Holger Schlüter

A role for pericytes as microenvironmental regulators of human skin tissue regeneration

The cellular and molecular microenvironment of epithelial stem and progenitor cells is poorly characterized despite well-documented roles in homeostatic tissue renewal, wound healing, and cancer progression. Here, we demonstrate that, in organotypic cocultures, dermal pericytes substantially enhanced the intrinsically low tissue-regenerative capacity of human epidermal cells that have committed to differentiate and that this enhancement was independent of angiogenesis. We used microarray analysis to identify genes expressed by human dermal pericytes that could potentially promote epidermal regeneration. Using this approach, we identified as a candidate the gene LAMA5, which encodes laminin alpha5, a subunit of the ECM component laminin-511/521 (LM-511/521). LAMA5 was of particular interest as we had previously shown that it promotes skin regeneration both in vitro and in vivo. Analysis using immunogold localization revealed that pericytes synthesized and secreted LAMA5 in human skin. Consistent with this observation, coculture with pericytes enhanced LM-511/521 deposition in the dermal-epidermal junction of organotypic cultures. We further showed that skin pericytes could also act as mesenchymal stem cells, exhibiting the capacity to differentiate into bone, fat, and cartilage lineages in vitro. This study suggests that pericytes represent a potent stem cell population in the skin that is capable of modifying the ECM microenvironment and promoting epidermal tissue renewal from non-stem cells, a previously unsuspected role for pericytes.

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  6bri CD71dim, α  6bri CD71bri, 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  6bri CD71dim subset was quiescent, the α  6bri CD71bri 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  6bri CD71dim 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  6bri CD71bri 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  6bri CD71dim) followed by cycling progenitors (i.e., α  6bri CD71bri) and finally early differentiating keratinocytes (i.e., α  6dim ). STEM CELLS 2011;29:1256–1268

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.

Bioengineered human skin from embryonic stem cells.

The therapeutic use of human embryonic stem cells (hESCs) is currently prevented by several important hurdles. These hurdles include the availability of wellcharacterised, appropriately monitored cell lines that can be specifi ed into the required lineages reproducibly and effi ciently, without resulting in tumour forma tion or eliciting an immune response resulting in rejection. However, recent progress has provided hope that these challenges are not insurmountable and might eventually result in the use of hESCs for regenerative therapies for various diseases, including spinal cord injury, Parkinson’s disease, and major tissue defi cits. Although the treatment of large skin-tissue losses, especially in severely burnt patients, is greatly assisted by regrafting of autologous keratinocytes amplifi ed in culture, substantial limitations remain, such as the time taken to grow enough cells to provide adequate coverage—a delay that increases the risk of mortality from infection and dehydration. The availability of stable frozen keratinocytes derived from hESCs that lack tumorigenic capacity, have low HLA-antigen expression, and can be stably stored in tissue banks ready to be thawed before grafting would be a valuable asset in the treatment of critically burnt patients. Both murine ESC and hESC lines (H9 and H1) can be converted to K14-positive epidermal cells in culture. An important advance was the ability to reconstitute skin tissue complete with a dermal and epidermal component from murine ESCs, by culturing with extracellular matrix proteins and bone morphogenetic protein 4 (BMP4) in a three-dimensional organotypic culture model. Importantly, the diff erentiation of hESCs into the skin lineage was achieved without the generation of teratomas. In The Lancet today, Hind Guenou and colleagues provide proof-of-principle for using existing hESC lines to reconstitute human skin tissue in mice for up to 12 weeks. This report takes research into regenerative skin stem cells to the next level. The investigators derived the keratinocyte lineage from human H9 and SA01 ESC lines (K-hESCs) by amending existing protocols (ie, the supplementation of culture medium with BMP4 and ascorbic acid), and culturing for the prolonged period of 40 days. This study shows the progressive loss of the pluripotency gene markers OCT4 and NANOG, the transient expression of cytokeratins K8 (KRT8) and K18 (KRT18), followed by the induction and maintenance of KRT5 and KRT14 expression in culture. These cells have characteristics of epidermal cells. Importantly, K-hESCs could be passaged and frozen over nine times without loss of proliferative potential and epidermal diff erentiation properties. Furthermore, K-hESCs showed low or no expression of HLA antigens, confi rming early reports that hESCs and their derivatives might be immunoprivileged. This fi nding suggests that keratinocyte allografts derived from hESCs could be transplanted onto burned patients awaiting autologous grafts with a reduced risk of rejection. However, further exploration of the eff ects of MHC–antigen-independent cell-mediated immunity (eg, via natural killer cells and T cells, or cytokines such as transforming growth factor β) is required. A legitimate concern with therapies based on ESCs is the tumorigenic capacity of these cells, restricting their potential usefulness for patients. However, no tumours were seen in Guenou and colleagues’ See Articles page 1745

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

BackgroundThe 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.MethodsTo 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.ResultsWe 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.ConclusionsFor 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.

