationship of sebocyte maturation to their ability to grow in culture.Single cell suspensions from adult rat preputial glands were prepared, and sebocyte stages were classified using lipid staining as one criterion of maturation. Cells were subjected to one-step, isokinetic discontinuous density gradient centrifugation in Percoll. Cells were cultured on 3T3 feeder layers in an epithelial cell medium containing 20% fetal calf serum, growth factors, and antibiotics.Growth in culture occurred from sebocytes in all gradient fractions. The least growth was found from cells in the lightest density fraction (1.020), which contained sebocytes with the most lipid, i.e., the most mature. Over fivefold more growth occurred from cells in the most dense fraction (1.080), which contained undifferentiated and immature sebocytes. Cell growth in culture was then correlated with the type of sebocyte in each fraction plated. Although cell growth in culture correlated significantly with the number of undifferentiated cells (r2= 0.460), epithelial colonies were found in the absence of discernible undifferentiated cells in most fractions. Growth in culture correlated much better with the number of early plus mid-differentiated sebocytes (r2=0.702).These data suggest that, whereas mature sebocytes are not capable of attachment/proliferation, early differentiation of sebocytes is compatible with retention of the capacity to proliferate. If this property is shared by human sebocytes, it would contribute to the intransigence and diversity of acne form lesions.

Differentiation of Cultured Epithelial Cells: Response to Toxic Agents

Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Great differences were evident even among those cells derived from stratified squamous epithelia (epidermal, esophageal, vaginal, forestomach) despite their expression of aryl hydrocarbon hydroxylase activities to similar degrees. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAMP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Although expressing keratinocyte character (transglutaminase activity and envelope forming ability), the cells thus retain some hormonal character that may be modulated by cAMP-dependent kinase activity. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents.

Differentiation of cultured epithelial cells: response to toxic agents.

Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Great differences were evident even among those cells derived from stratified squamous epithelia (epidermal, esophageal, vaginal, forestomach) despite their expression of aryl hydrocarbon hydroxylase activities to similar degrees. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAMP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Although expressing keratinocyte character (transglutaminase activity and envelope forming ability), the cells thus retain some hormonal character that may be modulated by cAMP-dependent kinase activity. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents.

Clone-forming ability and differentiation potential of migratory neural crest cells.

The neural crest of vertebrate embryos gives rise to a variety of differentiated cell types, including neuronal and non-neuronal cells of peripheral ganglia (sensory and autonomic), pigment cells, and mesectodermal derivatives. Neural crest cells were taken from quail embryos at the level encompassing mesencephalon and metencephalon and the developmental potentials were evaluated by culturing them as single cells on 3T3 feeder layers. Such conditions proved to be particularly favorable for survival, proliferation, and differentiation of quail neural crest cells. Two hundred and forty-three clones that contained from 1 to greater than 20,000 cells were analyzed after 7-10 days of culture. Phenotype analysis provided evidence for the existence of cells with extremely diverse developmental potencies. A few committed neuron progenitors were observed as well as some pluripotent cells, able to differentiate into several types of neurons, non-neuronal cells, and melanocytes, and many cells with intermediate developmental potencies. These cloning experiments revealed the striking heterogeneity of migrating neural crest cells in terms of their capacity for differentiation and their potential for proliferation.

Density-Dependent Variations in the Lipid Content and Metabolism of Cultured Human Keratinocytes

