Limbal fibroblast conditioned media: a non-invasive treatment for limbal stem cell deficiency.

PURPOSE: Limbal fibroblasts are known to regulate the maintenance and differentiation of the corneal epithelium including the limbal epithelial stem cells. This study examined the effect of limbal fibroblast conditioned media in a mouse model of limbal stem cell deficiency. METHODS: Limbal stem cell deficiency was created in C57/Bl6 mice by performing a limbus to limbus epithelial debridement. The mice were treated topically for 3 weeks with conditioned media derived from human limbal fibroblasts. The control mice were treated with skin fibroblast conditioned media or Dulbecco's serum-free medium. RESULTS: The mice treated with limbal fibroblast conditioned media demonstrated substantial growth of corneal type epithelial cells on the corneal surface with less conjunctival goblet cells. By contrast, the control treated corneas were found to be covered primarily by conjunctival type epithelium. CONCLUSIONS: Cell culture media conditioned by limbal fibroblasts appear to contain factor(s) that are therapeutically beneficial in a model of limbal stem cell deficiency. The current results further support the notion that the essential limbal stem cell niche is provided by limbal fibroblasts and suggest a new, non-invasive option in the treatment of limbal stem cell deficiency.

Formation of horny cells: the fate of cell organelles and differentiation products in ruminal epithelium.

Epithelial cells changing from the granular stage of differentiation to the horny stage are more numerous, and reveal sequential events of transformation in finer detail in the rumen epithelium than in other keratinizing epithelia thus far studied in the electron microscope. Studies of such cells indicate that transformation is initiated by the release of hydrolytic enzymes, as evidenced by the appearance of lysosomes. As lysosomes increase in number, the nucleus, ribosomes, mitochondria, Golgi apparatus, and mucous granules are gradually degraded. Furthermore, marked changes occur in permeability of the plasma membrane as voluminous amounts of the lysed cell components pass through and accumulate in the intercellular space in the form of an amorphous mass. Filaments, keratohyalin granules, and the content of the ER (ER-protein) are not lysed, revealing the action of released enzymes to be specific. During transformation, filaments become displaced toward the cell periphery and keratohyalin granules disperse and mix with the ER-protein in the cell center. Subsequently, the keratohyalin-ER-protein complex infiltrates the filament network whereby a fibrous-amorphous cell content is formed. Loss of fluids through the plasma membrane leads to reduction of cell volume and consolidation of the remaining cell content. The deep interdigitations formed between the cells ultimately interlock the outer part of the epithelium into a cohesive and protective stratum corneum.

Plakoglobin suppresses epithelial proliferation and hair growth in vivo.

Plakoglobin regulates cell adhesion by providing a modulatable connection between both classical and desmosomal cadherins and their respective cytoskeletal linker proteins. Both plakoglobin and the related protein beta-catenin are posttranscriptionally upregulated in response to Wnt-1 in cultured cells. Upregulation of beta-catenin has been implicated in potentiating hyperproliferation and tumor formation. To investigate the role of plakoglobin in these functions we expressed a full-length (PG) and an NH(2)-terminally truncated form of plakoglobin (DeltaN80PG) in mouse epidermis and hair follicles, tissues which undergo continuous and easily observed postnatal renewal and remodeling. Expression of these constructs results in stunted hair growth, a phenotype that has also been observed in transgenic mice expressing Wnt3 and Dvl2 (Millar et al. 1999). Hair follicles from PG and DeltaN80PG mice show premature termination of the growth phase (anagen) of the hair cycle, an event that is regulated in part by FGF5 (Hebert et al. 1994). The proliferative rate of the epidermal cells was reduced and apoptotic changes, which are associated with entry into the regressive phase of the hair follicle cycle (catagen), occurred earlier than usual.

Heterogeneity in epidermal basal keratinocytes: morphological and functional correlations.

Two structurally distinct populations of basal keratinocytes, nonserrated and serrated, were observed in cynomolgus monkey and human palm epidermis. Anatomical location, fine structural features, and kinetic properties suggest that nonserrated cells represent a stem cell population and that serrated cells help anchor the epidermis to the dermis.

