Binary organization of epidermal basal domains highlights robustness to environmental exposure.

Adulte interfollicular epidermis (IFE) renewal is likely orchestrated by physiological demands of its complex tissue architecture comprising spatial and cellular heterogeneity. Mouse tail and back skin display two kinds of basal IFE spatial domains that regenerate at different rates. Here, we elucidate the molecular and cellular states of basal IFE domains by marker expression and single-cell transcriptomics in mouse and human skin. We uncover two paths of basal cell differentiation that in part reflect the IFE spatial domain organization. We unravel previously unrecognized similarities between mouse tail IFE basal domains defined as scales and interscales versus human rete ridges and inter-ridges, respectively. Furthermore, our basal IFE transcriptomics and gene targeting in mice provide evidence supporting a physiological role of IFE domains in adaptation to differential UV exposure. We identify Sox6 as a novel UV-induced and interscale/inter-ridge preferred basal IFE-domain transcription factor, important for IFE proliferation and survival. The spatial, cellular, and molecular organization of IFE basal domains underscores skin adaptation to environmental exposure and its unusual robustness in adult homeostasis.

Investigating lymphangiogenesis in vitro and in vivo using engineered human lymphatic vessel networks.

The lymphatic system is involved in various biological processes, including fluid transport from the interstitium into the venous circulation, lipid absorption, and immune cell trafficking. Despite its critical role in homeostasis, lymphangiogenesis (lymphatic vessel formation) is less widely studied than its counterpart, angiogenesis (blood vessel formation). Although the incorporation of lymphatic vasculature in engineered tissues or organoids would enable more precise mimicry of native tissue, few studies have focused on creating engineered tissues containing lymphatic vessels. Here, we populated thick collagen sheets with human lymphatic endothelial cells, combined with supporting cells and blood endothelial cells, and examined lymphangiogenesis within the resulting constructs. Our model required just a few days to develop a functional lymphatic vessel network, in contrast to other reported models requiring several weeks. Coculture of lymphatic endothelial cells with the appropriate supporting cells and intact PDGFR-ß signaling proved essential for the lymphangiogenesis process. Additionally, subjecting the constructs to cyclic stretch enabled the creation of complex muscle tissue aligned with the lymphatic and blood vessel networks, more precisely biomimicking native tissue. Interestingly, the response of developing lymphatic vessels to tensile forces was different from that of blood vessels; while blood vessels oriented perpendicularly to the stretch direction, lymphatic vessels mostly oriented in parallel to the stretch direction. Implantation of the engineered lymphatic constructs into a mouse abdominal wall muscle resulted in anastomosis between host and implant lymphatic vasculatures, demonstrating the engineered construct's potential functionality in vivo. Overall, this model provides a potential platform for investigating lymphangiogenesis and lymphatic disease mechanisms.

Pericytes in Metastasis.

Pericytes have long been known to contribute indirectly to tumour growth by regulating angiogenesis. Thus, remodelling tumour blood vessels to maintain blood supply is critical for continued tumour growth. A role for pericytes in restricting leakage of tumour cells through blood vessels has also become evident given that adequate pericyte coverage of these blood vessels is critical for maintaining vascular permeability. Interestingly, the relocation of pericytes from blood vessels to the tumour microenvironment results in the emergence of different properties in these cells that actively promote tumour growth and metastasis-functions not associated with their well-studied role in vascular stability and permeability. These form the focus of this review.

Keratin 76 is required for tight junction function and maintenance of the skin barrier.

Keratins are cytoskeletal intermediate filament proteins that are increasingly being recognised for their diverse cellular functions. Here we report the consequences of germ line inactivation of Keratin 76 (Krt76) in mice. Homozygous disruption of this epidermally expressed gene causes neonatal skin flaking, hyperpigmentation, inflammation, impaired wound healing, and death prior to 12 weeks of age. We show that this phenotype is associated with functionally defective tight junctions that are characterised by mislocalization of the integral protein CLDN1. We further demonstrate that KRT76 interacts with CLDN1 and propose that this interaction is necessary to correctly position CLDN1 in tight junctions. The mislocalization of CLDN1 has been associated in various dermopathies, including the inflammatory disease, psoriasis. These observations establish a previously unknown connection between the intermediate filament cytoskeleton network and tight junctions and showcase Krt76 null mice as a possible model to study aberrant tight junction driven skin diseases.

The polarity protein Scrib mediates epidermal development and exerts a tumor suppressive function during skin carcinogenesis.

