Subpopulations of primary adult murine epidermal basal cells sedimented on density gradients

Epidermal cells were harvested from the dorsal skin of adult mice by trypsinization and were sedimented through continuous density gradients of Percoll, formulated to separate basal cells of different buoyant density. Five fractions from the gradients were characterized with regard to the number of cells present, their viability and morphology and their basal origin. Suprabasal keratinocytes remained primarily at the top of the gradient; basal keratinocytes sedimented throughout. With increasing density, a relative enrichment was observed: (i) for [3H]-thymidine and [3H]-benzo[alpha]pyrene label-retaining (slowly cycling) keratinocytes; (ii) for keratinocytes that could proliferate in vitro in the continuous presence of 0.1 micrograms ml-1 of 12-O-tetradecanoylphorbol-13-acetate; (iii) for cells from untreated as well as initiated epidermis able to proliferate under conditions where calcium induces terminal differentiation; and (iv) for primary in vitro clonogenic keratinocytes from normal epidermis. The relative enrichment for epidermal basal cells having characteristics thought to be associated with immaturity and with the initiation and promotion of skin carcinogenesis suggests that density gradient sedimentation could be used in conjunction with other methods for the eventual purification of epidermal progenitors.     

Subpopulations of primary adult murine epidermal basal cells sedimented on density gradients

Abstract. Epidermal cells were harvested from the dorsal skin of adult mice by trypsinization and were sedimented through continuous density gradients of Percoll, formulated to separate basal cells of different buoyant density. Five fractions from the gradients were characterized with regard to the number of cells present, their viability and morphology and their basal origin. Suprabasal keratinocytes remained primarily at the top of the gradient; basal keratinocytes sedimented throughout. With increasing density, a relative enrichment was observed: (i) for [³H]-thymidine and [³H]-benzo[a]pyrene label-retaining (slowly cycling) keratinocytes; (ii) for keratinocytes that could proliferate in vitro in the continuous presence of 0–1 μ g ml^-1 of 12-0-tetradecanoylphorbol-13-acetate; (iii) for cells from untreated as well as initiated epidermis able to proliferate under conditions where calcium induces terminal differentiation; and (iv) for primary in vitro clonogenic keratinocytes from normal epidermis. The relative enrichment for epidermal basal cells having characteristics thought to be associated with immaturity and with the initiation and promotion of skin carcinogenesis suggests that density gradient sedimentation could be used in conjunction with other methods for the eventual purification of epidermal progenitors.     

Single modified cilia displayed by cells of human internal stratified epithelia (oral cavity,vagina)

Summary Serial sections of human vaginal and keratinized oral-gingival epithelia were investigated for ciliary structures. Most melanocytes of the gingival epithelium lacked cilia, whereas almost all basal keratinocytes of the deeper portion of the epithelial ridges possessed one cilium each. In the suprabasal layers of the ridges only a few keratinocytes exhibited a single cilium. In the basal layer, at the top of the connective tissue papillae, approximately every second keratinocyte displayed a single cilium. In the suprabasal layers above the ridges no ciliated keratinocytes were observed. The basal cells of the vaginal epithelium were endowed with cilia, while cilia were absent from the suprabasal cells. In the human forearm epidermis most melanocytes and keratinocytes are supplied with a single cilium; it has been suggested that they may play a role in light reception. However, the widespread occurrence of 9 + 0 cilia in epithelial cells of internal epithelia and their coincidence with the sites of renewal of keratinocytes suggests that a relationship may exist between solitary cilia and mitotic activity.     