The tumor suppressor effect of the glucocorticoid receptor in skin is mediated via its effect on follicular epithelial stem cells.

Glucocorticoids are potent inhibitors of mouse skin tumorigenesis. The glucocorticoid control of cellular functions is mediated via the glucocorticoid receptor (GR), a well-known transcription factor. Recently, we generated transgenic mice overexpressing GR under control of the keratin5 (K5) promoter, and showed that K5.GR animals are resistant to skin carcinogenesis. Follicular epithelial stem cells (SCs), located in the bulge region of the hair follicle, are believed to be one of the target cells for skin carcinogenesis. We found that the number of putative hair follicle SC detected as label-retaining cells was significantly less in the K5.GR transgenics compared to wild type (w.t.) littermates. We also showed that GR overexpression led to a reduction in the clonogenicity of the follicular epithelial SCs. We evaluated the global effect of GR on gene expression in a population of follicular SC-enriched bulge keratinocytes isolated by fluorescence activated cell sorting. We found that GR affected the expression of numerous bulge SC 'signature' genes, genes involved in the maintenance of SC and progenitor cells of non-epidermal origin and proapoptotic genes. Our findings underscore the important role of GR signaling in the homeostasis of follicular epithelial SCs, and suggest that the reduction in their number may underlie the tumor suppressor effect of GR in the skin.

Down-regulation of Notch signaling during corneal epithelial proliferation.

PURPOSE: We evaluated the expression and activation of Notch pathway genes in the adult human and murine corneal epithelium during proliferation. METHODS: The expression of Notch pathway genes in the limbal and central human corneal epithelium was compared by reverse transcription polymerase chain reaction (RT-PCR). Their expression pattern was examined by immunofluorescence and in situ hybridization. The temporal expression of Notch1 during murine wound healing was assessed by RT-PCR. Notch activity was determined using western blot for the Notch intracellular domain (NotchIC). The expression of Hes1 was evaluated in cell culture. RESULTS: The expression of Notch1 and Jagged1 was higher in the human limbal epithelium while the expression of Hes1 and Hes5 was higher in the central cornea. Expression of Notch1, Jagged1, and Hes1 was found predominantly in the basal and immediate suprabasal cells. During neonatal corneal development, NotchIC was detected in occasional cells at P10 while at P15 and P90, it was found in the basal and early suprabasal layers. NotchIC was found to be lower in the limbal compared to central corneal epithelium. The expression of Notch1 was lower at 24 h post-wounding but was completely restored in six days. The levels of NotchIC were decreased at 24 h post-wounding and after application of topical phorbol myristate. In vitro, the expression of Hes1 was higher in confluent cells maintained under high calcium conditions. CONCLUSIONS: The inverse correlation between Notch signaling and the proliferative status of the corneal epithelium is consistent with the idea that Notch plays a role in corneal epithelial differentiation.

Lipid synthesis in chick epidermis.

Lipid synthesis in newborn chick epidermis was studied by electron microscopic autoradiography after injection of tritiated palmitate. The labeled lipid product in the tissue was identified as mostly triglyceride. At the earliest time after injection (6 hr), the radioactive precursor was taken up by all viable cells of the epidermis. Grain density was heaviest over basal cells, moderate over spinous cells, and slight over granular cells; thus lipid incorporation is highest in the basal and spinous regions of the chick epidermis. As time after injection progressed, the increasing amounts of grains over the granular and horny cells and decreasing amounts over the basal and spinous cells reflected the continuous upward displacement of cells from one layer into the next. From the distribution of silver grains within the epidermal cells, it has been concluded that, with the passage of time, triglycerides synthesized by the epidermal cells were mainly located in lipid droplets. The numerous grains associated with the elements of the endoplasmic reticulum indicated that this organelle is involved in aggregating triglyceride molecules into lipid droplets. The fact that grains were seen within the horny cells indicated that part of the horny cell consists of lipid probably derived from the lipid droplets retained by the cells during keratinization.

Chronic heliodermatitis: a morphologic evaluation of chronic actinic dermal damage with emphasis on the role of mast cells.