Cultured human foreskin keratinocytes have been utilized extensively to study modulations in protein content during epidermal differentiation. In this study we examined their usefulness as a model system for differentiation-linked changes in lipid content and metabolism. First-to-third passage keratinocytes were grown in 10% fetal calf serum on a mitomycin-treated 3T3 feeder layer and harvested at intervals before, during, and after reaching confluence for determination of lipid, protein, and DNA content. Lipid synthesis, determined as acetate incorporation into lipid, was most active in pre-confluent cultures and at all times closely paralleled the growth activity of the cultures. Post-confluent cultures were characterized by an increase in total lipid content and by increased triglyceride content and synthesis. Pulse-chase studies demonstrated that labeling of the triglyceride pool was labile and suggested that even in post-confluent cultures, triglycerides provide a fatty acid reservoir for phospholipid biosynthesis. A novel band, which co-migrated with monoalkyldiacylglycerol in two solvents systems was present in confluent and post-confluent cultures, but absent in pre-confluent cultures. Sphingolipids constituted less than 10% of total lipid at all stages of growth, and cholesterol sulfate was present only in small quantities. These studies illustrate the relationship of lipid synthesis to growth and demonstrate that human foreskin keratinocytes, cultured under standard conditions, reproduce incompletely the lipid composition of epidermis in vivo.

Organotypic culture of outer root sheath cells from human hair follicles using a new culture device.

A new culture vessel for the growth of cells on biological substrata and under organotypic conditions is described. This device, named Combi-ring-dish (CRD), is composed of four concentric rings designed to take up one or several substrata on which cells can be grown either immersed in culture medium or exposed to air and fed from underneath. Using the CRD, outer root sheath cells, isolated from plucked human hair follicles and plated on growth-arrested 3T3 feeder layers, were grown on native collagen lattices populated with living human fibroblasts. After reaching confluence, the immersed cultures were recombined (in vitro) with pieces of freeze-killed dermis and grown further, exposed to air. Thus by mimicking epidermal growth conditions, differentiation was dramatically improved, compared to control cultures on plastic substratum. Virtually all morphologic features of interfollicular epidermis developed. This seems a suitable model to investigate the differentiation potential of human hair follicle cells.

The effect of donor age on the proliferative response of human and mouse keratinocytes to phorbol, 12-myristate, 13-acetate

Epidermal keratinocytes grow clonally when provided with a 3T3 feeder layer and medium supplemented with 20% foetal bovine serum, hydrocortisone and cholera toxin. In this culture system the proliferative response of freshly isolated human and mouse keratinocytes to a short exposure (24 h) of the tumour promoter phorbol, 12-myristate, 13-acetate (PMA) was dependent on the donor age but was independent of the species or the biopsy site. Human keratinocytes from early (16-18 week) foetal donors displayed a strong proliferative response to PMA as determined by a 5- to 7-fold increase in colony number and an increase in the average colony size. In contrast, adult and juvenile keratinocytes of all ages from both mice and humans displayed an inhibition of colony forming efficiency and a reduction in colony size. When continuously passaged in culture human foetal keratinocytes gradually changed the pattern of their response to PMA so that they were inhibited from growing rather than being stimulated and after 21 days (three passages) their response was quantitatively similar to juvenile keratinocytes. Co-culture of juvenile keratinocytes with irradiated foetal keratinocytes did not alter their response to PMA so that the observed proliferative response of foetal keratinocytes to PMA is not readily explained by the autosecretion of mitogens or other regulatory molecules by these cells. Late foetal (17 days gestation), neonatal and post-neonatal (5-10 days old) mouse keratinocytes were also inhibited from growing by PMA but the magnitude of the effect was directly related to the age of the mouse and was in all cases less than that observed with adult mice. The relationship of these results to the mechanism of action of phorbol esters in epidermis is discussed.

Serial Cultivation of Single Keratinocytes from the Outer Root Sheath of Human Scalp Hair Follicles

A method for the isolation of outer root sheath keratinocytes from plucked human hair follicles and for their subsequent cultivation has been developed. The selective trypsinization of outer root sheath keratinocytes provided a single cell suspension of defined origin within the hair follicle. The 3T3 feeder layer technique supports sustained growth of these cells in that as little as one single plucked hair follicle (yielding approximately 1.5 X 10(4) cells) consistently gave rise to a confluent 35-mm culture dish (with approximately 1.5 X 10(6) cells) within about 2 weeks. The outer root sheath keratinocytes can be serially passaged for up to 3 times and also cryopreserved.