Pericytes Promote Malignant Ovarian Cancer Progression in Mice and Predict Poor Prognosis in Serous Ovarian Cancer Patients

Purpose: The aim of this study was to investigate the role of pericytes in regulating malignant ovarian cancer progression. Experimental Design: The pericyte mRNA signature was used to interrogate ovarian cancer patient datasets to determine its prognostic value for recurrence and mortality. Xenograft models of ovarian cancer were used to determine if co-injection with pericytes affected tumor growth rate and metastasis, whereas co-culture models were utilized to investigate the direct effect of pericytes on ovarian cancer cells. Pericyte markers were used to stain patient tissue samples to ascertain their use in prognosis. Results: Interrogation of two serous ovarian cancer patient datasets [the Australian Ovarian Cancer Study, n = 215; and the NCI TCGA (The Cancer Genome Atlas), n = 408] showed that a high pericyte score is highly predictive for poor patient prognosis. Co-injection of ovarian cancer (OVCAR-5 & -8) cells with pericytes in a xenograft model resulted in accelerated ovarian tumor growth, and aggressive metastases, without altering tumor vasculature. Pericyte co-culture in vitro promoted ovarian cancer cell proliferation and invasion. High αSMA protein levels in patient tissue microarrays were correlated with more aggressive disease and earlier recurrence. Conclusions: High pericyte score provides the best means to date of identifying patients with ovarian cancer at high risk of rapid relapse and mortality (mean progression-free survival time < 9 months). The stroma contains rare yet extremely potent locally resident mesenchymal stem cells—a subset of “cancer-associated fibroblasts” that promote aggressive tumor growth and metastatic dissemination, underlying the prognostic capacity of a high pericyte score to strongly predict earlier relapse and mortality. Clin Cancer Res; 22(7); 1813–24. ©2015 AACR.

In vivo transplantation assay at limiting dilution to identify the intrinsic tissue reconstitutive capacity of keratinocyte stem cells and their progeny.

This protocol describes an in vivo grafting approach to investigate the intrinsic long-term tissue reconstitutive capabilities of interfollicular keratinocyte stem cells and their committed progeny-the committed progenitors or transit amplifying and early differentiating cells. This approach utilizes the previously described skin reconstitution rat trachea assay, which has been adapted to investigate differences between stem cells and their more committed progeny. Limiting dilutions of each cell fraction reveal that both stem cells and their progeny are capable of skin tissue reconstitution, but at a limiting dilution of 100 cells per rat trachea only the keratinocyte stem cells maintain the reconstituted skin for as long as 10 weeks. The thorough analysis of reconstituted tissues using skin specific proliferation and stage-specific differentiation markers is also described because it provides qualitative distinction of the epithelial sheets reconstituted by stem cells and their progeny.

WIF1 is expressed by stem cells of the human interfollicular epidermis and acts to suppress keratinocyte proliferation

Ibusuki for excellent technical assistance. Jennifer E. Kloepper, Kazuhiro Kawai, Marta Bertolini, Takuro Kanekura and Ralf Paus Department of Dermatology, University of Luebeck, Luebeck, Germany; Department of Dermatology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan and The Dermatology Centre, Salford Royal NHS Foundation Trust, and Institute of Inflammation and Repair, University of Manchester, Manchester, UK These authors contributed equally to this work. E-mail: ralf.paus@uksh.de

Chapter 34 – Burns and Skin Ulcers

Burns and skin ulcers are major causes of morbidity and, in the case of burns, mortality in both the developed and developing world. Epidermal cells have been used in the therapy of these conditions for decades in the form of autologous skin grafts. For the last 20 years, advanced cell culture techniques have permitted the development of methods to identify and even isolate viable skin stem cells that could potentially be used in the treatment of these two conditions. These advances provide a strong foundation for building more advanced and exciting therapies based on stem cells to treat these challenging conditions. Furthermore, new developments in the use of gene therapy of keratinocyte stem cells and the differentiation of embryonic stem cells into immunoprivileged keratinocytes have real potential to treat burns and other skin disorders in the future.

Burns and Skin Ulcers

Publisher Summary Epidermal cells have been used in the therapy of burns and skin ulcers (which are major causes of morbidity and, in the case of burns, mortality) for decades in the form of autologous skin grafts. Advanced cell culture techniques have permitted the development of methods to identify and even isolate viable skin stem cells that could potentially be used in the treatment of these two conditions. These advances provide a strong foundation for building more advanced and exciting therapies based on stem cells to treat these challenging conditions. The epidermis of the skin is a constantly renewing stratified squamous epithelium. It consists mostly of keratinocytes, but also of Langerhans cells, melanocytes, and Merkel cells resting on a supporting dermis that contains the nerve and vascular networks, which nourish the epidermis. The dermis is also the location of epidermal appendages, fibroblasts, mast cells, macrophages, and lymphocytes. Epidermal stem cells are responsible for the ability of the epidermis to replace itself, both in normal circumstances and in traumatic skin loss, such as from burns and skin ulceration. The stem cells of the dermis have yet to be definitively identified and localized within the skin; this can be attributed to the complex cellular heterogeneity of this tissue. The expanding understanding of stem cells and their manipulation will build on these advances to enable even better treatment of these conditions in the future.