人表皮细胞分离培养的研究

目的探索来源于人的表皮细胞的分离培养方法,为其进一步作为皮肤组织工程中的种子细胞或临床应用奠定前期研究基础。方法取3~9岁健康儿童包皮环切术后包皮,经分离酶处理分离真表皮,再将表皮以胰蛋白酶消化为细胞悬液,分别接种于有血清培养基DMEM和无血清培养基K-SFM中进行细胞培养,观察表皮细胞生长融合情况及克隆形成率。结果表皮细胞在DMEM和K-SFM培养液中均能融合成片,但在K-SFM中的融合成片时间明显短于在DMEM中所需时间;在K-SFM中2周时克隆形成率显著高于在DMEM中的克隆形成率。结论两步酶法分离表皮细胞接种于K-SFM中培养,是一种简便有效的表皮细胞分离培养方法。     

Adhesion of Human Epidermal Keratinocytes to Laminin

We have examined the mechanism by which human epidermal keratinocytes adhere to the A/B1/B2 (α₁β₁γ₁) form of laminin. Adhesion could be completely inhibited with an antibody to the β₁ integrin subunit or a combination of antibodies recognising the α₂β₂ a₃β₁ and α₆β₄ integrins. Keratinocytes adhered in the presence of magnesium and manganese ions, but calcium ions did not support adhesion and inhibited adhesion when combined with magnesium and manganese. The effects of anti-integrin antibodies (including a stimulatory antibody to the β₁ subunit) were not influenced by specific cations, with the exception that inhibition by an antibody to α₂β₁ was abrogated by the presence of manganese ions. The E3 and E8 proteolytic fragments of laminin did not support keratinocyte adhesion and heat inactivation of the E8 site in intact laminin did not reduce adhesion. Three laminin fragments that did support adhesion were P1, E4 and E1X-Nd, P1 activity being attributable at least in part to the RGD site; antibody blocking experiments suggested that adhesion to these fragments was primarily via α₁β₃. The synthetic peptide GD-6, derived from the carboxy terminus of the laminin A chain (included within E3) did support adhesion, but the significance of this observation is unclear, since a scrambled control peptide could also support adhesion. In conclusion, keratinocyte adhesion to A/B1/B2 laminin involves three integrins and multiple binding sites that are different from those defined previously.     

人表皮生长因子基因转染原代角质形成细胞

目的:将带信号肽的人表皮生长因子基因转染原代成人角质形成细胞, 证实细胞活性及hEGF的有效表达, 为后期的皮肤修复研究打下基础.方法:先酶切验证pcDNA3.1-hEGF, 后消化成人皮肤组织, 以Defined Keratinocyte-SFM(DKSFM)传代培养角质形成细胞并鉴定, 脂质体转染质粒pcDNA3.1-hEGF入细胞, 转基因细胞培养48 h后作RT-PCR和Western-blot分析, 上清分别进行放射免疫测定和MTT测定.结果:质粒pcDNA3.1-hEGF上的hEGF序列经测序证实,双酶切后获得约230 bp和5.4 kb条带;成人角质形成细胞体外培养可快速稳定增殖, 转hEGF基因细胞经RT-PCR扩增出一条约230 bp的特异性条带, Western-blot检测到hEGF表达明显升高;放射免疫法和MTT实验证实转基因细胞有稳定的hEGF蛋白分泌.结论:质粒pcDNA3.1-hEGF在脂质体介导下成功转染成人皮肤角质形成细胞, 转基因细胞能分泌有生物活性的EGF;体外培养的成人皮肤角质形成细胞可见少量EGF分泌.     

Co-localization of Racl and E-Cadherin in Human Epidermal Keratinocytes

The Racl small GTP-binding protein is known to be involved in reorganization of the actin cytoskeleton and in regulation of intracellular signal transduction. The assembly and maintenance of cadherin-based cell-cell junctions in epidermal keratinocytes is thought to be dependent on activity of Racl. In this study we have generated green fluorescent protein (GFP)-tagged wild type, dominant negative and constitutively active Racl expression vectors and analyzed distribution of Racl following microinjection of human SCC12F epidermal keratinocytes. Wild type, dominant negative and constitutively active GFP-Racl proteins distribute to sites of cell-cell adhesion and co-localize with E-cadherin and the catenins. Disruption of cadherin-based junctions by reduction in extracellular calcium concentrations, or by use of antibodies to E-cadherin, results in redistribution of Racl away from sites of cell-cell interaction but the co-localization with E-cadherin is maintained. In addition, expression of constitutively active GFP-Racl results in formation of membrane ruffles on the apical surface of cells and intracellular vesicles. Interestingly, co-localization of Racl with E-cadherin is maintained in these structures. In contrast to previously published work we find that expression of dominant negative Racl neither disrupts cell-cell adhesion nor prevents assembly of new cadherin-based adhesion structures.     

