Recombinant adeno-associated virus type 2-mediated gene transfer into human keratinocytes is influenced by both the ubiquitin/proteasome pathway and epidermal growth factor receptor tyrosine kinase

Efficient gene delivery into keratinocytes is a prerequisite for successful skin gene therapy. Vectors based on recombinant adeno-associated virus type 2 (rAAV-2) offer several promising features that make them attractive for cutaneous applications. However, highly efficient gene delivery may be hampered by different cellular factors, including lack of viral receptors, impairment of cytoplasmic trafficking or limitations in viral second-strand synthesis. This study was undertaken to find factors that influence rAAV-2-mediated in vitro gene transfer into human keratinocytes and, consequently, ways to optimize gene delivery. Transduction experiments using rAAV-2 vectors expressing green fluorescent protein (GFP) demonstrated that impaired cellular trafficking of vector particles and high levels of autophosphorylation at epidermal growth factor receptor tyrosine kinase (EGF-R TK) have a negative influence on gene transfer into keratinocytes. Treatment of keratinocytes with proteasome inhibitor MG132 resulted in a transient augmentation of GFP expression in up to 37% of cells. Treatment with EGF-R TK inhibitors (quinazoline type) enhanced transgene expression in 10–14.5% of the cells. Gene expression was stable for more than 10 weeks and persisted until proliferative senescence occurred. This stable gene expression allows speculation that keratinocyte stem cells have initially been transduced. These findings might have relevance for the use of rAAV-2 vectors in skin gene therapy: transient enhancement of rAAV-2 transduction with proteasome inhibitors might be useful for genetic promotion of wound healing or skin-directed vaccination. Treatment with quinazolines may increase rAAV-2 transduction of keratinocyte stem cells, which is important for gene therapy approaches to inherited diseases.     

诱导多潜能干细胞在皮肤病治疗中的应用

诱导多潜能干细胞拥有胚胎干细胞所有特征,包括多能性和生成各种体细胞.用皮肤细胞产生诱导多潜能干细胞,不仅起始细胞易获取,而且这些诱导多潜能干细胞更容易定向分化为角质形成细胞、黑素细胞和成纤维细胞等多种功能性皮肤细胞.患者自体来源的诱导多潜能干细胞是细胞疗法理想的细胞库,用诱导多潜能干细胞分化后的细胞治疗皮肤病,不仅细胞量充足,且可避免伦理问题和免疫排斥反应.利用回复突变体嵌合体,结合诱导多潜能干细胞技术,能获得充分的患者特异性功能性回复体细胞而用于治疗遗传性皮肤病.该技术可避免常规基因治疗中出现的免疫排斥和插入诱变.     

Directing stem cells into the keratinocyte lineage in vitro

A major area of research in regenerative medicine is the potential application of stem cells in skin grafting and tissue engineering. This would require well defined and efficient protocols for directing the commitment and differentiation of stem cells into the keratinocyte lineage, together with their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying skin tissue biology, as well as facilitate the genetic manipulation of stem cells for therapeutic applications. The development of pharmacokinetic and cytotoxicity/genotoxicity screening tests for skin-related biomaterials and drugs could also utilize protocols developed for the commitment and differentiation of stem cells into the keratinocyte lineage. Hence, this review critically examines the various strategies that could be employed to direct the commitment and differentiation of stem cells into the keratinocyte lineage in vitro.     

诱导多能干细胞在皮肤病研究的应用进展

诱导多能干细胞是将一系列转录因子转入动物或人多种分化成熟的体细胞,经过重组的程序诱导获得多能干细胞,其在形态、增殖能力、表面抗原、基因和蛋白表达、分化能力等方面与胚胎干细胞相似,因此,科研工作者利用诱导多能干细胞代替胚胎干细胞来研究皮肤病.因诱导多能干细胞除具有多能性还具有无限增殖的能力,这些特性能使研究者获得无限的特定细胞,进而建立遗传性皮肤病模型.将致病的突变基因在诱导多能干细胞水平矫正,矫正后的诱导多能干细胞能产生基因正常的健康皮肤组织细胞,从而可望治疗遗传性皮肤病.     