Synthesis of cellular and extracellular glycoproteins by cultured human keratinocytes and their response to retinoids

The glycoproteins synthesized by human keratinocytes cultured on 3T3 feeder layers were studied by metabolic labelling. Keratinocytes freed of feeder cells synthesized a complex pattern of cellular and extracellular glycoproteins that was distinct from that of 3T3 cells, dermal fibroblasts and epidermal melanocytes. The effect of low concentrations of all- trans-retinoic acid and arotinoid ethyl ester on glycoprotein synthesis was examined in keratinocyte cultures depleted of vitamin A. Treatment with either retinoid resulted in a 2–3-fold increase in the amount of d-[ 3H]glucosamine-labelled material in the culture medium. Gel electrophoresis revealed increased incorporation of d-[ 3H]glucosamine into extracellular glycoproteins of M r 245 000, 170 000, 140 000, 130 000, 120 000 and 105 000 as well as into glycosaminoglycans in retinoid-treated cultures. The labelling of extracellular glycoproteins with l-[ 3H]leucine and l-[ 35S]methionine was also increased by retinoids suggesting increased synthesis of these components rather than an effect on their glycosylation. The M r 245 000 glycoprotein was identified as keratinocyte-derived fibronectin by immunoblotting, immunoprecipitation and specific binding to gelatin. The results show that retinoids increase the synthesis of glycoprotein as well as glycosaminoglycan components of the extracellular matrix in human keratinocyte cultures. It is suggested that retinoids select for a population of cells that synthesize relatively large amounts of glycosaminoglycan, fibronectin and other as yet unidentified extracellular glycoproteins.

Changing responsiveness to all-trans retinoic acid of rat tracheal epithelial cells at different stages of neoplastic transformation.

Retinoic acid (RA) treatment of rat tracheal epithelial (RTE) cells, pre-exposed to the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), inhibited transformation in a dose-dependent manner. Treatment with RA at concentrations ranging from 3-33 nM reduced the MNNG-induced transformation frequencies by 13-81% and in some experiments by greater than 90%. RA treatment for only 3 days caused 65-75% inhibition of transformation; treatments of longer duration resulted in greater inhibition of transformation. Delaying the onset of RA treatment reduced its effectiveness, but even when RA treatment was delayed for 3 weeks following MNNG exposure, 60% inhibition still occurred. The inhibition of transformation appeared to be irreversible. The colony forming efficiency of cells isolated from transformed colonies 5 weeks after MNNG exposure was drastically reduced when the replated cells were treated with RA either 1 day or 4 days after plating, indicating that RA blocked cell replication. However, cells isolated from transformed colonies at later times after MNNG exposure were increasingly resistant to the antiproliferative effects of RA. The RA concentration causing 50% inhibition (RA-IC50) of colony formation was 0.1-0.3 nM for cells isolated from 3-5 week-old transformed colonies; it increased greater than 100-fold for cells isolated from 12-week-old transformants. Five established RTE cell lines also showed a much increased resistance to the antiproliferative effects of RA; two of these cell lines were even slightly stimulated in their colony forming ability by RA. The RA-IC50 of colony forming efficiency of normal RTE cells was also determined and compared to that of cells isolated from 5-week-old transformed colonies. Since the normal RTE cells require 3T3 feeder layers for clonal growth, both cell types were grown on feeders. For both cell types, the RA-IC50 was similar (150-300 nM); the requirement for relatively high RA concentrations was attributed in part to the rapid RA metabolism by the feeder cells. These experiments show that early RTE cell transformants are growth-inhibited by RA; however, they increasingly lose their sensitivity to the growth controlling effects of RA as they progress to a more advanced stage of transformation. The inhibition of tracheal cell transformation by RA is probably due to the antiproliferative effects of the retinoid.