BACKGROUND: The establishment and maintenance of polarity is vital for embryonic development and loss of polarity is a frequent characteristic of epithelial cancers, however the underlying molecular mechanisms remain unclear. Here, we identify a novel role for the polarity protein Scrib as a mediator of epidermal permeability barrier acquisition, skeletal morphogenesis, and as a potent tumor suppressor in cutaneous carcinogenesis. METHODS: To explore the role of Scrib during epidermal development, we compared the permeability of toluidine blue dye in wild-type, Scrib heterozygous and Scrib KO embryonic epidermis at E16.5, E17.5 and E18.5. Mouse embryos were stained with alcian blue and alizarin red for skeletal analysis. To establish whether Scrib plays a tumor suppressive role during skin tumorigenesis and/or progression, we evaluated an autochthonous mouse model of skin carcinogenesis in the context of Scrib loss. We utilised Cre-LoxP technology to conditionally deplete Scrib in adult epidermis, since Scrib KO embryos are neonatal lethal. RESULTS: We establish that Scrib perturbs keratinocyte maturation during embryonic development, causing impaired epidermal barrier formation, and that Scrib is required for skeletal morphogenesis in mice. Analysis of conditional transgenic mice deficient for Scrib specifically within the epidermis revealed no skin pathologies, indicating that Scrib is dispensable for normal adult epidermal homeostasis. Nevertheless, bi-allelic loss of Scrib significantly enhanced tumor multiplicity and progression in an autochthonous model of epidermal carcinogenesis in vivo, demonstrating Scrib is an epidermal tumor suppressor. Mechanistically, we show that apoptosis is the critical effector of Scrib tumor suppressor activity during skin carcinogenesis and provide new insight into the function of polarity proteins during DNA damage repair. CONCLUSIONS: For the first time, we provide genetic evidence of a unique link between skin carcinogenesis and loss of the epithelial polarity regulator Scrib, emphasizing that Scrib exerts a wide-spread tumor suppressive function in epithelia.

Myb via TGFß is required for collagen type 1 production and skin integrity.

Skin integrity requires an ongoing replacement and repair orchestrated by several cell types. We previously investigated the architecture of the skin of avian myeloblastosis viral oncogene homolog (Myb) knock-out (KO) embryos and wound repair in Myb(+/)(-) mice revealing a need for Myb in the skin, attributed to fibroblast-dependent production of collagen type 1. Here, using targeted Myb deletion in keratin-14 (K14) positive cells we reveal further Myb-specific defects in epidermal cell proliferation, thickness and ultrastructural morphology. This was associated with a severe deficit in collagen type 1 production, reminiscent of that observed in patients with ichthyosis vulgaris and Ehlers-Danlos syndrome. Since collagen type 1 is a product of fibroblasts, the collagen defect observed was unexpected and appears to be directed by the loss of Myb with significantly reduced tumor growth factor beta 1 (Tgfß-1) expression by primary keratinocytes. Our findings support a specific role for Myb in K14+ epithelial cells in the preservation of adult skin integrity and function.

Dermal Contributions to Human Interfollicular Epidermal Architecture and Self-Renewal.

The human interfollicular epidermis is renewed throughout life by populations of proliferating basal keratinocytes. Though interfollicular keratinocyte stem cells have been identified, it is not known how self-renewal in this compartment is spatially organized. At the epidermal-dermal junction, keratinocytes sit atop a heterogeneous mix of dermal cells that may regulate keratinocyte self-renewal by influencing local tissue architecture and signalling microenvironments. Focusing on the rete ridges and complementary dermal papillae in human skin, we review the identity and organisation of abundant dermal cells types and present evidence for interactions between the dermal microenvironment and the interfollicular keratinocytes.

Functional characterization of quiescent keratinocyte stem cells and their progeny reveals a hierarchical organization in human skin epidermis.

