Separation of keratinocytes by density gradient centrifugation for DNA cytofluorometry

Summary A method for the separation of guinea pig epidermal keratinocytes, in which the Feulgen-stainable material suffers minimal damage, has been investigated. The principal stage involves trypsin treatment of the epidermal sheet, stripped from the dermis with ethylenediamine tetraacetic acid. The epidermal cells thus isolated are separated into three groups by centrifugation on a continuous colloidal silica (Percoll) density gradient. The resulting arrangement of the keratinocytes in the centrifuge tube corresponds to their arrangement in situ, with basal cells at the bottom and the more differentiated cells above. By morphological examination, it can be shown that relatively pure fractions of basal cells, spinous cells, and granular cells are obtained by this method. With respect to DNA distribution pattern, there was good agreement between that of keratinocytes separated by the microdissection-ultrasonic irradiation method, or by the chymotrypsin method as reported previously by us, and that obtained by the present method.     

Separation of keratinocytes by density gradient centrifugation for DNA cytofluorometry

A method for the separation of guinea pig epidermal keratinocytes, in which the Feulgen-stainable material suffers minimal damage, has been investigated. The principal stage involves trypsin treatment of the epidermal sheet, stripped from the dermis with ethylenediamine tetraacetic acid. The epidermal cells thus isolated are separated into three groups by centrifugation on a continuous colloidal silica (Percoll) density gradient. The resulting arrangement of the keratinocytes in the centrifuge tube corresponds to their arrangement in situ, with basal cells at the bottom and the more differentiated cells above. By morphological examination, it can be shown that relatively pure fractions of basal cells, spinous cells, and granular cells are obtained by this method. With respect to DNA distribution pattern, there was good agreement between that of keratinocytes separated by the microdissection-ultrasonic irradiation method, or by the chymotrypsin method as reported previously by us, and that obtained by the present method.     

Expression of the HB9 homeobox gene concomitant with proliferation accompanying epidermal stratification during development of chick embryonic tarsometatarsal skin

A homeobox gene, HB9, has been isolated from the tarsometatarsal skin of 13-day-old chick embryos using a degenerate RT-PCR-based screening method. In situ hybridization analysis revealed that, during development of chick embryonic skin, the HB9 gene was expressed in epidermal basal cells of the placodes, but not in those of interplacodes, and in the dermal cells under the placodes at 9 days before addition of an intermediate layer by proliferation of the basal cells in the placodes. With the onset of epidermal stratification, the direction of the basal cell mitosis changed, with the axis becoming vertical to the epidermal surface. Placodes and interplacodes form outer and inner scales, respectively, after they have elongated distally (Tanaka S, Kato Y (1983b) J Exp Zool 225: 271–283). During scale ridge elongation at 12–15 days, HB9 was strongly expressed in the epidermis of the outer scale face, where the cell proliferation is more active than in the epidermis of the inner scale face; hence, stratification of the outer scale face is more prominent than that of the inner scale face. After 16 days, when mitotic activity in the epidermal basal cells decreases and the thickness of the epidermis is maintained at a constant level, the HB9 expression decreases with the onset of epidermal keratinization. These results suggest that HB9 may be involved in the proliferation of the epidermal basal cells that accompanies epidermal stratification.     

Regulation of epidermal differentiation by a Distal-less homeodomain gene

The Distal-less-related homeodomain gene Dlx3 is expressed in terminally differentiated murine epidermal cells. Ectopic expression of this gene in the basal cell layer of transgenic skin results in a severely abnormal epidermal phenotype and leads to perinatal lethality. The basal cells of affected mice ceased to proliferate, and expressed the profilaggrin and loricrin genes which are normally transcribed only in the latest stages of epidermal differentiation. All suprabasal cell types were diminished and the stratum corneum was reduced to a single layer. These data indicate that Dlx3 misexpression results in transformation of basal cells into more differentiated keratinocytes, suggesting that this homeoprotein is an important regulator of epidermal differentiation.     

Immunomorphological analysis of G2-chalone localization during the process of rat epidermis histogenesis

The appearance of G2-chalone in the cytoplasm of the intermediate cell layer and partly in the periderm of 17-day-old rat embryo epidermis has been demonstrated by the indirect method of Coons using a monospecific antiserum. G2-chalone was absent from the basal cell layer of 17--21-day-old embryos and of the newborn rats. It was found in all the epidermal layers in 2--5-day-old postnatal rats, while in 6--9-day-old animals it was primarily detected in the cytoplasm of spinous and basal cells. Thus the localization of epidermal G2-chalone typical for defined tissue becomes stabilized at the end of epidermis histogenesis.     