The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies

The keratins are a highly heterogeneous group of proteins that form intermediate filaments in a wide variety of epithelial cells. These proteins can be divided into at least seven major classes according to their molecular weight and their immunological reactivity with monoclonal antibodies. Tissue-distribution studies have revealed a correlation between the expression of specific keratin classes and different morphological features of in vivo epithelial differentiation (simple vs. stratified; keratinized vs. nonkeratinized). Specifically, a 50,000- and a 58,000-dalton keratin class were found in all stratified epithelia but not in simple epithelia, and a 56,500- and a 65-67,000-dalton keratin class were found only in keratinized epidermis. To determine whether these keratin classes can serve as markers for identifying epithelial cells in culture, we analyzed cytoskeletal proteins from various cultured human cells by the immunoblot technique using AE1 and AE3 monoclonal antikeratin antibodies. The 56,500- and 65-67,000-dalton keratins were not expressed in any cultured epithelial cells examined so far, reflecting the fact that none of them underwent morphological keratinization. The 50,000- and 58,000-dalton keratin classes were detected in all cultured cells that originated from stratified squamous epithelia, but not in cells that originated from simple epithelia. Furthermore, human epidermal cells growing as a monolayer in low calcium medium continued to express the 50,000- and 58,000-dalton keratin classes. These findings suggest that the 50,000- and 58,000-dalton keratin classes may be regarded as "permanent" markers for stratified squamous epithelial cells (keratinocytes), and that the expression of these keratin markers does not depend on the process of cellular stratification. The selective expression of the 50,000- and 58,000-dalton keratin classes, which are synthesized in large quantities on a per cell basis, may explain the high keratin content of cultured keratinocytes.     

Immunolocalization of the neural cell adhesion molecule L1 in epithelia of rodents

The expression of the neural cell adhesion molecule L1 was analyzed in several non-neural tissues of the mouse using immunohistochemical and immunochemical techniques. In the adult mouse, L1 immunoreactivity was detectable in the basal and intermediate layers of epidermal and lingual epithelia, in the outer sheath of hair roots and in the single-layered endodermal epithelia of lung, small intestine, and colon. Epithelia of salivary glands also showed L1 immunoreactivity, while endothelial cells of blood vessels did not express detectable levels of L1. The epithelia of the kidney showed expression only in the collecting tubule system. In single-layered kidney epithelia and stratified epithelia, L1 expression was confined to lateral cell contacts and basal infoldings of the epithelial cells but was absent from apical and basal cell surface membranes. Also, in cultured keratinocytes L1 was confined to cell-cell contacts. During development of the epidermis, L1 immunoreactivity was first detectable at the onset of keratinization around embryonic day 16. At this age LI was detectable in the kidney on branching tubules of the ureter. Western blot analysis showed that L1 immunoreactivity in epidermis and kidney appeared as two bands of 190-210 and 210-230 kDa. Northern blot analysis of mRNA from the L1-immunopositive HEL-30 keratinocyte cell line revealed a single band with the expected size of 6 kb. The presence of L1 in epithelia indicates that this molecule may be involved in interactions between epithelial cells and thereby may affect differentiation and maintenance of epithelial tissues.     

Effects of subepithelial fibroblasts on epithelial differentiation in human skin and oral mucosa: heterotypically recombined organotypic culture model

The stratified squamous epithelia differ regionally in their patterns of morphogenesis and differentiation. Although some reports suggested that the adult epithelial phenotype is an intrinsic property of the epithelium, there is increasing evidence that subepithelial connective tissue can modify the phenotypic expression of the epithelium. The aim of this study was to elucidate whether the differentiation of cutaneous and oral epithelia is influenced by underlying mesenchymal tissues. Three normal skin samples and three normal buccal mucosa samples were used for the experiments. Skin equivalents were constructed in four ways, depending on the combinations of keratinocytes (cutaneous or mucosal keratinocytes) and fibroblasts (dermal or mucosal fibroblasts), and the effects of subepithelial fibroblasts on the differentiation of oral and cutaneous keratinocytes were studied with histological examinations and immunohistochemical analyses with anti-cytokeratin (keratins 10 and 13) antibodies. For each experiment, three paired skin equivalents were constructed by using single parent keratinocyte and fibroblast sources for each group; consequently, nine (3 x 3) organotypic cultures per group were constructed and studied. The oral and cutaneous epithelial cells maintained their intrinsic keratin expression. The keratin expression patterns in oral and cutaneous epithelia of skin equivalents were generally similar to their original patterns but were partly modified exogenously by the topologically different fibroblasts. The mucosal keratinocytes were more differentiated and expressed keratin 10 when cocultured with dermal fibroblasts, and the expression patterns of keratin 13 in cutaneous keratinocytes cocultured with mucosal fibroblasts were different from those in keratinocytes cocultured with cutaneous fibroblasts. The results suggested that the epithelial phenotype and keratin expression could be extrinsically modified by mesenchymal fibroblasts. In epithelial differentiation, however, the intrinsic control by epithelial cells may still be stronger than extrinsic regulation by mesenchymal fibroblasts.     