Although homeostatic renewal of human skin epidermis is achieved by the combined activity of quiescent stem cells (SCs) and their actively cycling progeny, whether these two populations are equipotent in their capacity to regenerate tissue has not been determined in biological assays that mimic lifelong renewal. Using fluorescence activated cell separation strategy validated previously by us, human epidermis was fractionated into three distinct subsets: that is, α 6briCD71(dim) , α 6briCD71(bri) , and α 6dim with characteristics of keratinocyte stem, transient amplifying, and early differentiating cells, respectively. The global gene expression profile of these fractions was determined by microarray, confirming that the α 6briCD71(dim) subset was quiescent, the α 6briCD71(bri) was actively cycling, and the α 6dim subset expressed markers of differentiation. More importantly, functional evaluation of these populations in an in vivo model for tissue reconstitution at limiting cell dilutions revealed that the quiescent α 6briCD71(dim) fraction was the most potent proliferative and tissue regenerative population of the epidermis, capable of long-term (LT) epidermal renewal from as little as 100 cells for up to 10 weeks. In contrast, the cycling α 6briCD71(bri) fraction was the first to initiate tissue reconstitution, although this was not sustained in the LT, while differentiating α 6dim cells possessed the lowest demonstrable tissue regenerative capacity. Our data suggest that in human skin, the epidermal proliferative compartment is not composed of equipotent cells, but rather is organized in a functionally hierarchical manner with the most potent quiescent SCs at its apex (i.e., α 6briCD71(dim) ) followed by cycling progenitors (i.e., α 6briCD71(bri) ) and finally early differentiating keratinocytes (i.e., α 6dim).

Assaying Candidate Human Skin Keratinocyte Stem Cells by Determining Their Long-Term Serial Proliferative Output in Culture.

Stem cells are found in niches around the body, including the epidermis of the skin, and can be distinguished from their more committed progeny by their high long-term proliferative capacity in vitro. Here we describe a technique used to isolate three main epidermal cell fractions from human neonatal foreskin termed early differentiating (ED), transient amplifying (TA) and keratinocyte stem cells (KSC) based on their differential expression of two cell surface markers: CD49f and CD71. These three fractions were cultivated in parallel in a serial proliferation assay to determine their long-term proliferative output. This assay demonstrates that the KSC fraction had the highest proliferative output (total cell yield) over a long experimental timeframe of 2-3 months, as well as a higher proliferative rate compared to the other two fractions (P > 0.05). This assay can be utilized under similar conditions to determine the proliferative capacity of other putative stem cells using novel stem cell markers for epidermal or other stem cell populations.

The interfollicular epidermal stem cell saga: sensationalism versus reality check.

Adult stem cells in rapidly renewing tissues have been classically defined as rare, relatively quiescent cells with the unique capacity to constantly self-renew and regenerate tissues during homeostasis. Although this view remains firmly embedded in the skin field, particularly in the area of hair follicle stem cell biology, it has been challenged by a number of notable publications in 2007. These papers leave an uncomfortable feeling with the reader if one believes that stem cells and transit amplifying cells are two polar opposites and 'never the twain shall meet.' Even if you do not subscribe to this extreme view, the implications appear to be far-reaching given that the majority of techniques devised for stem cell identification have used the fundamental tenet that the proliferating compartment is comprised of two distinct, mutually exclusive compartments, i.e. a minor proportion of long-lived quiescent stem cells with unlimited self-renewal and a large pool of rapidly cycling, short-lived transient amplifying cells with limited or no self-renewal capacity in normal steady-state conditions. However, these recent findings have resulted in papers that could be described as sensationalistic because they make little or no attempt to reconcile their observations with the large bulk of historical data with direct bearing on the interpretation of stem cell activity in normal steady-state conditions. Here, we offer some explanations that may help to integrate all of the data while presenting a case that both quiescent stem cells and cycling 'transit amplifying' cells contribute to epidermal replacement.

Dermal Pericytes Exhibit Declined Ability to Promote Human Skin Regeneration with Ageing in 3D Organotypic Culture Models.

The well documented decline in the regenerative ability of ageing human skin has been attributed to many factors including genomic instability, telomere shortening, poor nutrient sensing, cellular senescence, and stem cell exhaustion. However, a role for the dermal cellular and molecular microenvironment in skin ageing is just emerging. We previously showed that dermal pericytes co-operate with fibroblasts to improve human skin regeneration in an organotypic skin culture model, and even do so in the absence of fibroblasts. Here, we report that the number of dermal cells, particularly pericytes, declines significantly in human skin of donors aged > 50 years. Notably, aged pericytes promoted epidermal regeneration of neonatal keratinocytes in organotypic cultures and the resulting epithelium exhibited a Ki67+/ΔNp63+ basal layer and terminal differentiation. However, the epithelium lacked several features of homeostasis displaying lower levels of ΔNp63 expression, decreased LAMA5 deposition at the dermo-epidermal junction, and the absence of basement membrane and hemi-desmosome assembly. We conclude that a decline in pericyte incidence and function contribute to an impaired epidermal microenvironment and poor skin regeneration with ageing in the human skin.

Relationship between ovarian cancer stem cells, epithelial mesenchymal transition and tumour recurrence.