An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis

Despite the well-characterised role of sonic hedgehog (Shh) in promoting interfollicular basal cell proliferation and hair follicle downgrowth, the role of hedgehog signalling during epidermal stem cell fate remains largely uncharacterised. In order to determine whether the three vertebrate hedgehog molecules play a role in regulating epidermal renewal we overexpressed sonic (Shh), desert (Dhh) and Indian (Ihh) hedgehog in the basal cells of mouse skin under the control of the human keratin 14 promoter. We observed no overt epidermal morphogenesis phenotype in response to Ihh overexpression, however Dhh overexpression resulted in a range of embryonic and adult skin manifestations indistinguishable from Shh overexpression. Two distinct novel phenotypes were observed amongst Shh and Dhh transgenics, one exhibiting epidermal progenitor cell hyperplasia with the other displaying a complete loss of epidermal tissue renewal indicating deregulation of stem cell activity. These data suggest that correct temporal regulation of hedgehog activity is a key factor in ensuring epidermal stem cell maintenance. In addition, we observed Shh and Dhh transgenic skin from both phenotypes developed lesions reminiscent of human basal cell carcinoma (BCC), indicating that BCCs can be generated despite the loss of much of the proliferative (basal) compartment. These data suggest the intriguing possibility that BCC can arise outside the stem cell population. Thus the elucidation of Shh (and Dhh) target gene activation in the skin will likely identify those genes responsible for increasing the proliferative potential of epidermal basal cells and the mechanisms involved in regulating epidermal stem cell fate.     

A computer simulation of cell stacking for even thickness in mammalian epidermis

The method of even stacking of epidermal cells in mammalian skin was studied by computer simulation. The epidermis consists of neat vertical columns of stacked, flattened, tetrakaidecahedral cells. Cells which have been proliferated in a basal layer migrate upwards, occupy the bottom regions of vertical columns, and become members constituting columns. Computer simulations demonstrated that the column height becomes considerably varied if the cells are randomly supplied from the basal layer. In contrast, if the cells are assumed to have an ability to find the uppermost region among the column's bases consisting of one base where the cell has reached and its neighbouring bases, the cells stack into columns whose heights are remarkably uniform even if the cells are randomly supplied. The results indicated that an epidermal structure consisting of the flattened polyhedral cells could itself function as a control mechanism of the epidermal thickness.     

Epidermal ridge formation in the human fetus: a correlation to the appearance of basal cell heterogeneity and the expression of epidermal growth factor receptor and cytokeratin polypeptides in the epidermis.

This paper aims to clarify an expression of epidermal growth factor receptor (EGFR) and cytokeratin 10 and/or 11 in relation to primary and secondary epidermal ridge formation of the human fetus. Firstly, scanning electron microscopy revealed heterogeneity in basal cell morphology during epidermal ridge formation. Basal cells had a uniform, smooth, and polygonal dermal surface until formation of the primary epidermal ridges. Thereafter, the dermal surface became ruffled and elliptic except at the primary epidermal ridges. Secondly, EGFR was detected by monoclonal antibody and autoradiography using 125I-EGF. The antibody reacted with primary epidermal ridge, stratum basale, stratum intermedium, and outer layer of sweat duct. The reactivity became stronger at the primary epidermal ridge than at the secondary one. The binding of 125I-EGF was concentrated in the primary epidermal ridge and sweat duct. Thirdly, cytokeratin 10 and/or 11, a maturation marker of keratinocytes, was detected by monoclonal antibody. The antibody reacted only with the stratum intermedium before secondary epidermal ridge formation. Afterward, it also reacted with the stratum basale of the secondary epidermal ridge but never reacted with that of primary epidermal ridge. The results indicate that basal cells of the secondary epidermal ridge enter the maturation process and suggest a localization of epidermal stem cells on the primary epidermal ridges. Concerning epidermal ridge formation, we suppose that the formation of the primary epidermal ridge causes the segregation of the epidermal stem cells, and that the increased density of the basal cells between the two primary epidermal ridges brings about the change in their dermal surface shape and the formation of the secondary epidermal ridge.     