Epidermal stem cells: targets for carcinogenic chemicals

Epidermal stem cells long have been considered a target for carcinogenic chemicals, but these stem cells have never been identified or isolated. Toward this goal, this report examines two-stage carcinogenesis in light of the stem cell model for cellular replacement in the epidermis and considers characteristics that may be useful in the identification and the isolation of epidermal stem cells. Firstly, the carcinogenesis experiments in mice have indicated that the population of target cells normally remains in the epidermis for the life of the animal despite the continual cellular turnover. Hence, the slowly cycling (label-retaining) keratinocytes from the epidermis and hair follicles are potential targets. Secondly, the results of carcinogenesis experiments have also indicated that the target cells are necessarily ones with a high potential for proliferation relative the most of the proliferative population. The keratinocyte colony forming units (kCFU) from the epidermis of normal and treated adult mice are consequently a quantifiable indicator of proliferative potential and another possible target. Further application of the stem cell concepts of quiescence and of self-renewal is expected to yield additional tools for the identification and isolation of the epidermal targets for chemical carcinogens.     

Identification of label-retaining cells in nasopharyngeal epithelia and nasopharyngeal carcinoma tissues

Adult stem cells can be identified by label-retaining cell (LRC) approach based on their ability to retain nucleoside analog, such as bromodeoxyuridine (BrdU). We hypothesized that mouse nasopharynx contains a small population of epithelial stem/progenitor cells that may be detected by the LRC technique. To identify LRCs in mice nasopharyngeal epithelia, neonatal mice were intraperitoneally injected with BrdU twice daily for 3 consecutive days. After an 8-week chase, long-term BrdU-labeled LRCs (∼2% of cells) were detected in the adult mice nasopharyngeal epithelia by immunostaining with BrdU antibody and some of LRCs (∼12% of cells) were found to be recruited into the S phase of cell cycle with an additional radioactive thymidine-labeling technique, indicating that the stem cells also divide, most likely asymmetrically. To further investigate whether the LRCs existed in human nasopharyngeal carcinoma (NPC) tissues, three NPC cell lines (5-8F, 6-10B and TMNE) were labeled with BrdU in vitro and then individually engrafted into the back of nude mice, which developed tumors. Again, label-retaining stem cells were found in all the three kinds of NPC xenograft tumors (∼0.3% of cells), around 16% of which were also labeled with radioactive thymidine. Thus, this study has demonstrated for the first time the presence of epithelial LRCs in mouse nasopharyngx and human NPC tissues and these stem-like LRCs are not completely quiescent, as they will be recruited into the cell cycle to participate physiological or pathological process at any moment. More importantly, our data showed that NPC also contained stem cells, which are most likely the cause for NPC spread, metastasis and recurrence.     

mTOR Inhibition Prevents Epithelial Stem Cell Senescence and Protects from Radiation-Induced Mucositis

The integrity of the epidermis and mucosal epithelia is highly dependent on resident self-renewing stem cells, which makes them vulnerable to physical and chemical insults compromising the repopulating capacity of the epithelial stem cell compartment. This is frequently the case in cancer patients receiving radiation or chemotherapy, many of whom develop mucositis, a debilitating condition involving painful and deep mucosal ulcerations. Here, we show that inhibiting the mammalian target of rapamycin (mTOR) with rapamycin increases the clonogenic capacity of primary human oral keratinocytes and their resident self-renewing cells by preventing stem cell senescence. This protective effect of rapamycin is mediated by the increase in expression of?mitochondrial superoxide dismutase (MnSOD), and the consequent inhibition of ROS formation and oxidative stress. mTOR inhibition also protects from the loss of proliferative basal epithelial stem cells upon ionizing radiation in?vivo, thereby preserving the integrity of the oral mucosa and protecting from radiation-induced mucositis.     