Descriptions of actinically damaged human dermis have focused on the late stages of elastotic degeneration. This has diverted attention from preceding events, which are important for understanding the sequence of pathologic changes that culminate in the deranged fibrous structures of elastotic dermis. We studied specimens from the back of the necks (exposed) and inner arms (unexposed) of 24 individuals, aged 35-84 yr, by light and transmission electron microscopy. Intense sunlight exposure was common to all. A previously undescribed finding was the presence of a perivenular, histiocytic-lymphocytic infiltrate in which numerous mast cells, often in close apposition to fibroblasts, were observed. We have termed this "chronic heliodermatitis." We postulate that mast cell-derived mediators in conjunction with enzymes released by the infiltrating cells lead to breakdown of elastic and collagen fibers.

Urokinase is a positive regulator of epidermal proliferation in vivo.

The epidermis is a self-renewing tissue that must maintain a basal proliferative rate as well as respond to various perturbing stimuli. Regulation of keratinocyte proliferation involves diverse molecules, including growth factors, ions, and hormones. We recently proposed that a proteinase, urokinase-type plasminogen activator (uPA) may be added to this list, based on correlative evidence linking expression of uPA and murine epidermal hyperproliferation. Here we report that, during the first 3 d of life, the epidermis from mice that bear a targeted deletion of the uPA gene has a significantly lower proliferative rate than the epidermis from wild-type mice. In contrast, proliferation in the matrix keratinocytes of the hair follicles is not decreased in neonatal uPA-/- mice. Vertical migration of keratinocytes during terminal differentiation was not affected. We therefore conclude that lack of uPA is associated with a decrease in epidermal proliferation.

Rapid modulation of keratinocyte differentiation by the external environment.

The ultrastructural differentiation of epidermal keratinocytes cultured in the presence of 3T3 feeder cells is significantly different from that of the epidermis in vivo. Several markers of keratinization, including keratohyaline granules (KGs), membrane-coating granules (MCGs), and an enucleated stratum corneum are essentially absent from cultured cells. When cultured rabbit epidermal cells were trypsinized and injected subcutaneously into athymic mice, the cells reaggregated and formed cysts lined with a stratified squamous epithelium morphologically resembling the in vivo epidermis. In this paper, we examined the differentiation of the injected cells by electron microscopy. Within 24 h after injection, MCGs and KGs appeared in the reaggregated epidermal cells. Horny cells were noted within 48 h. Since basement membrane formation was not completed until much later (between 4-9 days), a direct contact between epidermal cells and a continuous basal lamina structure was not required for the formation of various keratinization markers. Glycogen and lipid droplets, which were abundant in the early (10-48 h) cystic epithelia, gradually disappeared from the basal through the granular layers during days 2-9. By 16 days, the ultrastructure of the cystic epithelium appeared similar to the in vivo rabbit epidermis including the formation of "rabbit-type" KGs, MCGs, and normal enucleated horny cells. These results provided further evidence that the differentiation of epidermal cells can be modulated significantly and reversibly by the external environment. Moreover, the expression of certain morphologic markers of keratinization (KGs and MCGs) can be modulated rapidly by the growth environment.

Epidermal stem cells.

Stem cells are by definition present in all self-renewing tissues and are believed to play a central role in cell growth and differentiation. Existing evidence suggests that a subpopulation of epidermal basal keratinocytes represents stem cells; however, these cells have never been positively identified. In this paper we review evidence that in monkey palm epidermis there exist two morphologically distinct subpopulations of basal keratinocytes that are spatially segregated. One population, located in the shallow rete ridges, is characterized by a cytoplasm filled with tonofilaments and a highly convoluted ("serrated") dermal-epidermal junction; these cells may play a role in anchoring the epidermis to the dermis. In contrast, the other population, located at the tips of deep rete ridges, is characterized by a "primitive" cytoplasm containing abundant melanosomes and a relatively flattened ("nonserrated") dermal-epidermal junction. Tritiated thymidine labeling experiments suggest that the nonserrated basal keratinocytes are slow-cycling; however, a highly proliferative population of keratinocytes can be identified immediately above these basal cells. These findings are consistent with the concept that the nonserrated basal keratinocytes may represent stem cells that give rise to suprabasally located, transient amplifying cells before undergoing terminal differentiation. Monkey palm epidermis provides a model system for further studies of primate epidermal stem cells.