Determination of epidermal G2-chalone in epidermal-type tissues

By means of monospecific immune serum, using the indirect method by Coons, the epidermal G2-chalone was revealed in the corneal epithelium, in the transitory epithelium of the urinary bladder, renal pelvis, as well as in stromal epithelial cells of the cortical substance and in thymic bodies, the facts that suggest epithelial nature of these tissues. In tracheal epithelium the method mentioned failed to reveal G2-chalone. Analysing localization of the epidermal G2-chalone in various tissues of the epidermal origin, it has been stated that in the non-cornified multistratified flat epithelium it is present in cellular cytoplasm of all layers, while in the cornified epithelium - it is predominantly detected in basal and scupular cells. A suggestion is made that distribution of the intratissue epidermal G2-chalone depends on the process of cornification. A possibility to use G2-chalone as a marker for tissues of the epidermal type is discussed.     

BMP-FGF Signaling Axis Mediates Wnt-Induced Epidermal Stratification in Developing Mammalian Skin

Epidermal stratification of the mammalian skin requires proliferative basal progenitors to generate intermediate cells that separate from the basal layer and are replaced by post-mitotic cells. Although Wnt signaling has been implicated in this developmental process, the mechanism underlying Wnt-mediated regulation of basal progenitors remains elusive. Here we show that Wnt secreted from proliferative basal cells is not required for their differentiation. However, epidermal production of Wnts is essential for the formation of the spinous layer through modulation of a BMP-FGF signaling cascade in the dermis. The spinous layer defects caused by disruption of Wnt secretion can be restored by transgenically expressed Bmp4. Non-cell autonomous BMP4 promotes activation of FGF7 and FGF10 signaling, leading to an increase in proliferative basal cell population. Our findings identify an essential BMP-FGF signaling axis in the dermis that responds to the epidermal Wnts and feedbacks to regulate basal progenitors during epidermal stratification.     

Changes in Soybean Agglutinin (SBA) and Peanut Agglutinin (PNA) Binding Pattern in the Epidermis of the Developing Chick Embryo

Lectin binding pattern in the developing chick embryonic epidermis was studied using peroxidase labeling method. The epidermis of the 13-day-old embryo is in an undifferentiated state. Little binding of soybean agglutinin (SBA), specific for N-acetyl-D-galactosamine, and peanut agglutinin (PNA), specific for β-D-galactose, was seen in such epidermal cells. As the epidermis developed toward keratinization, the cell membrane of the differentiating flattened cells was positively stained with SBA and PNA. The positive staining was also seen in the supranuclear region of the cells located between the flattened cells and the basal cells. The basal cells remained unstained in all the stages of development. Similar staining pattern with SBA and PNA was seen in the cultured skin explants during the epidermal differentiation in vitro. These observations show that the SBA- and PNA-reactive glycoconjugates accumulate during the epidermal cell differentiation, suggesting their important roles in the maintenance of the ordered structure of the epidermis.     

Basal Keratinocytes Contribute to All Strata of the Adult Zebrafish Epidermis

The epidermis of terrestrial vertebrates is a stratified epithelium and forms an essential protective barrier. It is continually renewed, with dead corneocytes shed from the surface and replaced from a basal keratinocyte stem cell population. Whilst mouse is the prime model system used for epidermal studies, there is increasing employment of the zebrafish to analyse epidermis development and homeostasis, however the architecture and ontogeny of the epidermis in this system are incompletely described. In particular, it is unclear if adult zebrafish epidermis is derived entirely from the basal epidermal stem cell layer, as in the mouse, or if the most superficial keratinocyte layer is a remnant of the embryonic periderm. Furthermore, a relative paucity of cellular markers and genetic reagents to label and manipulate the basal epidermal stem cell compartment has hampered research. Here we show that the type I keratin, krtt1c19e, is a suitable marker of the basal epidermal layer and identify a krtt1c19e promoter fragment able to drive strong and specific expression in this cell type. Use of this promoter to express an inducible Cre recombinase allowed permanent labelling of basal cells during embryogenesis, and demonstrated that these cells do indeed generate keratinocytes of all strata in the adult epidermis. Further deployment of the Cre-Lox system highlighted the transient nature of the embryonic periderm. We thus show that the epidermis of adult zebrafish, as in the mouse, derives from basal stem cells, further expanding the similarities of epidermal ontogeny across vertebrates. Future use of this promoter will assist genetic analysis of basal keratinocyte biology in zebrafish.     