Modulation of Heart Fibroblast Migration and Collagen Gel Contraction by IGF-I

Dynamic interactions between cells and the extracellular matrix are essential in the regulation of a number of cellular processes including migration, adhesion, proliferation and differentiation. A variety of factors have been identified which modulate these interactions including transforming growth factor+, platelet-derived growth factor and others. Insulin-like growth factors have been shown to regulate collagen production by heart fibroblasts; however, the effects of this growth factor on the interactions of heart fibroblasts with the extracellular matrix have not been examined. The present studies were carried out to determine the effects of IGF-I on the ability of fibroblasts to interact with the extracellular matrix and to begin to determine the mechanisms of this response. These experiments illustrate that IGF-I treatment results in increased migration, collagen reorganization and gel contraction by heart fibroblasts. IGF-I has been shown to activate both the mitogen-activated protein kinase and phophatidylinositol-3 kinase pathways in isolated cells. Experiments with pharmacological antagonists of these pathways indicate that the mitogen-activated protein kinase pathway is essential for IGF-I stimulated collagen gel contraction by fibroblasts. These studies illustrate that IGF-I modulates the ability of fibroblasts to interact with the collagen matrix and that activation of multiple signaling pathways by IGF-I may produce distinct downstream responses in these cells.     

Expression and Functional Role of Sox9 in Human Epidermal Keratinocytes

In this study, we investigated the expression and putative role of Sox9 in epidermal keratinocyte. Immunohistochemical staining showed that Sox9 is predominantly expressed in the basal layer of normal human skin epidermis, and highly expressed in several skin diseases including psoriasis, basal cell carcinoma, keratoacanthoma and squamous cell carcinoma. In calcium-induced keratinocyte differentiation model, the expression of Sox9 was decreased in a time dependent manner. When Sox9 was overexpressed using a recombinant adenovirus, cell growth was enhanced, while the expression of differentiation-related genes such as loricrin and involucrin was markedly decreased. Similarly, when rat skin was intradermally injected with the adenovirus expressing Sox9, the epidermis was thickened with increase of PCNA positive cells, while the epidermal differentiation was decreased. Finally, UVB irradiation induced Sox9 expression in cultured human epidermal keratinocytes, and keratinocytes are protected from UVB-induced apoptosis by Sox9 overexpression. Together, these results suggest that Sox9 is an important regulator of epidermal keratinocytes with putative pro-proliferation and/or pro-survival functions, and may be related to several cutaneous diseases that are characterized by abnormal differentiation and hyperproliferation.     

Hepatocytic Cells Form Bile Duct-like Structures within a Three-Dimensional Collagen Gel Matrix

It has been suggested that hepatocytes have the ability to form bile ductal structures during normal development and in various pathological conditions of the liver. In the present study, we attempted to establish anin vitromodel of ductal morphogenesis of hepatocytic cells by combining an aggregate culture and a type I collagen gel culture. When spheroidal aggregates of rat or mouse primary hepatocytes were embedded within the collagen gel matrix and then cultured with a medium containing a fibroblast-conditioned medium, the aggregates extended many dendritic processes composed of a trabecular arrangement of cells. Dendritic morphogenesis was also seen in embedded aggregates of immortal liver epithelial cell lines, which spontaneously emerged during long-term cultures of mouse primary hepatocytes. A similar morphogenesis was induced by the presence of insulin in the medium. Although epidermal growth factor (EGF) and hepatocyte growth factor (HGF) showed only a small effect on the morphogenesis of most of the hepatocytic cells when used alone, these factors, especially EGF, enhanced the morphogenetic effect of insulin. Electron microscopical observations revealed luminal structures lined by microvilli within these dendritic processes, indicating ductal differentiation. Immunocytochemically, the dendritic processes were positive for cytokeratin 19, a marker for bile duct cells. On the other hand, an H-ras-transformed mouse liver epithelial cell line and rat hepatocellular carcinoma cell lines did not demonstrate the organized morphogenesis. Our results indicate that hepatocytic cells can produce bile duct-like structures in the presence of the type I collagenous matrix and soluble morphogenetic factors.     