Profilaggrin, a High-Molecular-Weight Precursor of Filaggrin in Human Epidermis and Cultured Keratinocytes

Filaggrin is a histidine-rich matrix protein of keratinized epidermis. Filaggrin is synthesized as a high-Mr, phosphorylated precursor, profilaggrin, that is processed to form the lower-Mr product present in cornified cells. This study reports the identification of profilaggrin in human epidermis and in unusually well-differentiated cultured human keratinocytes with the use of a polyclonal antihuman filaggrin antiserum. Polyclonal antiserum raised against human filaggrin stained keratohyaline granules and stratum corneum in tissue sections of human skin. Analysis of epidermal extracts showed an immunoreactive low-Mr band (37,000), previously identified as filaggrin, and an immunoreactive high-Mr band (greater than 220,000). Both [32P] phosphate and [3H]histidine were incorporated into the high-Mr band after pulse labeling for 3 h. After a 15-h chase, [3H]histidine, but not [32P]phosphate appeared in filaggrin. Human foreskin keratinocytes cultured on a 3T3 feeder layer were unusually well differentiated. Numerous well-formed keratohyaline granules, complete desmosomes, lamellar granules, and cornified cell envelopes were observed. Immunofluorescence with antihuman filaggrin antiserum showed a granular staining pattern in the more stratified cells. Extracts of cultured cells contained a diffuse high-Mr immunoreactive band but no immunoreactive equivalent of filaggrin. These studies suggest that human skin filaggrin, like rodent filaggrin, is synthesized as a high-Mr, phosphorylated, histidine-rich precursor (profilaggrin) that is processed via posttranslational modification to filaggrin. In human keratinocyte cultures a similar high-Mr precursor is present, but evidence of processing to the lower-Mr product, filaggrin, is lacking.

Estrogen receptor expression in serially cultivated rat endometrial cells: stimulation by forskolin and cholera toxin.

Serially propagated with 3T3 feeder layer support, epithelial cells derived from normal rat endometrium expressed estrogen receptor activity. Specific binding of 17-beta-estradiol was in the range of 30-60 fmol/mg of protein and was of high affinity (Kd = 0.3 nM). A survey of cell lines derived from several other normal epithelia showed that rat vaginal and human cervical cultures also had high-affinity estrogen receptors (6-13 fmol/mg of protein), while rat epidermal and esophageal cells had no detectable activity. In the endometrial cultures, receptor levels were elevated nearly two- to fourfold by cholera toxin or forskolin in the medium. This effect was detectable after 4 hr but not 1 hr of treatment and did not occur in the presence of cycloheximide. We conclude that serially cultivated rat endometrial cells retain hormonal properties expressed in vivo while exhibiting some keratinocyte character. These cells may provide a useful model for study of receptor modulation.

Optimised growth of human epidermal cells in vitro without the use of a feeder layer or collagen substrate.

A culture method has been developed which enables a reasonably large quantity of human epidermal cells to be grown in vitro without the use of irradiated 3T3 feeder layers or collagen substrates. These cultures become confluent within 2 or 3 weeks of initiation, and may be sub-cultured several times over their 16-week average lifespan provided that the calcium levels in the cultured medium are controlled. Some of the potential uses of these cultures are briefly discussed.