Aged skin: a study by light, transmission electron, and scanning electron microscopy.

The fine structural organization of the epidermis, dermal/epidermal junction, and dermis from an unexposed site (upper inner arm) of elderly people was compared with the organization of a similar region of young people. Despite an overall thinning of the epidermis and focal areas of cytologic atypia, the characteristic morphological markers associated with the keratinization process are not markedly altered in appearance or amount. A well-formed stratum corneum consisting of flattened, enucleated horny cells enveloped by a thickened membrane, and intracellular spaces filled with electron-dense material provide structural evidence that barrier ability is not compromised in senile skin. The dermal/epidermal changes in aged skin are marked and have significant physiologic implications. The major change is a relatively flat dermal/epidermal junction resulting from the retraction of the epidermal papillae as well as the microprojections of basal cells into the dermis. This flattening results in a more fragile epidermal/dermal interface and, consequently, the epidermis is less resistant to shearing forces. Retraction of the epidermal downgrowths (preferential sites of the putative epidermal stem cell) may also explain the loss in proliferative capacity associated with the aged epidermis. The three-dimensional arrangements of collagen and elastic fibers showed marked alterations with age. Both fibrous components appear more compact because of a decrease in spaces between the fibers. Collagen bundles appear to unravel, and the individual elastic fibers show signs of elastosis. These changes may contribute to the loss of resilience that is one of the salient features of senile skin.

Upper human hair follicle contains a subpopulation of keratinocytes with superior in vitro proliferative potential.

We and others have shown previously that corneal keratinocyte stem cells can proliferate in vitro better than their progeny cells. In this paper, we applied this approach to the identification of hair follicular stem cells. When human scalp hair follicles were placed in explant culture, the bulge area yielded best outgrowths. In another experiment, we isolated different subpopulations of human follicular keratinocytes by micro-dissection, dispersed them by trypsin/EDTA into single cells, and grew them in the presence of 3T3 feeder cells. The keratinocytes were then subcultured under identical conditions to compare their in vitro life span. Our results indicate that the life span of keratinocytes of the upper follicle (containing mainly the isthmus area) > sebaceous gland > lower follicle (between the bulge and bulb) > bulb (containing the matrix cells). The cultured upper follicular keratinocytes tend to be small and relatively uniform in size. The poor in vitro growth of matrix cells may reflect their non-stem cell nature and/or special growth requirement(s) satisfied in vivo by the neighboring dermal papilla cells. Unexpectedly, we found that the upper follicular keratinocytes grow even better than epidermal keratinocytes. The existence of a subpopulation of keratinocytes with an in vitro growth potential superior than other known keratinocytes of the skin supports the hypothesis that follicular stem cells reside in the upper follicle. Our data also raise the possibility that putative follicular stem cells are involved not only in forming the follicle, but also in the long-term maintenance of the epidermis. Finally, we discuss the possibility that keratinocyte stem cells, as defined by their in vivo slow-cycling nature, are absent in culture.

Corticosteroid atrophy in human skin. A study by light, scanning, and transmission electron microscopy.

Steroid atrophy was induced in 3 volunteers by the continuous, occlusive application of clobetasol propionate to the forearms for 6 weeks. The changes were followed sequentially by light, scanning, and transmission electron microscopy. a 59% decrease in viable epidermal thickness was noted after the sixth week of treatment, as well as a flattening of the dermal-epidermal junction. The 3-dimensional architecture of the dermis was strikingly reorganized. This was largely brought about by resorption of the ground substance as revealed by a progressive diminution of Hale's stain for acid mucopolysaccharides. Loss of ground substance resulted in decreased spaces between collagen and elastic fibers as shown by scanning and transmission electron microscopy. The fibrous network consequently collapsed, yielding a more compact papillary and reticular dermis. This compression caused the reorientation of both collagen and elastic fibers. However, no differences in collagen and elastin fine structure were noted. Fibroblasts were shrunken but not reduced in density. A marked decrease in number of mast cells was noted in 3-week specimens and virtually no mast cells were observed after 6 weeks. We found that the primary effect of short-term steroid use was a rearrangement of the geometry of the dermal fibrous network. This was not due to alterations in the fibers themselves but a secondary consequence of the loss of ground substance.