Quercetin-Induced Melanogenesis in a Reconstituted Three-Dimensional Human Epidermal Model

Quercetin (3,5,7,3',4'-pentahydroxyflavone) is one of the most abundant natural flavonoids. It is present in various common vegetables and fruits. In this report, we examined the effect of quercetin on melanogenesis using a three-dimensional reconstituted human epidermal culture model, MelanoDerm, which is a new commercially-available cultured human epidermis containing functional melanocytes. Treatment with 10 microM quercetin induced an increase of tyrosinase activity in cultured epidermis after 3-5 days in time-dependent manner. In the quercetin-treated epidermis, furthermore, melanin content and tyrosinase expression were markedly increased, as shown by immunohistochemistry after a 7-day culture period. Ultrastructural studies clearly indicated an accumulation of mature melanosomes (stages III and IV) inside the basal layer of the cultured epidermis after the quercetin treatment. In addition, the dendrites of melanocytes extended further towards the adjacent keratinocytes after quercetin treatment. These results suggest that quercetin has an effect on maturation of melanosomes and that quercetin has the potential to induced melanogenesis in human epidermis.     

Recombinant Human Collagen XV Regulates Cell Adhesion and Migration

The C-terminal end of collagen XV, restin, has been the focus of several studies, but the functions of full-length collagen XV have remained unknown. We describe here studies on the production, purification, and function of collagen XV and the production of a monoclonal N-terminal antibody to it. Full-length human collagen XV was produced in insect cells using baculoviruses and purified from the cell culture medium. The yield was 15 mg/liter of cell culture medium. The collagen XV was shown to be trimeric, with disulfide bonds in the collagenous region. Rotary shadowing electron microscopy revealed rod-like molecules with a mean length of 241.8 nm and with a globular domain at one end. The globular domain was verified to be the N-terminal end by N-terminal antibody binding. The molecules show flexibility in their conformation, presumably due to the many interruptions in their collagenous domains. The ability of collagen XV to serve as a substrate for cells was tested in cell adhesion assays, and it was shown that cells did not bind to collagen XV-coated surfaces. When added to the culture medium of fibroblasts and fibrosarcoma cells, however, collagen XV rapidly bound to their fibronectin network. Solid phase assays showed that collagen XV binds to fibronectin, laminin, and vitronectin and that it binds to the collagen/gelatin-binding domain of fibronectin. No binding was detected to fibrillar collagens, fibril-associated collagens, or decorin. Interestingly, collagen XV was found to inhibit the adhesion and migration of fibrosarcoma cells when present in fibronectin-containing matrices.     

Infection by Toxoplasma gondii Induces Amoeboid-Like Migration of Dendritic Cells in a Three-Dimensional Collagen Matrix

Toxoplasma gondii, an obligate intracellular parasite of humans and other warm-blooded vertebrates, invades a variety of cell types in the organism, including immune cells. Notably, dendritic cells (DCs) infected by T. gondii acquire a hypermigratory phenotype that potentiates parasite dissemination by a ‘Trojan horse’ type of mechanism in mice. Previous studies have demonstrated that, shortly after parasite invasion, infected DCs exhibit hypermotility in 2-dimensional confinements in vitro and enhanced transmigration in transwell systems. However, interstitial migration in vivo involves interactions with the extracellular matrix in a 3-dimensional (3D) space. We have developed a collagen matrix-based assay in a 96-well plate format that allows quantitative locomotion analyses of infected DCs in a 3D confinement over time. We report that active invasion of DCs by T. gondii tachyzoites induces enhanced migration of infected DCs in the collagen matrix. Parasites of genotype II induced superior DC migratory distances than type I parasites. Moreover, Toxoplasma-induced hypermigration of DCs was further potentiated in the presence of the CCR7 chemotactic cue CCL19. Blocking antibodies to integrins (CD11a, CD11b, CD18, CD29, CD49b) insignificantly affected migration of infected DCs in the 3D matrix, contrasting with their inhibitory effects on adhesion in 2D assays. Morphological analyses of infected DCs in the matrix were consistent with the acquisition of an amoeboid-like migratory phenotype. Altogether, the present data show that the Toxoplasma-induced hypermigratory phenotype in a 3D matrix is consistent with integrin-independent amoeboid DC migration with maintained responsiveness to chemotactic and chemokinetic cues. The data support the hypothesis that induction of amoeboid hypermigration and chemotaxis/chemokinesis in infected DCs potentiates the dissemination of T. gondii.     