Lineage of anuran epidermal basal cells and their differentiation potential in relation to metamorphic skin remodeling

The anuran remodels the larval epidermis into the adult one during metamorphosis. Larval and adult epidermal cells of the bullfrog were characterized by determining the presence of huge cytoplasmic keratin bundles and the expression profiles of specific marker genes, namely colalpha1 (collagen alpha1 (I)), rlk (larval keratin) and rak (adult keratin). We identified four types of epidermal basal cells: (i) basal skein cells that have keratin bundles and express colalpha1 and rlk; (ii) rak+-basal skein cells that have keratin bundles and express colalpha1, rlk, and rak; (iii) larval basal cells that express rlk and rak; and (iv) adult basal cells that express rak. These traits suggested that these basal cells are on the same lineage in which basal skein cells are the original progenitor cells that consecutively differentiate into rak+-basal skein cells into larval basal cells, and finally into adult basal cells. To directly verify the differentiation potential of larval basal cells into adult ones, the mono-layered epidermis composed of larval basal cells was cultured in the presence of aldosterone and thyroid hormone. In this culture, larval basal cells differentiated into adult basal cells that reconstituted the adult epidermis. Thus, it was concluded that larval basal cells are the direct progenitor cells of the adult epidermal stem cells.     

Altered distribution of phospholipase C-gamma 1 in benign hyperproliferative epidermal diseases.

Phospholipase C-gamma 1 (PLC-gamma 1) is a well characterized substrate for the epidermal growth factor receptor tyrosine kinase and has been implicated in the intracellular biochemical signaling cascade which occurs following stimulation of cells with epidermal growth factor. The in vivo localization of PLC-gamma 1 was examined by immunohistochemistry in sections of normal human skin and in skin sections from a diverse series of hyperproliferative epidermal conditions (psoriasis, seborrheic keratoses, acrochordons, and margins near second-degree burns). Immunoreactive PLC-gamma 1 was detected only in the basal compartment of normal skin but was readily detectable in both the basal and outer epidermal compartment in hyperproliferative skin conditions. In addition, immunoreactive PLC-gamma 1 colocalizes with immunoreactive epidermal growth factor receptor in both normal and hyperproliferative epidermis.     

Fluorochrome-coupled lectins reveal distinct cellular domains in human epidermis

The distribution of saccharide moieties in human interfollicular epidermis was studied with fluorochrome-coupled lectins. In frozen sections Concanavalin A (Con A), Lens culinaris agglutinin (LCA), Ricinus communis agglutinin I (RCAI), and wheat germ agglutinin (WGA) stained intensively both dermis and viable epidermal cell layers, whereas peanut agglutinin (PNA) bound only to living epidermal cell layers. Ulex europaeus agglutinin I (UEAI) bound to dermal endothelial cells and upper cell layers of the epidermis but left the basal cell layer unstained. Dolichos biflorus agglutinin (DBA) bound only to basal epidermal cells, whereas both soybean agglutinin (SBA) and Helix pomatia agglutinin (HPA) showed strong binding to the spinous and granular cell layers. On routinely processed paraffin sections, a distinctly different staining pattern was seen with many lectins, and to reveal the binding of some lectins a pretreatment with protease was required. All keratin-positive cells in human epidermal cell suspensions, obtained with the suction blister method, bound PNA, whereas only a fraction of the keratinocytes bound either DBA or UEAI. Such a difference in lectin binding pattern was also seen in epidermal cell cultures both immediately after attachment and in organized cell colonies. This suggests that in addition to basal cells, more differentiated epidermal cells from the spinous cell layer are also able to adhere and spread in culture conditions. Gel electrophoretic analysis of the lectin-binding glycoproteins in detergent extracts of metabolically labeled primary keratinocyte cultures revealed that the lectins recognized both distinct and shared glycoproteins. A much different lectin binding pattern was seen in embryonic human skin: fetal epidermis did not show any binding of DBA, whereas UEAI showed diffuse binding to all cell layers but gave a bright staining of dermal endothelial cells. This was in contrast to staining results obtained with a monoclonal cytokeratin antibody, which showed the presence of a distinct basal cell layer in fetal epidermis also. The results indicate that expression of saccharide moieties in human epidermal keratinocytes is related to the stage of cellular differentiation, different cell layers expressing different terminal saccharide moieties. The results also suggest that the emergence of a mature cell surface glycoconjugate pattern in human epidermis is preceded by the acquisition of cell layer-specific, differential keratin expression.     

