Cell-Extracellular Matrix Interactions in Normal and Diseased Skin

Mammalian skin comprises a multi-layered epithelium, the epidermis, and an underlying connective tissue, the dermis. The epidermal extracellular matrix is a basement membrane, whereas the dermal ECM comprises fibrillar collagens and associated proteins. There is considerable heterogeneity in ECM composition within both epidermis and dermis. The functional significance of this extends beyond cell adhesion to a range of cell autonomous and nonautonomous processes, including control of epidermal stem cell fate. In skin, cell-ECM interactions influence normal homeostasis, aging, wound healing, and disease. Disturbed integrin and ECM signaling contributes to both tumor formation and fibrosis. Strategies for manipulating cell-ECM interactions to repair skin defects and intervene in a variety of skin diseases hold promise for the future.The focus of this review is the role of cell-ECM interactions in the physiology of normal and diseased mammalian skin. The skin has epithelial and mesenchymal components and contains ECM comprising both fibrillar collagen and basement membrane. Experimentally, it is a highly tractable tissue, and a range of in vitro and in vivo approaches are available to explore cell-ECM interactions. Such studies are of medical importance because of the wide variety of benign and malignant skin diseases. Research on skin therefore provides an integrated, in vivo, context for understanding the functional significance of specific molecular interactions and signaling pathways involved in cell-ECM adhesion.     

表皮干细胞研究进展

哺乳动物表皮中包含有多种不同类型的表皮干细胞, 它们共同维持了表皮组织结构的稳态并在皮肤创伤的修复中起重要作用。表皮干细胞具备干细胞两大基本特征: 自我更新和分化, 两者间平衡的破坏通常是皮肤肿瘤和其他皮肤疾病的根源。文章着重叙述了表皮干细胞存在的证据、两大基本特征、分裂模式、调节表皮干细胞的信号通路以及维持其稳态的微观和宏观环境。     

Biochemistry of epidermal stem cells

Background

The epidermis is an important protective barrier that is essential for maintenance of life. Maintaining this barrier requires continuous cell proliferation and differentiation. Moreover, these processes must be balanced to produce a normal epidermis. The stem cells of the epidermis reside in specific locations in the basal epidermis, hair follicle and sebaceous glands and these cells are responsible for replenishment of this tissue.

Scope of review

A great deal of effort has gone into identifying protein epitopes that mark stem cells, in identifying stem cell niche locations, and in understanding how stem cell populations are related. We discuss these studies as they apply to understanding normal epidermal homeostasis and skin cancer.

Major conclusions

An assortment of stem cell markers have been identified that permit assignment of stem cells to specific regions of the epidermis, and progress has been made in understanding the role of these cells in normal epidermal homeostasis and in conditions of tissue stress. A key finding is the multiple stem cell populations exist in epidermis that give rise to different structures, and that multiple stem cell types may contribute to repair in damaged epidermis.

General significance

Understanding epidermal stem cell biology is likely to lead to important therapies for treating skin diseases and cancer, and will also contribute to our understanding of stem cells in other systems. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Epidermal stem cells - role in normal, wounded and pathological psoriatic and cancer skin

In this review we focus on epidermal stem cells in the normal regeneration of the skin as well as in wounded and psoriatic skin. Furthermore, we discuss current data supporting the idea of cancer stem cells in the pathogenesis of skin carcinoma and malignant melanoma. Epidermal stem cells present in the basal layer of the interfollicular epidermis and in the bulge region of the hair follicle play a critical role for normal tissue maintenance. In wound healing, multipotent epidermal stem cells contribute to re-epithelization. It is possible that defects in growth control of either epidermal stem cells or transit amplifying cells constitute a primary pathogenetic factor in the epidermal hyperproliferation seen in psoriasis. In cutaneous malignancies mounting evidence supports a stem cell origin in skin carcinoma and malignant melanoma and a possible existence of cancer stem cells.     

Extracellular matrix: A dynamic microenvironment for stem cell niche

Background

Extracellular matrix (ECM) is a dynamic and complex environment characterized by biophysical, mechanical and biochemical properties specific for each tissue and able to regulate cell behavior. Stem cells have a key role in the maintenance and regeneration of tissues and they are located in a specific microenvironment, defined as niche.

Scope of review

We overview the progresses that have been made in elucidating stem cell niches and discuss the mechanisms by which ECM affects stem cell behavior. We also summarize the current tools and experimental models for studying ECM–stem cell interactions.

Major conclusions

ECM represents an essential player in stem cell niche, since it can directly or indirectly modulate the maintenance, proliferation, self-renewal and differentiation of stem cells. Several ECM molecules play regulatory functions for different types of stem cells, and based on its molecular composition the ECM can be deposited and finely tuned for providing the most appropriate niche for stem cells in the various tissues. Engineered biomaterials able to mimic the in vivo characteristics of stem cell niche provide suitable in vitro tools for dissecting the different roles exerted by the ECM and its molecular components on stem cell behavior.