Use of Collagen Gel as a Three-Dimensional In Vitro Model to Study Electropermeabilization and Gene Electrotransfer

Gene electrotransfer is a promising nonviral method that enables transfer of plasmid DNA into cells with electric pulses. Although many in vitro and in vivo studies have been performed, the question of the implied gene electrotransfer mechanisms is largely open. The main obstacle toward efficient gene electrotransfer in vivo is relatively poor mobility of DNA in tissues. Since cells are mechanically coupled to their extracellular environment and act differently compared to standard in vitro conditions, we developed a three-dimensional (3-D) in vitro model of CHO cells embedded in collagen gel as an ex vivo model of tissue to study electropermeabilization and different parameters of gene electrotransfer. For this purpose, we first used propidium iodide to detect electropermeabilization of CHO cells embedded in collagen gel. Then, we analyzed the influence of different concentrations of plasmid DNA and pulse duration on gene electrotransfer efficiency. Our results revealed that even if cells in collagen gel can be efficiently electropermeabilized, gene expression is significantly lower. Gene electrotransfer efficiency in our 3-D in vitro model had similar dependence on concentration of plasmid DNA and pulse duration comparable to in vivo studies, where longer (millisecond) pulses were shown to be more optimal compared to shorter (microsecond) pulses. The presented results demonstrate that our 3-D in vitro model resembles the in vivo situation more closely than conventional 2-D cell cultures and, thus, provides an environment closer to in vivo conditions to study mechanisms of gene electrotransfer.     

Polarized Cell Migration during Cell-to-Cell Transmission of Herpes Simplex Virus in Human Skin Keratinocytes

In addition to transmission involving extracellular free particles, a generally accepted model of virus propagation is one wherein virus replicates in one cell, producing infectious particles that transmit to the next cell via cell junctions or induced polarized contacts. This mechanism of spread is especially important in the presence of neutralizing antibody, and the concept underpins analysis of virus spread, plaque size, viral and host functions, and general mechanisms of virus propagation. Here, we demonstrate a novel process involved in cell-to-cell transmission of herpes simplex virus (HSV) in human skin cells that has not previously been appreciated. Using time-lapse microscopy of fluorescent viruses, we show that HSV infection induces the polarized migration of skin cells into the site of infection. In the presence of neutralizing antibody, uninfected skin cells migrate to the initial site of infection and spread over infected cells to become infected in a spatially confined cluster containing hundreds of cells. The cells in this cluster do not undergo cytocidal cell lysis but harbor abundant enveloped particles within cells and cell-free virus within interstitial regions below the cluster surface. Cells at the base and outside the cluster were generally negative for virus immediate-early expression. We further show, using spatially separated monolayer assays, that at least one component of this induced migration is the paracrine stimulation of a cytotactic response from infected cells to uninfected cells. The existence of this process changes our concept of virus transmission and the potential functions, virus, and host factors involved.     

Strain-Dependent Augmentation of Tight-Junction Barrier Function in Human Primary Epidermal Keratinocytes by Lactobacillus and Bifidobacterium Lysates

In this study, we investigated whether probiotic lysates can modify the tight-junction function of human primary keratinocytes. The keratinocytes were grown on cell culture inserts and treated with lysates from Bifidobacterium longum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus fermentum, or Lactobacillus rhamnosus GG. With the exception of L. fermentum (which decreased cell viability), all strains markedly enhanced tight-junction barrier function within 24 h, as assessed by measurements of transepithelial electrical resistance (TEER). However, B. longum and L. rhamnosus GG were the most efficacious, producing dose-dependent increases in resistance that were maintained for 4 days. These increases in TEER correlated with elevated expression of tight-junction protein components. Neutralization of Toll-like receptor 2 abolished both the increase in TEER and expression of tight-junction proteins induced by B. longum, but not L. rhamnosus GG. These data suggest that some bacterial strains increase tight-junction function via modulation of protein components but the different pathways involved may vary depending on the bacterial strain.     