Identification of epithelial label-retaining cells at the transition between the anal canal and the rectum in mice

In certain regions of the body, transition zones exist where stratified squamous epithelia directly abut against other types of epithelia. Certain transition zones are especially prone to tumorigenesis an example being the anorectal junction, although the reason for this is not known. One possibility is that the abrupt transition of the simple columnar epithelium of the colon to the stratified squamous epithelium of the proximal portion of the anal canal may contain a unique stem cell niche. We investigated whether the anorectal region contained cells with stem cell properties relative to the adjacent epithelium. We utilized a tetracycline-regulatable histone H2B-GFP transgenic mice model, previously used to identify hair follicle stem cells, to fluorescently label slow-cycling anal epithelial cells (e.g. prospective stem cells) in combination with a panel of putative stem cell markers. We identified a population of long-term GFP label-retaining cells concentrated at the junction between the anal canal and the rectum. These cells are BrdU-retaining cells and expressed the stem cell marker CD34. Moreover, tracking the fate of the anal label-retaining cells in vivo revealed that the slow-cycling cells only gave rise to progeny of the anal epithelium. In conclusion, we identified a unique population of cells at the anorectal junction which can be separated from the other basal anal epithelial cells based upon the expression of the stem cell marker CD34 and integrin a6, and thus represent a putative anal stem cell population.     

Involucrin synthesis and tissue assembly by keratinocytes in natural and cultured human epithelia

Different stratified squamous epithelia, whether they bear a stratum corneum or not, are shown by immunofluorescence to possess the precursor protein of the cross-linked envelope that is characteristic of epidermal s. corneum. This protein, involucrin, is not present in the deepest epithelial cells but appears in the course of their outward migration. The boundary at which involucrin first appears can sometimes by correlated with a visible boundary between zones of large and small cells. Cultured keratinocytes, derived from all stratified squamous epithelia (epidermal, corneal, conjuctival, esophageal, lingual, and vaginal), form colonies that grow together to form a stratified epithelium. The cells of the basal layer are nearly always free of detectable involucrin, but, in contrast to the natural epithelium, this protein usually makes its appearance in the cells immediately above the basal layer. When a cultured epithelium derived from epidermal keratinocytes is detached and applied as a graft to animals, the cells flatten and the distinctness of the basal layer is at first reduced; but with time the organization of the epithelium becomes more characteristic of epidermis. Cell size and shape become more orderly along the cell migration pathway, and involucrin first appears at some distance from the basal layer, instead of in immediately suprabasal cells, as in the cultured epithelium. The progeny of dissociated and cultured keratinocytes are therefore able, when grafted, to reassemble an epidermis in which the timing of specific gene expression is restored to that of the original tissue.     

Differentiating cells of murine stratified squamous epithelia constitutively express plasminogen activator inhibitor type 2 (PAI-2)

 In stratified squamous epithelia a critical balance among cell proliferation, differentiation, and death must be maintained in order for these tissues to fulfill their barrier function. Previous studies have demonstrated that plasminogen activator inhibitor 2 (PAI-2) is a product of differentiating epidermal keratinocytes, suggesting a role for this inhibitor during squamous differentiation. Furthermore, in certain tumor cell lines, overexpression of PAI-2 confers resistance to the induction of programmed cell death, suggesting cytoprotective function(s). In the present study we demonstrate that PAI-2 mRNA and protein are constitutively and uniquely expressed in differentiating cells of murine stratified squamous epithelia, including epidermis, esophagus, vagina, oral mucosa, and tongue. PAI-2 immunohistochemical localization patterns suggest a predominantly cytosolic distribution, consistent with biochemical identification of the major PAI-2 species as a 43-kDa, presumably non-glycosylated protein. Functional analysis shows that the majority of epithelial PAI-2 is active. In contrast to the high levels of PAI-2 expression in stratified squamous epithelia, little or no PAI-2 is detectable in simple epithelia. These findings suggest that epithelial PAI-2 may mediate inhibition of intracellular proteinases associated with events during terminal differentiation and death that are unique to stratified squamous epithelia. Accepted: 29 June 1998     