General significance

ECM is a key component of stem cell niches and is involved in various aspects of stem cell behavior, thus having a major impact on tissue homeostasis and regeneration under physiological and pathological conditions. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Epidermal Stem Cells

Epidermis contains a compartment of stem cells but currently there is no common criterion to recognize individual stem cells with any confidence. Epidermis appears to contain stem cells of different levels of maturity and it is very likely that the main repository of epidermal stem cells is located in the hair follicle from which cells can emigrate into epidermis and also give rise to follicular and sebaceous keratinocytes. Epidermis consists of proliferative units containing stem and transit-amplifying cells, but the exact size of a proliferative unit cannot be measured accurately. The available data suggest that populations of stem and transit-amplifying cells are not discrete but represent a continuum from cells with a high self-renewal capacity and a low probability of differentiation to those with low self-renewal capacities and high commitments to differentiation. Stem cells occupy a special niche that provides a microenvironment, including an adhesion of stem cells to the basal membrane and their paracrine interactions with neighbor epidermal and mesenchymal cells. The fate of an epidermal stem cell depends on its prehistory and microenvironment.     

Reprogramming adult dermis to a neonatal state through epidermal activation of β-catenin

Hair follicle formation depends on reciprocal epidermal-dermal interactions and occurs during skin development, but not in adult life. This suggests that the properties of dermal fibroblasts change during postnatal development. To examine this, we used a PdgfraEGFP mouse line to isolate GFP-positive fibroblasts from neonatal skin, adult telogen and anagen skin and adult skin in which ectopic hair follicles had been induced by transgenic epidermal activation of β-catenin (EF skin). We also isolated epidermal cells from each mouse. The gene expression profile of EF epidermis was most similar to that of anagen epidermis, consistent with activation of β-catenin signalling. By contrast, adult dermis with ectopic hair follicles more closely resembled neonatal dermis than adult telogen or anagen dermis. In particular, genes associated with mitosis were upregulated and extracellular matrix-associated genes were downregulated in neonatal and EF fibroblasts. We confirmed that sustained epidermal β-catenin activation stimulated fibroblasts to proliferate to reach the high cell density of neonatal skin. In addition, the extracellular matrix was comprehensively remodelled, with mature collagen being replaced by collagen subtypes normally present only in developing skin. The changes in proliferation and extracellular matrix composition originated from a specific subpopulation of fibroblasts located beneath the sebaceous gland. Our results show that adult dermis is an unexpectedly plastic tissue that can be reprogrammed to acquire the molecular, cellular and structural characteristics of neonatal dermis in response to cues from the overlying epidermis.     

Tgm2/Gh, Gbx1 and TGF-beta are involved in retinoic acid-induced transdifferentiation from epidermis to mucosal epithelium

We previously demonstrated that retinoic acid (RA) induces epidermis to transdifferentiate to mucosal epithelium with goblet cells in chick embryonic cultured skin. To characterize the molecular mechanism of this transdifferentiation process, we used rat embryonic cultured skin and immunohistochemistry to confirm that RA-induced epidermal transdifferentiation accompanies the expression of markers of esophagus epithelium. Because Gbx1, TG2/Gh (transglutaminase2) and TGF-beta2 are reported individually to be induced by RA in cultures of chick embryonic skin, mouse epidermal cells and human hair follicles respectively, here, we investigated whether cooperative interplay of Gbx1, TG2/Gh and TGF-beta2 is required for the transdifferentiation of epidermal cells to mucosal cells. We have shown that expression of Gbx1, TG2/Gh and TGF-beta proteins were all upregulated in RA-induced transdifferentiated skin and that the former two were expressed in the epidermis, while TGF-beta was expressed in the dermis. Inhibitors of the TGF-beta signal pathway partially inhibited transdifferentiation. Overexpression of both hTG2/Gh and mGbx1 together in the epidermis by electroporation resulted in cuboidal cells in the upper cell layers of the epidermis without keratinized layers, although epidermal keratinization was observed in skin by overexpression of either of them. Labeling DNA with BrdU indicated that RA directly transdifferentiated transient amplifying epidermal cells, not stem cells, to mucosal cells. This study showed that coexpression of TG/2 and Gbx1 in the epidermis was required for esophagus-like mucosal transdifferentiation, and that increase in TGF-beta2 expression by RA in the dermis was essential to induce transdifferentiation through epithelial-mesenchymal interaction.     