Generation of melanocytes from induced pluripotent stem cells

Epidermal melanocytes have an important role in protecting skin from UV rays, and are implicated in a variety of skin diseases. Here, we developed an efficient method for differentiating induced pluripotent stem cells (iPSCs) into melanocytes. We first generated iPSCs from adult mouse tail-tip fibroblasts (TTFs) using retroviral vectors or virus-free piggyBac transposon vectors carrying murine Sox2, Oct3/4, c-Myc, and Klf4. The TTF-derived iPSC clones exhibited similar morphology and growth properties as mouse embryonic stem (ES) cells. The iPSCs expressed ES cell markers, displayed characteristic epigenetic changes, and formed teratomas with all three germ layers. The iPSCs were used to generate embryonic bodies and were then successfully differentiated into melanocytes by treatment with growth factors. The iPSC-derived melanocytes expressed characteristic melanocyte markers and produced melanin pigment. Electron microscopy showed that the melanocytes contained mature melanosomes. We manipulated the conditions used to differentiate iPSCs to melanocytes and discovered that Wnt3a is not required for mouse melanocyte differentiation. This report shows that melanocytes can be readily generated from iPSCs, providing a powerful resource for the in vitro study of melanocyte developmental biology and diseases. By inducing iPSCs without viruses, the possibility of integration mutagenesis is alleviated, and these iPSCs are more compatible for cell replacement therapies.     

Human umbilical cord blood cells form epidermis in the skin equivalent model

Please cite this paper as: Human umbilical cord blood cells form epidermis in the skin equivalent model. Experimental Dermatology 2010. Abstract: Recently, human embryonic stem cells have been differentiated in vitro into functional epidermal keratinocytes. Here, we demonstrated that these cells can be generated also from non‐embryonic, human umbilical cord blood (hUCB) cells that have the potential to differentiate into cells of non‐hematopoietic lineage. Human UCB mono‐nucleated cells were cultivated in monolayer and in three‐dimensional skin equivalent cultures and assayed for the presence of phenotype‐specific markers. Our results determined that after one month of culturing in serum containing medium, the hUCB cells produced morphologically homogeneous colonies of epithelial cells expressing keratinocyte‐specific markers. They also formed stratified epidermis in organ cultures that contained sporadic CD1a‐positive cells within the accurate strata. We concluded that hUCB cells have the capacity to differentiate into functional epidermal keratinocytes and may serve as a source of high‐quality keratinocytes for clinical applications.     

The effect of pH in modulating skin cell behaviour

Background  Variations occur in the pH of cutaneous wounds which may affect wound closure, graft take and microbial infection rates.
Objectives  To determine how pH modulates cell behaviour in order to optimize wound care.
Methods  The effects of pH on the attachment, proliferation and migration of keratinocytes and fibroblasts were investigated in vitro and in an ex vivo skin growth model. In addition, the effect of pH on keratinocyte differentiation as measured by the expression of cytokeratins 1 (K1) and K5 was studied.
Results  We demonstrated that the optimal pH for both keratinocyte and fibroblast proliferation is between pH 7·2 and 8·3. The optimal pH for growth from ex vivo skin explants was pH 8·43 which correlates with a previously reported improvement in skin graft take at higher pHs. Expression of K1 was found to be elevated in keratinocytes at a low pH.
Conclusions  These results demonstrate that skin cells and explants proliferate and migrate at pHs higher than the physiological pH and that at lower pH keratinocytes express a differentiated keratinocyte phenotype. A better understanding of the responses of the cellular components of skin to fundamental physiological variables such as pH may help inform improved clinical wound care.     