Morphologic investigations on the rebound phenomenon after corticosteroid-induced atrophy in human skin.

Cutaneous atrophy was induced on the forearms of 4 volunteers by continuous occlusive application of clobetasol-17-propionate for 6 weeks, after which time the steroid was discontinued. Epidermal and dermal changes during the subsequent rebound "flare" were monitored for 2 weeks by light and transmission electron microscopy. An exuberant hyperplasia characterized the epidermal response. Within 2 days poststeroid, most basal cells displayed fine structural features typical of highly proliferating cells. "Dark"-staining keratinocytes appeared in large numbers 4 days poststeroid, preceding a 4-fold maximal increase of viable epidermal thickness which occurred at 7 days. The stratum corneum, initially very thin, increased markedly in thickness and displayed the typical basket-weave appearance. By 14 days, Langerhans cells, which were absent immediately poststeroid, were again present. At this time, the epidermis returned to a nearly normal state. Dermal restitution was similarly rapid. Initially, fibroblasts appeared very active as evidenced by widely dilated endoplasmic reticulum filled with flocculent material. Ground substance increased continuously, reaching normal levels by 14 days. An increase in postcapillary venules was noted during the rebound flare. Swift epidermal and dermal changes are evidence that topical corticosteroids are rapidly cleared from the skin. The vigorous epidermal hyperplasia reflects repair of the atrophic, suppressed epidermis as well as a response to desiccation consequent to the loss of the stratum corneum.

The relationship between bacteria and the abnormal follicular keratinization in acne vulgaris.

The structural organization and bacteriological profile of follicular casts and early comedones in prepuberal children were investigated. Follicular casts were present in all samples but were not as abundant as usually seen in older individuals with acne. When examined with the light microscope, all casts and comedones were composed of numerous layers of horny cells and sebum, but were devoid of bacteria. Neither follicular casts nor comedones yielded bacteria when cultured. Electron microscopy of the follicular casts revealed the presence of small round, discrete lipid droplets, and alternating dense and less dense lamellar configurations within the horny cells. Some horny cells closest to the sebum-filled lumen contained large lipid masses, resulting in "balloon-shaped" regions. Prepuberal follicular horny cells contained all of the abnormalities usually seen in follicular casts and biopsy material from acne patients, which suggests that these casts are potential comedones. These aberrations occurred in the complete absence of bacteria indicating that bacteria are not essential to the formation of casts or comedones. Furthermore, our findings indicate that bacteria play little if any role in the initial events of pathological keratinization.

Substance P induces the expression of an endothelial-leukocyte adhesion molecule by microvascular endothelium.

Leukocyte trafficking in normal and diseased skin appears to be initially governed by endothelial surface glycoproteins that promote adhesive interactions with circulating leukocytes. In a separate study, we have demonstrated that one of these glycoproteins, endothelial-leukocyte adhesion molecule-1 (ELAM-1), is rapidly induced on postcapillary dermal venules as a direct consequence of experimentally-elicited degranulation of adjacent mast cells (Proc Natl Acad Sci USA 86:8972-8976, 1989). A principle endogenous mediator of mast cell degranulation is the neuropeptide substance P. In this study, we exposed organ cultures of neonatal human foreskins for 45 min to substance P or to a substance P analogue (D-pro4, D-trp7,9)SP(4-11) that binds to the identical mast cell surface receptor but which does not provoke histamine release. Dermal mast cells were uniformly degranulated only in explants exposed to substance P, as judged by ultrastructural analysis. After subsequent culture in medium alone for 6 h, superficial venules of explants exposed to substance P showed evidence of ELAM-1 induction, as documented histochemically using H4/18 monoclonal antibody. ELAM-1 was not induced by substance P analogue. Furthermore, preincubation of explants with analogue or with the mast cell inhibitor, cromolyn sodium, abrogated the ability of substance P to induce ELAM-1. From these results we suggest that substance P endogenously released by dermal nerve fibers upon physiologic or electrical stimulation may be important in the regulation of endothelial-leukocyte interactions in vivo. This concept provides further evidence for a neurogenic and psychogenic modulation of the immune response, and may be relevant to the course of naturally occurring dermatoses (e.g., psoriasis) that are commonly exacerbated by emotional stress.