Nerve-derived sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells

In adult skin, stem cells in the hair follicle bulge cyclically regenerate the follicle, whereas a distinct stem cell population maintains the epidermis. The degree to which all bulge cells have equal regenerative potential is not known. We found that Sonic hedgehog (Shh) from neurons signals to a population of cells in the telogen bulge marked by the Hedgehog response gene Gli1. Gli1-expressing bulge cells function as multipotent stem cells in their native environment and repeatedly regenerate the anagen follicle. Shh-responding perineural bulge cells incorporate into healing skin wounds where, notably, they can change their lineage into epidermal stem cells. The perineural niche (including Shh) is dispensable for follicle contributions to acute wound healing and skin homeostasis, but is necessary to maintain bulge cells capable of becoming epidermal stem cells. Thus, nerves cultivate a microenvironment where Shh creates a molecularly and phenotypically distinct population of hair follicle stem cells.     

Dendritic cell migrations involving the pilosebaceous unit in the development of murine skin

Over the lint week of postnatal life, dermal dendritic cells stream upwards to invade the epidermis of the mouse tail and back skin. Their migrations seem associated with the development of distinct types of epidermal physiology:ortho- and parakeratosis. Changes from neonatal epidermal morphology occur at similar times in both back and tail skin. The hairv mouse back skin is alwavs orthokeratotic, but the initially orthokeratotic tail epidermis later becomes parakeratolir in the scale regions, remaining orthokeratotic in areas of hair production.
Dermal cells studied were adenosine triphosphatase (ATPase)-, non-specific esterase (NSE)-, naphthvl AS-D chloroacetate-, and dihydroxyphenvlalanine (dopa)-positive dendritic cells. The results are discussed in connection with hair growth and glabrous epidermal kcratinization. Dendritic cell regulation of epidermal physiology involving the dermis and pilosebaceous unit is discussed in relation to reviewed work on mesenchymal-epithelial interactions in animal and human skin.     

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.     

The adult hair follicle: cradle for pluripotent neural crest stem cells

This review focuses on the recent identification of two novel neural crest-derived cells in the adult mammalian hair follicle, pluripotent stem cells, and Merkel cells. Wnt1-cre/R26R compound transgenic mice, which in the periphery express beta-galactosidase in a neural crest-specific manner, were used to trace neural crest cells. Neural crest cells invade the facial epidermis as early as embryonic day 9.5. Neural crest-derived cells are present along the entire extent of the whisker follicle. This includes the bulge area, an epidermal niche for keratinocyte stem cells, as well as the matrix at the base of the hair follicle. We have determined by in vitro clonal analysis that the bulge area of the adult whisker follicle contains pluripotent neural crest stem cells. In culture, beta-galactosidase-positive cells emigrate from bulge explants, identifying them as neural crest-derived cells. When these cells are resuspended and grown in clonal culture, they give rise to colonies that contain multiple differentiated cell types, including neurons, Schwann cells, smooth muscle cells, pigment cells, chondrocytes, and possibly other types of cells. This result provides evidence for the pluripotentiality of the clone-forming cell. Serial cloning showed that bulge-derived neural crest cells undergo self-renewal, which identifies them as stem cells. Pluripotent neural crest cells are also localized in the back skin hair of adult mice. The bulge area of the whisker follicle is surrounded by numerous Merkel cells, which together with innervating nerve endings form slowly adapting mechanoreceptors that transduce steady skin indentation. Merkel cells express beta-galactosidase in double transgenic mice, which confirms their neural crest origin. Taken together, our data indicate that the epidermis of the adult hair follicle contains pluripotent neural crest stem cells, termed epidermal neural crest stem cells (eNCSCs), and one newly identified neural crest derivative, the Merkel cell. The intrinsic high degree of plasticity of eNCSCs and the fact that they are easily accessible in the skin make them attractive candidates for diverse autologous cell therapy strategies.     

Role of epidermal stem cells in repair of partial-thickness burn injury after using Moist Exposed Burn Ointment (MEBO) histological and immunohistochemical study

Moist Exposed Burn Ointment (MEBO^®) is widely used topical agent applied on skin burn. This study investigated the effect of MEBO topical application on activation and proliferation of epidermal stem cells through the immunohistochemical localization of cytokeratin 19 (CK19) as a known marker expressed in epidermal stem cells. Biopsies from normal skin and burn wounds were taken from 21 patients with partial thickness burn 1, 4, 7, 14, 21, and 28 days after treatment with MEBO. Tissue sections were prepared for histological study and for CK19 immunohistochemical localization. In control skin, only few cells showed a positive CK19 immune-reaction. Burned skin showed necrosis of full thickness epidermis that extended to dermis. Gradual regeneration of skin accompanied with an enhancement in CK19 immune-reactivity was noted 4, 7, 14 and 21 days after treatment with MEBO. On day 28, a complete regeneration of skin was observed with a return of CK19 immune-reactivity to the basal pattern again. In conclusion, the enhancement of epidermal stem cell marker CK19 after treatment of partial thickness burn injuries with MEBO suggested the role of MEBO in promoting epidermal stem cell activation and proliferation during burn wound healing.     