Cutaneous gene delivery

Over the past decade, many approaches to transferring genes into the skin have been investigated. However, most such approaches have been specifically aimed against genodermatosis, and have not produced sufficient results. The goal of such research is to develop a method in which genes are transferred easily, efficiently and stably into keratinocytes, especially into keratinocyte stem cells, and in which the transgene expression persists without a reaction from the host immune response. Although accidental development of cancer has occurred in trials of gene therapy for X-linked severe combined immunodeficiency (X-SCID), resulting in slowing of the progress of this research, the lessons of these setbacks have been applied to further research. Moreover, combined with the techniques acquired from tissue engineering, recent developments in our knowledge about stem cells will lead to new treatments for genodermatoses. The present review summarizes the methods by which therapeutic genes can be transferred into keratinocytes, with discussion of how gene transfer efficiency can be improved, with particular emphasis on disruption of the skin barrier function. It concludes with discussion of the challenges and prospects of keratinocyte gene therapy, in terms of achieving efficient and long-lasting therapeutic effects.     

Production of retroviral vectors in primary human keratinocytes after DNA-mediated gene transfer leads to prolonged gene expression

Prolonged stability and controlled expression of gene constructs transferred directly to human skin improve the possibility of using this tissue in somatic gene therapy. We aim to develop a simple transfection method resulting in retroviral mediated gene transfer to keratinocyte stem cells in situ. We here show that after DNA-mediated gene transfer into primary human keratinocytes it is possible to achieve production of retroviral vectors, leading to the transduction of co-cultured keratinocytes and prolonged reporter gene expression. The method is a first step in a strategy to generate retroviral producer cells in situ in the skin furthermore the method can be used for rapid analysis of the possible effects of transgenes in cultured human keratinocytes without preparatory retroviral vector production in packaging cell lines.     

Neurofibromatosis type 1 tumour suppressor gene expression is deficient in psoriatic skin in vivo and in vitro: a potential link to increased Ras activity

BACKGROUND: Neurofibromatosis type 1 (NF1) protein (neurofibromin) accelerates the inactivation of Ras-GTP in various cell types. Somatic mutations of the NF1 gene may lead to malignant transformation and uncontrolled proliferation. We have previously shown that NF1 protein expression is downregulated in psoriasis in vivo. OBJECTIVES: To study the functional expression and distribution of NF1 mRNA and protein in vivo and in psoriatic and normal keratinocyte cultures. METHODS: Immunohistochemistry and in situ hybridization were used to study NF1 gene and protein expression in psoriasis in vivo. Furthermore, Northern and in situ hybridizations, immunoblot and localization analyses were utilized to study NF1 mRNA and protein in vitro in keratinocyte cultures. RESULTS: NF1 tumour suppressor gene expression was reduced in lesional psoriatic skin compared with perilesional and normal skin in vivo. The in vitro results showed that the levels of NF1 mRNA and protein were reduced in cultured psoriatic keratinocytes during cellular differentiation even after multiple passaging of the cells. Moreover, cultured nonlesional psoriatic keratinocytes were almost equally defective as lesional cells with respect to NF1 expression. CONCLUSIONS: Our findings demonstrate that psoriatic keratinocytes maintain an altered phenotype and gene expression profile even when isolated from interaction with lymphocytes and fibroblasts, which are known to increase proliferation of keratinocytes. As NF1 protein is regarded as a Ras proto-oncogene regulator, the aberrant expression and distribution of NF1 protein and mRNA found in the present study may be causative to the previously described increased activation of Ras in psoriatic lesions, and relate to altered cellular behaviour.     

