Keratinocyte cultures have been used for the treatment of severe burn patients. Here, we describe a new cultured bioengineered skin based on (1) keratinocytes and fibroblasts obtained from a single skin biopsy and (2) a dermal matrix based on human plasma. A high expansion capacity achieved by keratinocytes grown on this plasma-based matrix is reported. In addition, the results of successful preclinical and clinical tests are presented. Keratinocytes and fibroblasts were obtained by a double enzymatic digestion (trypsin and collagenase, respectively). In this setting, human fibroblasts are embedded in a clotted plasma-based matrix that serves as a three-dimensional scaffold. Human keratinocytes are seeded on the plasma-based scaffold to form the epidermal component of the skin construct. Regeneration performance of the plasma-based bioengineered skin was tested on immunodeficient mice as a preclinical approach. Finally, this skin equivalent was grafted on two severely burned patients. Keratinocytes seeded on the plasma-based scaffold grew to confluence, allowing a 1,000-fold cultured-area expansion after 24 to 26 days of culture. Experimental transplantation of human keratinocytes expanded on the engineered plasma scaffold yielded optimum epidermal architecture and phenotype, including the expression of structural intracellular proteins and basement-membrane components. In addition, we report here the successful engraftment and stable skin regeneration in two severely burned patients at 1 and 2 years follow-up. Our data demonstrate that this new dermal equivalent allows for (1) generation of large bioengineered skin surfaces, (2) restoration of both the epidermal and dermal skin compartments, and (3) functional epidermal stem-cell preservation.

Cultures of growth-arrested feeder cells have been used for years to promote cell proliferation, particularly with low-density inocula. Basically, feeder cells consist in a layer of cells unable to divide, which provides extracellular secretions to help another cell to proliferate. It differs from a coculture system because only one cell type is capable to proliferate. It is known that feeder cells support the growth of target cells by releasing growth factors to the culture media, but this is not the only way that feeder cells promote the growth of target cells. In this work, we discuss the different mechanisms of action of feeder cells, tackling questions as to why for some cell cultures the presence of feeder cell layers is mandatory, while in some other cases, the growth of target cells can be achieved with just a conditioned medium. Different treatments to avoid feeder cells to proliferate are revised, not only the classical treatments as mitomycin or γ-irradiation but also the not so common treatments as electric pulses or chemical fixation. Regenerative medicine has been gaining importance in recent years as a discipline that moves biomedical technology from the laboratory to the patients. In this context, human stem and pluripotent cells play an important role, but the presence of feeder cells is necessary for these progenitor cells to grow and differentiate. This review addresses recent specific applications, including those associated to the growth of embryonic and induced pluripotent stem cells. In addition, we have also dealt with safety issues, including feeder cell sources, as major factors of concern for clinical applications.

Background and purpose:  The activation of CB₂ receptors induces analgesia in experimental models of chronic pain. The present experiments were designed to study whether the activation of peripheral or spinal CB₂ receptors relieves thermal hyperalgesia and mechanical allodynia in two models of bone cancer pain.Experimental approach:  NCTC 2472 osteosarcoma or B16‐F10 melanoma cells were intratibially inoculated to C3H/He and C57BL/6 mice. Thermal hyperalgesia was assessed by the unilateral hot plate test and mechanical allodynia by the von Frey test. AM1241 (CB₂ receptor agonist), AM251 (CB₁ receptor antagonist), SR144528 (CB₂ receptor antagonist) and naloxone were used. CB₂ receptor expression was measured by Western blot.Key results:  AM1241 (0.3–10 mg·kg⁻¹) abolished thermal hyperalgesia and mechanical allodynia in both tumour models. The antihyperalgesic effect was antagonized by subcutaneous, intrathecal or peri‐tumour administration of SR144528. In contrast, the antiallodynic effect was inhibited by systemic or intrathecal, but not peri‐tumour, injection of SR144528. The effects of AM1241 were unchanged by AM251 but were prevented by naloxone. No change in CB₂ receptor expression was found in spinal cord or dorsal root ganglia.Conclusions and implications:  Spinal CB₂ receptors are involved in the antiallodynic effect induced by AM1241 in two neoplastic models while peripheral and spinal receptors participate in the antihyperalgesic effects. Both effects were mediated by endogenous opiates. The use of drugs that activate CB₂ receptors could be a useful strategy to counteract bone cancer‐induced pain symptoms.

Background Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three cancer‐prone genodermatoses whose causal genetic mutations cannot fully explain, on their own, the array of associated phenotypic manifestations. Recent evidence highlights the role of the stromal microenvironment in the pathology of these disorders. Objectives To investigate, by means of comparative gene expression analysis, the role played by dermal fibroblasts in the pathogenesis of RDEB, KS and XPC. Methods We conducted RNA‐Seq analysis, which included a thorough examination of the differentially expressed genes, a functional enrichment analysis and a description of affected signalling circuits. Transcriptomic data were validated at the protein level in cell cultures, serum samples and skin biopsies. Results Interdisease comparisons against control fibroblasts revealed a unifying signature of 186 differentially expressed genes and four signalling pathways in the three genodermatoses. Remarkably, some of the uncovered expression changes suggest a synthetic fibroblast phenotype characterized by the aberrant expression of extracellular matrix (ECM) proteins. Western blot and immunofluorescence in situ analyses validated the RNA‐Seq data. In addition, enzyme‐linked immunosorbent assay revealed increased circulating levels of periostin in patients with RDEB. Conclusions Our results suggest that the different causal genetic defects converge into common changes in gene expression, possibly due to injury‐sensitive events. These, in turn, trigger a cascade of reactions involving abnormal ECM deposition and underexpression of antioxidant enzymes. The elucidated expression signature provides new potential biomarkers and common therapeutic targets in RDEB, XPC and KS. What's already known about this topic? Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three genodermatoses with high predisposition to cancer development. Although their causal genetic mutations mainly affect epithelia, the dermal microenvironment likely contributes to the physiopathology of these disorders. What does this study add? We disclose a large overlapping transcription profile between XPC, KS and RDEB fibroblasts that points towards an activated phenotype with high matrix‐synthetic capacity. This common signature seems to be independent of the primary causal deficiency, but reflects an underlying derangement of the extracellular matrix via transforming growth factor‐β signalling activation and oxidative state imbalance. What is the translational message? This study broadens the current knowledge about the pathology of these diseases and highlights new targets and biomarkers for effective therapeutic intervention. It is suggested that high levels of circulating periostin could represent a potential biomarker in RDEB.