Circulating MicroRNA Biomarkers in Melanoma: Tools and Challenges in Personalised Medicine.

Effective management of melanoma depends heavily on early diagnosis. When detected in early non-metastatic stages, melanoma is almost 100% curable by surgical resection, however when detected in late metastatic stages III and IV, 5-year survival rates drop to ~50% and 10⁻25%, respectively, due to limited efficacy of current treatment options. This presents a pressing need to identify biomarkers that can detect patients at high risk of recurrence and progression to metastatic disease, which will allow for early intervention and survival benefit. Accumulating evidence over the past few decades has highlighted the potential use of circulating molecular biomarkers for melanoma diagnosis and prognosis, including lactate dehydrogenase (LDH), S100 calcium-binding protein B (S100B) and circulating tumor DNA (ctDNA) fragments. Since 2010, circulating microRNAs (miRNAs) have been increasingly recognised as more robust non-invasive biomarkers for melanoma due to their structural stability under the harsh conditions of the blood and different conditions of sample processing and isolation. Several pre-analytical and analytical variables challenge the accurate quantification of relative miRNA levels between serum samples or plasma samples, leading to conflicting findings between studies on circulating miRNA biomarkers for melanoma. In this review, we provide a critical summary of the circulating miRNA biomarkers for melanoma published to date.

The effect of isolation and culture methods on epithelial stem cell populations and their progeny-toward an improved cell expansion protocol for clinical application.

BACKGROUND AIMS: The use of cultured epithelial keratinocytes in the treatment of burns and skin graft donor sites is well established in clinical practice. The most widely used culture method for clinical use was originally developed by Rheinwald and Green 40 years ago. This system uses irradiated mouse dermal fibroblasts as a feeder cell layer to promote keratinocyte growth, a process that is costly and labor-intensive for health care providers. The medium formulation contains several components of animal origin, which pose further safety risks for patients. Improvements and simplification in the culturing process would lead to clear advantages: improved safety through reduction of xenobiotic components and reduction in cost for health care providers by dispensing with feeder cells. METHODS: We compared the Rheinwald and Green method to culture in three commercially available, feeder-free media systems with defined/absent components of animal origin. RESULTS: During the isolation process, short incubation times in high-strength trypsin resulted in increased numbers of liberated keratinocyte stem cells compared with longer incubation times. All three commercially available media tested in this study could support the expansion of keratinocytes, with phenotypes comparable to cells expanded using the established Rheinwald and Green method. Growth rates varied, with two of the media displaying comparable growth rates, whereas the third was significantly slower. DISCUSSION: Our study demonstrates the suitability of such feeder-free media systems in clinical use. It further outlines a range of techniques to evaluate keratinocyte phenotype when assessing the suitability of cells for clinical application.

Novel macro-microporous gelatin scaffold fabricated by particulate leaching for soft tissue reconstruction with adipose-derived stem cells.

The restoration of body contours as shaped by adipose tissue remains a clinical challenge specifically in patients who have experienced loss of contour due to trauma, surgical removal of tumours or congenital abnormalities. We have developed a novel macro-microporous biomaterial for use in soft tissue re-bulking and augmentation. Alginate beads provided the pore template for the construct. Incorporation, and subsequent dissolution, of the beads within a 7 % (w/v) gelatin matrix, produced a highly porous scaffold with an average pore size of 2.01 ± 0.08 mm. The ability of this scaffold to support the in vitro growth and differentiation of human adipose-derived stem cells (ADSCs) was then investigated. Histological analysis confirmed that the scaffold itself provided a suitable environment to support the growth of ADSCs on the scaffold walls. When delivered into the macropores in a fibrin hydrogel, ADSCs proliferated and filled the pores. In addition, ADSCs could readily be differentiated along the adipogenic lineage. These results therefore describe a novel scaffold that can support the proliferation and delivery of ADSCs. The scaffold is the first stage in developing a clinical alternative to current treatment methods for soft tissue reconstruction.

Strategies Demonstrating Efficacy in Reducing Wound Contraction In Vivo.

SIGNIFICANCE: Scarring continues to present a significant clinical problem. Wound contraction leads to scarring and is mediated by myofibroblasts and contractile forces across the wound bed. Contracture formation can have a significant impact on the quality of life of the patient, particularly where function and appearance are affected. RECENT ADVANCES: Novel tissue-engineered matrices, cell-based therapies, and medicinal therapeutics have shown significant reduction in wound contraction in in-vivo models, particularly at early time points. These have been accompanied in many cases by reduced numbers of myofibroblasts, and in some by increased angiogenesis and improved neodermal architecture. CRITICAL ISSUES: There are no animal models that replicate all aspects of wound healing as seen in patients. Therefore, information obtained from in vivo studies should be assessed critically. Additional studies, in particular those that seek to elucidate the mechanisms by which novel therapies reduce contraction, are needed to gain sufficient confidence to move into clinical testing. FUTURE DIRECTIONS: The use of knockout mouse models in particular has generated significant advances in knowledge of the mechanisms behind myofibroblast conversion and other factors involved in generating tension across the wound. Medicinal therapeutics and tissue-engineering approaches that seek to disrupt/alter these pathways hold much promise for future development and translation to clinical practice.

Wound contraction is significantly reduced by the use of microcarriers to deliver keratinocytes and fibroblasts in an in vivo pig model of wound repair and regeneration.

In full-thickness injuries caused by extensive burns or penetrating traumatic injuries, the natural epidermal stem cell niche is destroyed, and wound healing occurs through migration of cells from the wound edges and wound contraction. This can lead to significant contracture formation, especially in large full-thickness injuries, causing lack of mobility and pain. Contraction is reduced when wounds are treated using split-thickness skin grafts (STSG) or dermal substitutes, particularly in combination with cultured autologous keratinocytes, delivered as confluent sheets or sprayed as a single cell suspension (SAK). Here, we show that the application of keratinocytes alone or keratinocytes with fibroblasts, delivered on microcarriers, in combination with STSG or a dermal substitute, significantly reduces contraction of wounds in vivo in a porcine model of wound repair and regeneration. A decrease in alpha-smooth muscle actin-positive myofibroblasts, the cell type responsible for wound contraction, accompanies the reduction in contraction. These findings demonstrate the potential for a significant clinical advantage in the treatment of full-thickness injuries.

Microcarriers and their potential in tissue regeneration.

Microcarriers are a versatile tool with applications across a wide range of disciplines within tissue engineering. Large numbers of cells of appropriate phenotypes are required in engineering the many different tissues of the body, and microcarriers facilitate not only the expansion of many cell types but also the investigation of cell behavior in vitro. Microcarriers can also be used to directly deliver cells in vivo to repair and regenerate tissues. This review summarizes and discusses the use of microcarriers in diverse applications of tissue repair, including bone, cartilage, skin, vascular, central nervous system, adipose tissue, and liver repair. It also considers how microcarriers can be used to bulk-culture and deliver stem cells for tissue regeneration. Microcarriers thus have multidisciplinary use and advances in their use are of benefit to the entire tissue engineering field.

Topology and endoplasmic reticulum retention signals of the lysosomal storage disease-related membrane protein CLN6.

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.