A novel role of fibrin in epidermal healing: plasminogen-mediated migration and selective detachment of differentiated keratinocytes

Recent studies have shown that fibrin promotes epidermal regeneration in vitro and maintains the stem cell population after transplantation of keratinocytes in vivo. As epidermal keratinocytes do not express integrin alpha(v)beta3, the receptor for fibrin and fibrinogen, the mechanism through which fibrin affects epidermal cells remains elusive. To investigate the role of fibrin in epidermal wound healing, we developed an in vitro model in which fibrin was added to the top of wounded keratinocyte monolayers grown on collagen. With this matrix topology, keratinocytes migrate between the collagen on their basal side and fibrin on their apical side mimicking migration of the epidermis in vivo. Using this model, we found that fibrin promoted keratinocyte migration in low and high calcium concentrations by exposing the cells to plasminogen. The migration rate depended strongly on the concentration of fibrinogen and the rate of plasmin-mediated fibrin degradation. Surprisingly, fibrin and fibrinogen caused significant detachment of keratinocytes which was prevented by the addition of calcium. Further examination using flow cytometry revealed that the detached cells were larger, more granular, and had very low levels of beta1 integrin, which are all signs of differentiated keratinocytes. Our results suggest a novel dual role of fibrin in epidermal healing. First, fibrin promotes keratinocyte migration indirectly by exposing plasminogen to migrating cells, and second, fibrin selectively disrupts adhesion of differentiated keratinocytes. Our data are novel and may have important implications in understanding wound healing and in the use of fibrin as a biomaterial for protein and gene delivery.     

Markers for Ca++-induced terminal differentiation of keratinocytes in vitro under defined conditions

Differentiation of normal human keratinocytes (NHK) grown in vitro as a monolayer to confluency can be triggered with an acute increase in concentration of extracellular Ca⁺⁺. Over several days, induced by Ca⁺⁺, the cells form pseudostratified sheets that somewhat resemble the basic organization of the intact skin. This experimental system is widely used in studies of keratinocyte biology and skin pathology. However, expression pattern of the genes considered as markers for cells in specific layers of epidermis in vivo does not always match the specific pattern observed in vitro and might lead to misinterpretation of data. Here, we demonstrate that among 18 markers of terminally differentiated keratinocytes of stratum granulosum (SG) and stratum corneum (SC) in vivo, only four (CDSN, KPRP, LCE1C and SPRR4) have reproduced their expression pattern in vitro. Our data suggest that findings based on two-dimensional (2D) Ca⁺⁺-induced terminal differentiation of NHK in vitro should be subjected to additional scrutiny before conclusions could be made and, if possible, verified in other experimental system that might more faithfully represent the in vivo microenvironment.     

Use of enhanced green fluorescent protein to monitor retroviral-mediated gene therapy in human keratinocytes

Keratinocytes have great promise as targets for gene therapy involving both skin as well as for systemic disorders due to their availability and potential long life span. Improvement of gene transfer into keratinocytes will be greatly facilitated by markers that will allow both rapid detection and efficient selection of transduced cells. For these purposes, a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP) was placed into a replication-deficient retroviral vector. High-titer retrovirus was used to transduce both primary cultures of neonatal foreskin-derived human keratinocytes (HK) as well as the immortalized keratinocyte-derived cell line HaCaT. Both cell types stably expressed the EGFP, and this marker allowed rapid purification of transduced cells by fluorescence-activated cell sorting. EGFP expression was seen in HaCaT keratinocytes for at least 40 passages, and the presence of this construct did not effect cell growth, or apoptosis in response to UVB or etoposide. Transduced populations of HK were grafted into SCID mice, resulting in a functional epidermis. EGFP expression was readily seen in vivo by exposing the xenografts to an ultraviolet light source. These studies demonstrate the feasibility of using EGFP as a convenient and rapid marker to monitor keratinocyte gene transfer both in vitro and in vivo.     