Cyclic AMP in relation to proliferation of the epidermal cell: a new view.

Four agents known to increase the level of cellular cAMP by different means (cholera toxin, dibutyryl cAMP, methyl isobutyl xanthine and isoproterenol) increase the growth of colonies of cultured human epidermal cells and of keratinocytes derived from other stratified squamous epithelia. This effect is due to an increase in the overall rate of cell proliferation in the colonies. When added to cultures under hitherto optimum conditions for epidermal cell growth [in the presence of supporting 3T3 cells and epidermal growth factor (EGF)], most of the agents exert an effect of considerable magnitude, the toxin being the most potent. Since the toxin exerts an effect in the absence of supporting 3T3 cells, it must be able to act directly on the keratinocytes. It can also act in the absence of ECF and of medium conditioned by 3T3 cells, although proliferation is greatest when supporting 3T3 cells and EGF are present. The increased proliferation in the presence of the toxin is associated with an increased proportion of small cells known to include the multiplying fraction. The use of toxin makes the cultivation of keratinocytes from epidermis and other stratified squamous epithelia much easier and prolong the culture life of the cells. Whether cell proliferation in the intact epidermis is regulated through agents affecting cAMP (in a direction opposite to that suggested by much of the earlier literature) remains to be elucidated, but the existence of such a mechanism in cultured cells suggests that it may function in the intact epithelium.     

miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration

During keratinocyte differentiation and stratification, cells undergo extensive remodeling of their actin cytoskeleton, which is important to control cell mobility and to coordinate and stabilize adhesive structures necessary for functional epithelia. Limited knowledge exists on how the actin cytoskeleton is remodeled in epithelial stratification and whether cell shape is a key determinant to trigger terminal differentiation. In this paper, using human keratinocytes and mouse epidermis as models, we implicate miR-24 in actin adhesion dynamics and demonstrate that miR-24 directly controls actin cable formation and cell mobility. miR-24 overexpression in proliferating cells was sufficient to trigger keratinocyte differentiation both in vitro and in vivo and directly repressed cytoskeletal modulators (PAK4, Tks5, and ArhGAP19). Silencing of these targets recapitulated the effects of miR-24 overexpression. Our results uncover a new regulatory pathway involving a differentiation-promoting microribonucleic acid that regulates actin adhesion dynamics in human and mouse epidermis.     

Epiplakin is dispensable for skin barrier function and for integrity of keratin network cytoarchitecture in simple and stratified epithelia

Epiplakin, a giant epithelial protein of >700 kDa, belongs to the plakin family of cytolinker proteins. It represents an atypical family member, however, as it consists entirely of plakin repeat domains but lacks any of the other domains commonly shared by plakins. Hence, its putative function as a cytolinker protein remains to be shown. To investigate epiplakin's biological role, we generated epiplakin-deficient mice by gene targeting in embryonic stem cells. Epiplakin-deficient mice were viable and fertile, without developing any discernible phenotype. Ultrastructurally, their epidermis revealed no differences compared to wild-type littermates, and cornified envelopes isolated from skin showed no alterations in shape or stability. Furthermore, neither embryonal formation nor later function of the epithelial barrier was affected. In primary cultures of epiplakin-deficient keratinocytes, the organization of actin filaments, microtubules, and keratin networks was found to be normal. Similarly, no alterations in keratin network organization were observed in simple epithelia of small intestine and liver or in primary hepatocytes. We conclude that, despite epiplakin's abundant and highly specific expression in stratified and simple epithelia, its absence in mice does not lead to severe skin dysfunctions, nor has it detectable consequences for keratin filament organization and cytoarchitecture of cells.     