Reconstituted skin from murine embryonic stem cells

Embryonic stem (ES) cell lines can be expanded indefinitely in culture while maintaining their potential to differentiate into any cell type. During embryonic development, the skin forms as a result of reciprocal interactions between mesoderm and ectoderm. Here, we report the in vitro differentiation and enrichment of keratinocytes from murine ES cells seeded on extracellular matrix (ECM) in the presence of Bone Morphogenic Protein-4 (BMP-4) or ascorbate. The enriched preparation of keratinocytes was able to form an epidermal equivalent composed of a stratified epithelium when cultured at the air-liquid interface on a collagen-coated acellular substratum. Interestingly, an underlying cellular compartment that belongs to the fibroblast lineage was systematically formed between the reconstituted epidermis and the inert membrane. The resulting tissue displayed morphological patterns similar to normal embryonic skin, as evidenced by light and transmission electron microscopy. Immunohistochemical studies revealed expression patterns of cytokeratins, basement membrane (BM) proteins and late differentiation markers of epidermis, as well as fibroblast markers, similar to native skin. The results demonstrate the capacity of ES cells to reconstitute in vitro a fully differentiated skin. This ES-derived bioengineered skin provides a powerful tool for studying the molecular mechanisms controlling epidermal and dermal commitments.     

Tumor-Like Stem Cells Derived from Human Keloid Are Governed by the Inflammatory Niche Driven by IL-17/IL-6 Axis

Background

Alterations in the stem cell niche are likely to contribute to tumorigenesis; however, the concept of niche promoted benign tumor growth remains to be explored. Here we use keloid, an exuberant fibroproliferative dermal growth unique to human skin, as a model to characterize benign tumor-like stem cells and delineate the role of their “pathological” niche in the development of the benign tumor.

Methods and Findings

Subclonal assay, flow cytometric and multipotent differentiation analyses demonstrate that keloid contains a new population of stem cells, named keloid derived precursor cells (KPCs), which exhibit clonogenicity, self-renewal, distinct embryonic and mesenchymal stem cell surface markers, and multipotent differentiation. KPCs display elevated telomerase activity and an inherently upregulated proliferation capability as compared to their peripheral normal skin counterparts. A robust elevation of IL-6 and IL-17 expression in keloid is confirmed by cytokine array, western blot and ELISA analyses. The altered biological functions are tightly regulated by the inflammatory niche mediated by an autocrine/paracrine cytokine IL-17/IL-6 axis. Utilizing KPCs transplanted subcutaneously in immunocompromised mice we generate for the first time a human keloid-like tumor model that is driven by the in vivo inflammatory niche and allows testing of the anti-tumor therapeutic effect of antibodies targeting distinct niche components, specifically IL-6 and IL-17.

Conclusions/Significance

These findings support our hypothesis that the altered niche in keloids, predominantly inflammatory, contributes to the acquirement of a benign tumor-like stem cell phenotype of KPCs characterized by the uncontrolled self-renewal and increased proliferation, supporting the rationale for in vivo modification of the “pathological” stem cell niche as a novel therapy for keloid and other mesenchymal benign tumors.     

Dorsal skin responses to subchronic ultraviolet B (UVB)-irradiation in Wistar-derived hypotrichotic WBN/ILA-Ht rats

Dorsal skin responses to a subchronic UVB-irradiation (10kJ/m2/rat /day), were examined in Wistar-derived hypotrichotic WBN/ILA-Ht rats for up to 3 months. Hyperplasia of epidermal cells and hair follicle epithelial cells as well as parakeratosis developed at 1 month and progressed thereafter, resulting in a prominent epidermis thickening and formation of epidermal ingrowths projecting into the dermis. At the same time, the percentage of proliferating cell nuclear antigen (PCNA)-positive epidermal cells significantly increased after I month. In some portions of the hyperplastic epidermis, especially of the epidermal ingrowths, keratinocytes were somewhat pleomorphic and migrated into the dermis. In the upper dermis, edema with capillary congestion, mast cell infiltration and fibroblast proliferation developed at I month, and the intensity of edema and the number of dermal mast cells was most prominent at 3 months. Edema spread to the epidermis, resulting in intercellular edema and subsequent dissociation of epidermal cells. Degeneration of collagen fibers was also detected in the upper dermis, especially beneath the epidermis. In addition, although not significant because of a large individual difference, the serum IgE concentration, showed a tendency to increase after 2 months. The present study clarified the characteristics of the dorsal skin responses to a subchronic UVB-irradiation in rats.