Complexity of VEGF responses in skin carcinogenesis revealed through ex vivo assays based on a VEGF-A null mouse keratinocyte cell line

Vascular endothelial growth factor (VEGF-A) is a critical player in cutaneous angiogenesis. However, the relative contribution of VEGF-A from different sources including epithelial and mesenchymal cells has not been fully characterized during skin repair and tumorigenesis. Moreover, the actual involvement of other vascular-specific acting molecules has remained elusive in part due to the masking and/or overlapping effects of VEGF-A. To shed light on these uncertainties we generated and characterized a clonal VEGF-null mouse keratinocyte cell line, through in vitro adenoviral gene transfer of Cre recombinase to VEGF-LoxP primary keratinocytes followed by repeated cell passaging under controlled conditions and cloning. In vitro and in vivo assays demonstrated that VEGF-null keratinocytes were nontumorigenic and expressed normal differentiation markers after calcium switch. Hras-induced tumorigenesis of immortalized VEGF-null keratinocytes upon subcutaneous injection was markedly reduced but not fully suppressed. However, the metastatic ability of Hras-transformed VEGF-null keratinocytes was abolished. These ex vivo approaches suggest the existence of VEGF-dependent and independent angiogenic stimuli in skin carcinogenesis. The VEGF-null mouse keratinocyte cell line arises as an important tool to assess the actual contribution of keratinocyte-derived VEGF with respect to other angiogenic factors in skin homeostasis and malignancy.     

Conversion from human haematopoietic stem cells to keratinocytes requires keratinocyte secretory factors

Background. Recent studies have reported that bone‐marrow‐derived stem cells (BMSCs), including haematopoietic stem cells (HSCs) and mesenchymal stromal cells, differentiate in order to regenerate various cellular lineages. Based on these findings, it is known that BMSCs can be used clinically to treat various disorders, such as myocardial infarction and neurotraumatic injuries. However, the mechanism of HSC conversion into organ cells is incompletely understood. The mechanism is suspected to involve direct cell–cell interaction between BMSCs, damaged organ cells, and paracrine‐regulated soluble factors from the organ, but to date, there have been no investigations into which of these are essential for keratinocyte differentiation from HSCs. Aim. To elucidate the mechanism and necessary conditions for HSC differentiation into keratinocytes in vitro. Methods. We cultured human (h)HSCs under various conditions to try to elucidate the mechanism and necessary conditions for hHSCs to differentiate into keratinocytes. Result. hHSCs cocultured with mouse keratinocytes induced expression of human keratin 14 and transglutaminase I. Only 0.1% of the differentiated keratinocytes possessed multiple nuclei indicating cell fusion. Coculture of hHSCs with fixed murine keratinocytes (predicted to stabilize cellular components) failed to induce conversion into keratinocytes. Conversely, keratinocyte‐conditioned medium from both human and mouse keratinocytes was found to mediate hHSC conversion into keratinocytes. Conclusions. Human HSCs are capable of differentiation into keratinocytes, and cell fusion is extremely rare. This differentiating is mediated by the plasma environment rather than by direct cell–cell interactions.     

Stem cells and the skin

Stem cells live long lives, renew themselves, and differentiate into more mature, less potent, specialized cells, such as epidermal keratinocytes and dermal fibroblasts. Stem cells can be embryonic, if derived from an embryo, or adult/somatic if derived from postembryonic tissue. By producing new skin cells, stem cell division and differentiation can potentially rejuvenate skin and restore hair. To reproduce, stem cells can undergo symmetric nondifferentiative or differentiative divisions, or asymmetric differentiative divisions. Asymmetric divisions reproduce the stem cell and provide a more differentiated, but less potent transient amplifying cell. Divisions and differentiation of transient amplifying cells regenerate tissues by producing cells of a specific lineage, for example, keratinocytes. Epidermal stem cells lie in niches in the interfollicular epidermis, sebaceous gland, and in the bulge regions of hair follicles. These epidermal stem cells renew the epidermis, the sebaceous glands, and hair follicles after mature cells die. Dermal stem cells lie in the hair papillae, around pericytes, and elsewhere among other dermal cells. These form pericytes, myoblasts, fibroblasts, chondrocytes, and other specialized dermal cells. Along with other signaling pathways, the Wnt signaling pathway controls stem cell fate. Wnt signals enlist two functionally and chemically different gene coactivators to direct the time and type of replicative divisions. Stem cells may help to heal wounds, repair damaged tissues, regenerate aged skin, and reinvigorate growth of skin, hair, nails, and mucous membranes.     