The Effect of Hydrocortisone on the Epidermal Proliferation in an Organ Culture of Chick Embryonic Skin

Hydrocortisone is regarded as an initiator of keratinization in embryonic skin. The present investigation dealt with the effect of hydrocortisone on the proliferation of epidermal cells during early development: Cell kinetic analyses using ³H-thymidine autoradiography were applied to a skin organ culture prepared from a 13-day chick embryo.
Hydrocortisone at a concentration between 0.01 and 1.0 μg/ml was effective in initiating a morphological change leading to the epidermal keratinization in vitro and caused a marked decrease in the mitotic and labeling indices of epidermal basal cells, the decrease being maximum at 2 days of culture previous to the morphological change.
During continuous labeling with ³H-thymidine, the number of labeled basal cells reached 100% within 2 days in the control and 4 days in the culture treated with hydrocortisone. This confirmed that the growth fraction of epidermal basal cells was 1.0 even after the administration of hydrocortisone.
The duration of each cell cycle phase at 2 days of culture was determined by percent labeled mitoses and double-labeling analyses. It was concluded that hydrocortisone extended the generation time of epidermal basal cells at this time point about three fold over the control. This extension was mainly due to the elongation of the G ₁ phase.     

小鼠表皮干细胞的分离与培养

目的:探讨体外分离和培养小鼠表皮干细胞和分析表皮干细胞克隆形成能力的方法。方法:采用中性蛋白酶和胰酶消化新生小鼠表皮基底层细胞,将细胞直接接种在细胞瓶中,在无滋养层条件下培育;利用表皮干细胞标记物K15和α6整联蛋白进行免疫荧光鉴定;以小鼠胚胎成纤维细胞作为滋养层与成年小鼠角质细胞共培养,进而分析表皮干细胞的克隆形成能力。结果:新生小鼠表皮干细胞克隆在培养2～3 d后开始形成,细胞核质较小,细胞呈小而圆的形态特征;传代后的细胞可以被K15和α6整联蛋白特异性标记。结论:利用该方法能够实现对小鼠表皮干细胞的体外培养和传代。     

A preformed basal lamina alters the metabolism and distribution of hyaluronan in epidermal keratinocyte "organotypic" cultures grown on collagen matrices

A rat epidermal keratinocyte (REK) line which exhibits histodifferentiation nearly identical to the native epidermis when cultured at an air-liquid interface was used to study the metabolism of hyaluronan, the major intercellular macromolecule present in basal and spinous cell layers. Two different support matrices were used: reconstituted collagen fibrils with and without a covering basal lamina previously deposited by canine kidney cells. REKs formed a stratified squamous, keratinized epithelium on both support matrices. Hyaluronan and its receptor, CD44, colocalized in the basal and spinous layers similar to their distribution in the native epidermis. Most (approximately 75%) of the hyaluronan was retained in the epithelium when a basal lamina was present while most (approximately 80%) diffused out of the epithelium in its absence. While REKs on the two matrices synthesized hyaluronan at essentially the same rate, catabolism of this macromolecule was much higher in the epithelium on the basal lamina (half-life approximately 1 day, similar to its half-life in native human epidermis). The formation of a true epidermal compartment in culture bounded by the cornified layer on the surface and the basal lamina subjacent to the basal cells provides a good model within which to study epidermal metabolism.     

Structure and regeneration of the planarian basal lamina: An ultrastructural study

The structure and regeneration of the planarian subepidermal basement membrane or basal lamina have been electron microscopically examined, particularly in relation to the changes of extracellular products at the wounded area. The intact basal lamina consists of three structural elements; namely, an electron-lucent zone, a limiting layer and a microfibrillar layer. Ultrastructural changes during wound healing have suggested that the amorphous material secreted in the interspace between the epidermal cells and blastema contains precursors of the basal lamina. Within the amorphous zone two distinct phases of the basal lamina regeneration are observed: one is a reconstitution of the limiting layer and the other is a polymerization of the microfibrils. The limiting layer arises from areas subjacent to newly developed hemidesmosomes of epidermal cells. The unit microfibrils are formed from an accumulation of the precursors through transitional smaller microfibrils. At the late stage, individual mature microfibrils are regularly lined with the limiting layer and cell membranes of the newly differentiated muscle fibres. On the basis of these observations we suggest that the planarian basal lamina is regenerated by the interaction between epidermal cells and myoblasts.     

Collagen type IV of the basal membrane of the epithelium of the skin in certain forms of pathology

Using immunofluorescence, attenuation or complete disappearance of type IV collagen antiserum reaction were shown in the epidermal basal membrane zone of SLE patients. Some positive material was revealed in the intercellular spaces of the epidermis. There is an inverse correlation between the amount of immune complexes in the dermo-epidermal region and the intensity of anticollagen serum reaction in the basal membrane zone. This can be explained by the toxic effect of immune complexes on collagen-synthesizing cells and the disturbance of this protein fixation in the basal membrane followed by its sequestration from the affected regions. Complex therapy including hemoperfusion restores normal collagen location in the epidermal basal membrane.