Use of a serum-free epidermal culture model to show deleterious effects of epidermal growth factor on morphogenesis and differentiation.

The presence of serum has limited the utility of many culture models for the study of cytokine effects because its complexity and variability can confound the interpretation of data. In the present study, a serum-free skin co-culture model was used to investigate the effect of exogenous epidermal growth factor (EGF) on epidermal proliferation and differentiation. Human keratinocytes cultured on collagen rafts at the air-liquid interface produced a well-differentiated epithelium that resembled normal epidermis. Keratin filaments, membrane-coating granules, and keratohyalin granules were all observed. Epidermal differentiation markers keratin K1/K10, involucrin, and transglutaminase were localized in most of the suprabasal layers, whereas profilaggrin/filaggrin was confined to the granular layers and stratum corneum. In the continual presence of 10-20 ng/mL EGF, the epidermis was less organized, thinner, and less proliferative. EGF also depressed several indicators of differentiation: The number of keratohyalin granules and membrane-coating granules was greatly decreased; antigen expression of profilaggrin/filaggrin appeared diminished by immunocytochemical staining; frequent nuclear retention was noted in the relatively thickened stratum corneum-like layers. As detected by immunohistochemical staining, the expression of EGF receptor in the epidermis was reduced by exogenous EGF. These data illustrate that EGF cannot be considered a simple mitogen. Our findings also underscore the importance of using sophisticated culture models to assess complex cytokine effects that may be dependent on the architecture of a differentiating epidermis.

Mouse skin is particularly susceptible to tumor initiation during early anagen of the hair cycle: possible involvement of hair follicle stem cells.

Stem cells are believed to be a necessary target of chemical carcinogens. Based on autoradiographic, ultrastructural, and biologic criteria, we have recently proposed that hair follicle stem cells reside not in the bulb, but in the upper outer root sheath in an area called the bulge. Proliferating cells have been shown to be more susceptible to tumor initiation, and we have recently demonstrated that cells in the bulge undergo transient proliferation during early anagen. Therefore, we theorized that mouse skin should be particularly susceptible to carcinogen application during early anagen phase. In this paper, we show that early anagen Swiss and Sencar mouse skin is indeed particularly susceptible to one- and two-stage chemical carcinogenesis, resulting in tumor yields one to five times those obtained with telogen-timed carcinogen application. Our findings implicate a possible involvement of the bulge cells as precursors to some of the skin cancers, and support the concept that these are stem cells. These observations also raise important questions about the cellular origins and biologic behavior of chemically induced murine skin tumors.

Extracellular localization of human connective tissue mast cell granule contents.

In early phases of cutaneous inflammation, connective tissue mast cell degranulation is associated with apparent secretion and externalization of immunoreactive chymotryptic serine proteinase. To determine whether this event is associated with structural evidence of granule externalization, we studied the sequential evolution of IgE-mediated hypersensitivity in vivo, as well as mast cell degranulation provoked by a variety of stimuli in cultured explants of human skin. By 1 min after intradermal antigen challenge with ragweed extract, mast cell degranulation was associated with apparent extrusion of intragranule constituents into the pericellular connective tissue. Similar features typified cultured skin explants exposed for 45 min to anti-IgE and other mast cell secretagogues (morphine sulfate, calcium ionophore A23187, compound 48/80, and substance P). Once externalized, granule constituents could be identified within the dermal matrix by their rounded contour and structural similarity to solubilized granule matrices remaining within actively secreting cells. These data indicate that externalization of connective tissue mast cell granule contents occurs early after secretagogue exposure, potentially accounting for infrequent documentation of this event in naturally occurring dermatoses. The ability to recognize externalized granule products at a morphologic level should facilitate the understanding of interactions between mast cell-derived mediators and target structures of the dermal microvasculature.