Enhancement of Keratinocyte Differentiation by Rose Absolute Oil

Background

Through differentiation processes, keratinocytes provide a physical barrier to our bodies and control skin features such as moisturization, wrinkles and pigmentation. Keratinocyte differentiation is disturbed in several skin diseases such as psoriasis and atopic dermatitis.

Objective

The aim of this study is to evaluate the keratinocyte differentiation-enhancing effect of rose absolute oil (RAO).

Methods

Primary cultured human normal keratinocytes were treated with RAO, and differentiation then checked by the expression of marker genes.

Results

RAO did not induce cytotoxicity on cultured keratinocytes at a dose of 10µM. The level of involucrin, an early marker for keratinocyte differentiation, was significantly increased by RAO. Concomitantly, RAO increased involucrin promoter activity, indicating that RAO increased involucrin gene expression at the mRNA level. Furthermore, RAO increased the level of filaggrin in cultured keratinocytes, and in the granular layer of mouse skin. In line with these results, RAO decreased the proliferation of keratinocytes cultured in vitro. When RAO was applied topically on the tape-stripped mouse skins, it accelerated the recovery of disturbed barrier function.

Conclusion

These results suggest that RAO may be applicable for the control of skin texture and keratinocyte differentiation-related skin diseases.     

Analysis of adenovirus early and late gene expression in cultured epidermal keratinocytes

Previous studies have shown that expression of adenovirus type 2 (Ad2) is restricted in epidermal keratinocytes in what appears to be a differentiation specific manner. We have analyzed the relationship between keratinocyte differentiation and Ad2 early and late gene expression. Cultured epidermal keratinocytes infected with Ad2 were fractionated in density gradients of Ficoll 400 to enrich for populations of nondifferentiated cells and differentiated cells. Analysis of these populations revealed that both populations supported early Ad2 gene expression but restricted Ad2 late gene expression. The restriction to late gene expression differed in the two cell populations: Nondifferentiated keratinocytes did not support production of high levels of Ad2 capsid proteins, whereas differentiated keratinocytes supported synthesis of Ad2 capsid proteins but restricted Ad2 expression at a later step that normally leads to production of high titers of progeny virus. The changing restriction to Ad2 expression during keratinocyte differentiation may have resulted from changes in cellular components that play a role in cell differentiation.     

Side population keratinocytes resembling bone marrow side population stem cells are distinct from label-retaining keratinocyte stem cells

Very primitive hematopoietic stem cells have been identified as side population cells based on their ability to efflux a fluorescent vital dye, Hoechst 33342. In this study we show that keratinocytes with the same side population phenotype are also present in the human epidermis. Although side population keratinocytes have the same dye-effluxing phenotype as bone marrow side population cells and can be blocked by verapamil, they do not express increased levels of the ABCG2 transporter that is believed to be responsible for the bone marrow side population phenotype. Because bone marrow side population cells have stem cell characteristics, we sought to determine if side population keratinocytes represent a keratinocyte stem cell population by comparing side population keratinocytes with a traditional keratinocyte stem cell candidate, label-retaining keratinocytes. Flow cytometric analyses demonstrated that side population keratinocytes have a different cell surface phenotype (low beta1 integrin and low alpha6 integrin expression) than label-retaining keratinocytes and represent a unique population of keratinocytes distinctly different from the traditional keratinocyte stem cell candidate. Future in vivo studies will be required to analyze the function of side population keratinocytes in epidermal homeostasis and to determine if side population keratinocytes have characteristics of keratinocyte stem cells.