Fibroblasts Stimulate Epithelization of Collagen Gel

We studied the influence of human embryonic fibroblasts on epithelization of collagen gel. Introduction of the fibroblasts into the gel stimulates proliferation of keratinocytes plated on the gel surface. The presence of fibroblasts in the gel also affects the pattern of keratinocyte migration on the gel and induces pronounced polarization of the migrating colonies.     

Infection of Keratinocytes with Trichophytum rubrum Induces Epidermal Growth Factor-Dependent RNase 7 and Human Beta-Defensin-3 Expression

Human keratinocytes are able to express various antimicrobial peptides (AMP) to protect the skin from exaggerated microbial colonization and infection. Recently, in vitro growth-inhibiting activity of the skin-derived AMP psoriasin, RNase 7 and human beta-defensin (hBD)-2 against dermatophytes such as Trichophyton (T.) rubrum have been reported. To evaluate whether keratinocytes are able to respond to T. rubrum infection by an induced expression of AMP we exposed primary keratinocytes to living conidia of T. rubrum. This led to conidia germination and mycelial growth which was paralleled by a strong gene induction of the skin-derived AMP RNase 7 and hBD-3. Gene expression of the AMP psoriasin (S100A7) and hBD-2 were only slightly induced. The T. rubrum-mediated RNase 7 gene induction was accompanied by increased secretion of RNase 7. Parallel treatment of the keratinocytes with T. rubrum and the cytokine combination IL-17A/IFN-γ resulted in synergistic induction of RNase 7 and hBD-3 expression. Since patients receiving therapy by inhibition of the epidermal growth factor receptor (EGFR) more often suffer from dermatophytoses we investigated whether EGFR may be involved in the T. rubrum-mediated RNase 7 and hBD-3 induction. Primary keratinocytes incubated with an EGFR blocking antibody as well as with the EGFR antagonist AG1478 showed a significantly diminished RNase 7 and hBD-3 induction upon exposure of the keratinocytes to T. rubrum indicating that EGFR is involved in the T. rubrum-mediated induction of RNase 7 and hBD-3. The growth of T. rubrum in vitro was inhibited by hBD-3 in a dose-dependent manner suggesting that hBD-3 may contribute to cutaneous innate defense against T. rubrum. Taken together our data indicate that keratinocytes are able to initiate a fast defense response towards T. rubrum by the increased expression of AMP active against T. rubrum. A dysregulation of AMP may contribute to chronic and recurring dermatophytoses.     

人的原代上皮角质形成细胞和永生化的上皮角质形成细胞HaCaT的增殖能力的比较

目的：筛选适合充当组织工程皮肤的种子细胞,比较原代培养的人原代上皮角质形成细胞和永生化的上皮角质形成细胞HaCaT的增值能力。方法：将两种细胞（小儿包皮环切术后的组织培养表皮角质形成细胞,永生化的上皮角质形成细胞HaCaT）,分别接种于96孔板,通过MTT检测细胞1,3,5,7,9,11天的生长情况;当两种细胞融合至60%时分别取1×106个细胞,通过流式细胞检测细胞的周期。结果：永生化的上皮角质形成细胞HaCaT每隔一天即可传代一次,原代上皮角质形成细胞每3天传代一次;细胞周期：永生化上皮角质形成细胞HaCaT的G1期和S期的比例高于原代上皮角质形成细胞。生长曲线：MTT检测两种细胞1,3,5,7,9,11的生长情况,永生化的上皮角质形成细胞HaCaT的生长速度明显高于原代上皮角质形成细胞。结论：永生化上皮角质形成细胞HaCaT的增殖能力要高于原代培养的上皮角质形成细胞。     

A New Space-Time Computer Simulation Method for Human Migration

Migration is a universal but poorly understood human behavior, and new analytic tools for studying migration are badly needed. In this article, I describe a new computer simulation method for migrating human populations, which moves simulated human actors" on a lattice of points according to simple probability rules. The method includes models of birth, death, crowding, and competition, as well as of migration. Simulations are developed for a number of specific problems, including migration into empty continents, the effect of competition and migration on the spatial distribution of populations, the effect of biased migration on urban population structure, and the distribution of city sizes determined by migration. Direct comparison of simulations with measured population distributions demonstrates the plausibility of the models. The simulations suggest that seemingly complex problems of human space-time population dynamics may be resolved into simple behavioral rules and that migration phenomena can be subjected to scientific analysis. [Key words: migration, computer simulation, random walk, urban structures]     