Identification of epithelial label-retaining cells at the transition between the anal canal and the rectum in mice

In certain regions of the body, transition zones exist where stratified squamous epithelia directly abut against other types of epithelia. Certain transition zones are especially prone to tumorigenesis an example being the anorectal junction, although the reason for this is not known. One possibility is that the abrupt transition of the simple columnar epithelium of the colon to the stratified squamous epithelium of the proximal portion of the anal canal may contain a unique stem cell niche. We investigated whether the anorectal region contained cells with stem cell properties relative to the adjacent epithelium. We utilized a tetracycline-regulatable histone H2B-GFP transgenic mice model, previously used to identify hair follicle stem cells, to fluorescently label slow-cycling anal epithelial cells (e.g., prospective stem cells) in combination with a panel of putative stem cell markers. We identified a population of long-term GFP label-retaining cells concentrated at the junction between the anal canal and the rectum. These cells are BrdU-retaining cells and expressed the stem cell marker CD34. Moreover, tracking the fate of the anal label-retaining cells in vivo revealed that the slow-cycling cells only gave rise to progeny of the anal epithelium. In conclusion, we identified a unique population of cells at the anorectal junction which can be separated from the other basal anal epithelial cells based upon the expression of the stem cell marker CD34 and integrin α6, and thus represent a putative anal stem cell population.Key words: stem cells, transitional epithelium, keratinocyte, slow-cycling, label retaining cell     

EEDA: a protein associated with an early stage of stratified epithelial differentiation

Using suppressive subtractive hybridization, we have identified a novel gene, which we named early epithelial differentiation associated (EEDA), which is uniquely associated with an early stage of stratified epithelial differentiation. In epidermis, esophageal epithelium, and tongue epithelium, EEDA mRNA, and antigen was abundant in suprabasal cells, but was barely detectable in more differentiated cells. Consistent with the limbal location of corneal epithelial stem cells, EEDA was expressed in basal corneal epithelial cells that are out of the stem cell compartment, as well as the suprabasal corneal epithelial cells. The strongest EEDA expression occurred in suprabasal precortical cells of mouse, bovine, and human anagen follicles. Developmental studies showed that the appearance of EEDA in embryonic mouse epidermis (E 15.5) coincided with morphological keratinization. Interestingly, EEDA expression is turned off when epithelia were perturbed by wounding and by cultivation under both low and high Ca2+ conditions. Our results indicate that EEDA is involved in the early stages of normal epithelial differentiation, and that EEDA is important for the "normal" differentiation pathway in a wide range of stratified epithelia.     

Taurine levels and localisation in the stratified squamous epithelia

The content and distribution of the amino acid taurine in squamous epithelia were studied using high-performance liquid chromatography and immunohistochemical methods. Quantitative analysis demonstrated that taurine was highly concentrated in the epidermis (5.49 mumol/g fresh tissue in the hairless skin of the hind footpad of the rat), although the values in the isolated stratum corneum were extremely low (< 0.073 mumol/g in the horny layer of the same skin area). No other analysed amino acid (such as glutamate, glutamine, glycine or alanine) showed this specific pattern of distribution. The immunohistochemical study revealed that in the dog and rat epidermis, taurine was present in the keratinocytes of the granular and upper spinous layers. The basal layer, lower spinous layer and stratum corneum were immunonegative. A similar immunostaining pattern was found in the epithelia of the different organs studied: the mouth, tongue and oesophagus of the dog and rat, the rat forestomach and the rat corneal epithelium. Other cell types, such as sebaceous and muscle cells, were immunolabelled. The existence of a circulating pool of taurine in the epidermis (via taurine release from keratinocytes before they reach the horny layer and its uptake by nearby cells) and its possible roles in these cells are discussed.