Investigating the biological processes that occur to enable recurrence and the development of chemoresistance in ovarian cancer is critical to the research and development of improved treatment options for patients. The lethality of ovarian cancer is largely attributed to the recurrence of disease with acquired chemoresistance. Cancer stem cells are likely to be critical in ovarian cancer progression, contributing to tumour malignancy, metastasis and recurrence by persisting in the body despite treatment with anti-cancer drugs. Moreover, cancer stem cells are capable of mediating epithelial-to-mesenchymal transition traits and secrete extracellular vesicles to acquire therapy resistance and disease dissemination. These attributes merit in depth research to provide insight into the biological role of ovarian cancer stem cells in disease progression and chemotherapy response, leading to the development of improved biomarkers and innovative therapeutic approaches.

Mechanisms underlying acquired platinum resistance in high grade serous ovarian cancer - a mini review.

BACKGROUND: Advanced epithelial ovarian cancer is one of the hardest human malignancies to treat. Standard treatment involves cytoreductive surgery and platinum-based chemotherapy, however, median progression-free survival for patients diagnosed with advanced stage disease (FIGO stages III and IV) is approximately 18 months. There has been little improvement in overall survival over the past decade and less than half of women with advanced stage disease will be living 5 years after diagnosis. A majority of patients initially have a favourable response to platinum-based chemotherapy, but most will eventually relapse and their disease will become platinum resistant. SCOPE OF REVIEW: Here, we review our current understanding of mechanisms that promote recurrence and acquired resistance in epithelial ovarian cancer with particular focus on studies that describe differences observed between untreated primary tumors and recurrent tumors, post-first-line chemotherapy. Multiple molecular mechanisms contribute to recurrence in patients following initial treatment for advanced epithelial ovarian cancer including those involving the tumor microenvironment, tumor immune status, cancer stem cells, DNA repair/cell survival pathways and extracellular matrix. MAJOR CONCLUSIONS: Due to the adaptive nature of recurrent tumors, the major contributing and specific resistance pattern may largely depend on the nature of the primary tumor itself. GENERAL SIGNIFICANCE: Future work that aims to elucidate the complex pattern of acquired resistance will be useful for predicting chemotherapy response/recurrence following primary diagnosis and to develop novel treatment strategies to improve the survival of patients with advanced epithelial ovarian cancer, especially in tumors not harbouring homologous DNA recombination repair deficiencies.

Ovarian cancer stem cells and their role in drug resistance.

Ovarian cancer is typically diagnosed at advanced stages (III or IV), with metastasis ensuing at stage III. Complete remission is infrequent and is not achieved in almost half of the women diagnosed with ovarian cancer. Consequently, management and treatment of this disease is challenging as many patients are faced with tumour recurrence disseminating to surrounding organs further complicated with acquired chemo-resistance. The cancer stem cell theory proposes the idea that a drug resistant subset of tumour cells drive tumour progression, metastasis and ultimately, recurrent disease. In the ovarian cancer field, cancer stem cells remain elusive with significant gaps in our knowledge. The characteristics and specific role of ovarian cancer stem cells in recurrence still requires further research since different studies often arrive at contradictory conclusions. Here we present a review and critical analysis of current research conducted in the field of ovarian cancer stem cells and their potential role in drug resistance including several signalling pathways within these cells that affect the viability of targeted therapies.

Reconstitution of stratified murine and human oesophageal epithelia in an in vivo transplant culture system.

OBJECTIVE: The molecular and cellular events responsible for regulating development of the oesophageal epithelium are not well understood. At least in part, this is due to the lack of a suitable model system with which to study the process. Here, we report development of a manipulable in vivo transplant model for mouse or human oesophageal epithelium. MATERIAL AND METHODS: Epithelial cells were isolated from mouse or human oesophagus and inoculated into de-epithelialized and devitalized rat tracheas. The rat trachea, containing cells, was placed subcutaneously under the dorsal skin of immunodeficient mice. RESULTS: We show that a multilayered stratified squamous epithelium can be generated in 4-6 weeks from as few as 5 x 10(4) isolated oesophageal epithelial cells. The reconstituted epithelium recapitulates many of the structural and histological features of the normal oesophageal epithelium, including a basal layer of cuboidal-like cells, suprabasal layers of differentiating squamous cells and, in the case of murine cells, a superficial layer of cornified material. CONCLUSION: Our model can be used to generate a multilayered normal murine or human epithelium from a single cell suspension of oesophageal epithelial cells. The ability to genetically manipulate the cells prior to growth in the model is a powerful tool with which to study the molecular mechanisms involved in the development of normal oesophagus or in pathogenic processes such as Barrett's metaplasia or tumorigenesis.