A Three-Dimensional Computational Model of Collagen Network Mechanics

Extracellular matrix (ECM) strongly influences cellular behaviors, including cell proliferation, adhesion, and particularly migration. In cancer, the rigidity of the stromal collagen environment is thought to control tumor aggressiveness, and collagen alignment has been linked to tumor cell invasion. While the mechanical properties of collagen at both the single fiber scale and the bulk gel scale are quite well studied, how the fiber network responds to local stress or deformation, both structurally and mechanically, is poorly understood. This intermediate scale knowledge is important to understanding cell-ECM interactions and is the focus of this study. We have developed a three-dimensional elastic collagen fiber network model (bead-and-spring model) and studied fiber network behaviors for various biophysical conditions: collagen density, crosslinker strength, crosslinker density, and fiber orientation (random vs. prealigned). We found the best-fit crosslinker parameter values using shear simulation tests in a small strain region. Using this calibrated collagen model, we simulated both shear and tensile tests in a large linear strain region for different network geometry conditions. The results suggest that network geometry is a key determinant of the mechanical properties of the fiber network. We further demonstrated how the fiber network structure and mechanics evolves with a local formation, mimicking the effect of pulling by a pseudopod during cell migration. Our computational fiber network model is a step toward a full biomechanical model of cellular behaviors in various ECM conditions.     

Human EGF Receptor (HER) Family and Heregulin Members Are Differentially Expressed in Epidermal Keratinocytes and Modulate Differentiation

Human EGF receptor (HER), also designated HER1, is an activatable tyrosine kinase receptor. HER1 activation regulates cell growth and differentiation in epidermal keratinocytes. Expression of other HER family members was investigated in human keratinocytes cultured under autocrine conditions. HER2 and HER3 are expressed and upregulated by confluence, concurrent with induction of epidermal differentiation. HER4 is not expressed by keratinocytes. Maximum expression of the cognate ligand, heregulin, is observed in subconfluent keratinocytes, and the expression of both heregulin alpha and beta isoforms is downregulated with confluence. Recombinant heregulin isoforms have no effect on colony formation and keratinocyte proliferation, but heregulin beta activates tyrosine phosphorylation of HER2 and HER3, with no effect on HER1, in confluent differentiating keratinocyte cultures. Also, heregulin beta increases HER2/HER3 heterodimerization under those conditions. Treatment of confluent cultures by heregulin beta correlates with cell signaling and inhibition of epidermal differentiation. Together, HER2, HER3, and heregulin constitute a potential autocrine-paracrine system involved in epidermal homeostasis and repair, as well as in hyperproliferative pathologies.     

Extracellularly Extruded Syntaxin-4 Is a Potent Cornification Regulator of Epidermal Keratinocytes

In the skin epidermis, keratinocytes undergo anchorage-dependent cornification, which gives rise to stratified multilayers, each with a distinct differentiation feature. The active formation of the cornified cell envelope (CCE), an important element in the skin barrier, occurs in keratinocytes of the upper epidermal layers and impacts their terminal differentiation. In the present study, we identified the extracellularly extruded syntaxin-4 as a potent differentiation regulator of epidermal keratinocytes. We found that differentiation stimuli led to the acceleration of syntaxin-4 exposure at the keratinocyte cell surface and that the artificial control of extracellular syntaxin-4, either by the forced expression of several syntaxin-4 mutants with structural alterations at the putative functional core site (AIEPQK), or by using antagonistic circular peptides containing this core sequence, dramatically influenced the CCE formation, with spatial misexpression of TGase1 and involucrin. We also found that the topical application of a peptide that exerted the most prominent antagonistic activity for syntaxin-4, named ST4n1, evidently prevented the formation of the hyperplastic and hyperkeratotic epidermis generated by physical irritation in HR-1 mice skin. Collectively, these results demonstrate that extracellularly extruded syntaxin-4 is a potent regulator of CCE differentiation, and that ST4n1 has potential as a clinically applicable reagent for keratotic skin lesions.