Cultured epidermal autografts

In the early 1950s Billingham and Reynolds 1 demonstrated that epidermal sheets and epidermal cell suspensions obtained after trypsinization could be used to cover superficial wounds in experimental animals. Subsequent experiments in the late 1960s and early 1970s by Karasek. 2 Karasek and Charlton, 3 Freeman and co-workers, 4 and Igel et al. 5 proposed that epidermal cells grown in tissue culture could feasibly be transplanted onto wounds and that such cells would continue to grow and differentiate in vivo. In the late 1970s, Rheinwald and Green 6 and Green et al. 7 developed a system of culturing difficult-to-grow keratinocytes on lethally irradiated fibroblasts derived from a well-known mouse line (3T3). This technique for growing keratinocytes in culture made it possible to successfully grow and expand relatively small numbers of these cells into multilayered epithelium suitable for grafting. Growing keratinocytes in the absence of feeder layers, collagen substrates, or adhesive glycoproteins was achieved in 1979 by Eisinger and colleagues. 8 Keratinocytes could be grown into differentiated multilayered epidermal sheets provided that the pH of the culture medium was decreased (≅ 5.6) and the seeding density was optimal (2.5 × 10 5 cells/cm 2). In 1980, Eisinger et al. 9 demonstrated that cultured epidermal autografts could be used to cover deep partial-thickness wounds on dogs, pigs, and mice. Although it has been demonstrated that cultured epidermal autografts can be successfully transplanted onto partial-thickness wounds, there is very little, if any, objective data evaluating their potential effect on the healing process. This study was undertaken to evaluate objectively and to compare (1) the effect of epidermal autografts (grown on 3T3 feeder layers or on plastic) and wound occlusion on the healing of controlled partial-thickness wounds and (2) the effect of epidermal autografts. a collagen matrix (with or without keratinocytes), and occlusion alone on the healing and contraction of full-thickness wounds in swine.

Differentiation specific functions in cultured and transplanted mouse keratinocytes:: Environmental influences on ultrastructure and keratin expression

Keratinocytes from neonatal mouse back skin, growing in primary culture (PEC) under conventional conditions (immersed), exerted a reduced programme of differentiation as indicated by cell morphology and organisation, ultrastructure and keratin composition. Four major keratins were found in cytoskeletal extracts (mol. wt. 60K, 59K, 53K, 49K) of primary cultures, together with a minor 51K protein and some residual actin. This “culture-type” keratin profile remained stable and little variation was observed after repeated treatment with various agents, such as 12-0-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA) or dimethylsulphoxide (DMSO). The profile was unaltered by long-term growth of primary cultures in low Ca 2+ (0.1 m M) medium or on 3T3 feeder-layers. Nevertheless, TPA, RA and low Ca 2+ did alter the morphology and filament architecture (as observed by indirect immunofluorescence microscopy with anti-keratin antibodies). Comparison of keratinocytes from primary culture with basal cells isolated from epidermis revealed similarities in electrophoretic profile, with the 60K and 53K keratins being common to both. The other in vivo keratins had a characteristic spatial distribution; 67K and 58K keratins were present in supra basal cells (spinous and granular), while 64K, 62K, 58.5K and 57.5K keratins were present only in stratum corneum. None of these keratins were found in cultured cells grown under regular conditions. Several morphological features of epidermal differentiation could be restored by the growth of PEC on collagen gels exposed to the atmosphere (“organotypic” culture) without influencing the keratin profile. Almost complete restoration of epidermal function was achieved after transplantation of PEC onto adult syngeneic mice. In this in vivo environment, well-structured epithelia developed which resembled interfollicular epidermis. Restoration of both typical ultrastructure and in vivo type keratin expression occurred within 2 or 3 weeks. Thus, although keratinocytes in primary culture differ considerably from those in vivo, they have not irreversibly lost the capacity for complete differentiation.

Culture of uninvolved and involved psoriatic and normal epidermis with 3T3 feeder layers: morphological and biochemical comparison.

ABSTRACTIsolated cells of uninvolved and involved psoriatic and normal epidermis have been cultured with 3T3 feeder layers. The efficiency of colony formation in the involved psoriatic epidermal cells was 50% as high as that in the uninvolved. No differences were observed between the uninvolved and involved psoriatic epidermal cell growth assessed by measuring the size of the growing colonies. Histologically, the structure of psoriatic colonies as revealed by electron microscopy did not differ from that of healthy control skin, having several layers of stratified epidermal cells with incomplete keratinization. The cyclic AMP level stimulated by the addition of isoproterenol to the involved psoriatic cell culture was about 50% as high as that in the uninvolved.