Qualitative in vivo bioluminescence imaging.

Bioluminescence imaging (BLI) technology is an advanced method of carrying out molecular imaging on live laboratory animals in vivo. This powerful technique is widely-used in studying a variety of biological processes, and it has been an ideal tool in exploring tumor growth and metastatic spread in real-time. This technique ensures the optimal use of laboratory animal resources, particularly the ethical principle of reduction in animal use, given its non-invasive nature, ensuring that ongoing biological processes can be studied over time in the same animal, without the need to euthanize groups of mice at specific time points. In this protocol, the luciferase imaging technique was developed to study the effect of co-inoculating pericytes (contractile, αSMA+ mesenchymal stem cell-like cells, located abluminally in microvessels) on the growth and metastatic spread of ovarian cancers using an aggressive ovarian cancer cell line-OVCAR-5-as an example.

Pericytes promote skin regeneration by inducing epidermal cell polarity and planar cell divisions.

The cellular and molecular microenvironment of epithelial stem/progenitor cells is critical for their long-term self-renewal. We demonstrate that mesenchymal stem cell-like dermal microvascular pericytes are a critical element of the skin's microenvironment influencing human skin regeneration using organotypic models. Specifically, pericytes were capable of promoting homeostatic skin tissue renewal by conferring more planar cell divisions generating two basal cells within the proliferative compartment of the human epidermis, while ensuring complete maturation of the tissue both spatially and temporally. Moreover, we provide evidence supporting the notion that BMP-2, a secreted protein preferentially expressed by pericytes in human skin, confers cell polarity and planar divisions on epidermal cells in organotypic cultures. Our data suggest that human skin regeneration is regulated by highly conserved mechanisms at play in other rapidly renewing tissues such as the bone marrow and in lower organisms such as Drosophila. Our work also provides the means to significantly improve ex vivo skin tissue regeneration for autologous transplantation.

Extensive tissue-regenerative capacity of neonatal human keratinocyte stem cells and their progeny.

Given our recent discovery that it is possible to separate human epidermal stem cells of the skin from their more committed progeny (i.e., transit-amplifying cells and early differentiating cells) using FACS techniques, we sought to determine the comparative tissue regeneration ability of these keratinocyte progenitors. We demonstrate that the ability to regenerate a fully stratified epidermis with appropriate spatial and temporal expression of differentiation markers in a short-term in vitro organotypic culture system is an intrinsic characteristic of both epidermal stem and transit-amplifying cells, although the stem cell fraction is most capable of achieving homeostasis. Early differentiating keratinocytes exhibited limited short-term tissue regeneration under specific experimental conditions in this assay, although significant improvement was obtained by manipulating microenvironmental factors, that is, coculture with minimally passaged dermal cells or exogenous supply of the ECM protein laminin-10/11. Importantly, transplantation of all classes of keratinocyte progenitors into an in vivo setting demonstrated that tissue regeneration can be elicited from stem, transit-amplifying, and early differentiating keratinocytes for up to 10 weeks. These data illustrate that significant proliferative and tissue-regenerative capacity resides not only in keratinocyte stem cells as expected, but also in their more committed progeny, including early differentiating cells.

The transcription factors c-rel and RelA control epidermal development and homeostasis in embryonic and adult skin via distinct mechanisms.

Determining the roles of Rel/NF-kappaB transcription factors in mouse skin development with loss-of-function mutants has been limited by redundancy among these proteins and by embryonic lethality associated with the absence of RelA. Using mice lacking RelA and c-rel, which survive throughout embryogenesis on a tumor necrosis factor alpha (TNF-alpha)-deficient background (rela(-/-) c-rel(-/-) tnfalpha(-/-)), we show that c-rel and RelA are required for normal epidermal development. Although mutant fetuses fail to form tylotrich hair and have a thinner epidermis, mutant keratinocyte progenitors undergo terminal differentiation to form an outer cornified layer. Mutant basal keratinocytes are abnormally small, exhibit a delay in G(1) progression, and fail to form keratinocyte colonies in culture. In contrast to the reduced proliferation of mutant keratinocytes during embryogenesis, skin grafting experiments revealed that the mutant epidermis develops a TNF-alpha-dependent hyperproliferative condition. Collectively, our findings indicate that RelA and c-rel control the development of the epidermis and associated appendages during embryogenesis and regulate epidermal homeostasis in a postnatal environment through the suppression of innate immune-mediated inflammation.