BackgroundAdipose tissue-derived stem cells are considered to be a promising source in the field of cell therapy and regenerative medicine. In addition to direct cell replacement using adipose tissue or purified stem cells, intercellular molecule exchange by the adipose tissue complex, a vast array of bioactive secretory factors, demonstrated beneficial effects by reducing tissue damage and stimulation of endogenous repair. However, for therapeutic purposes, the use of secretome derivatives, such as full conditioned media or purified exosomes generated in vitro, may present considerable disadvantages for cell manufacturing, storage, product safety, and their potential as a ready-to-go therapeutic product.MethodsIn this study, the effect of a liquid fraction of lipoaspirates isolated intraoperatively from 28 healthy donors was evaluated for their protective effect against oxidative stress and senescence, proliferation, and migration in vitro on normal human melanocytes, keratinocytes, and fibroblasts. Immunoenzymatic quantification of several growth factors and important signal molecules was used to define the biological profile of physiological adipose tissue secretome.ResultsAdipose tissue extracellular fraction (AT-Ex), isolated from lipoaspirate, exhibited significant potential for skin repair. AT-Ex augmented dermal and epidermal cell proliferation in a dose-dependent manner without promoting cancer cell growth. Moreover, migration of dermal fibroblasts, an important phenomenon implicated in endogenous repair, was enhanced by AT-Ex treatment. AT-Ex has a positive impact on oxidative stress damage when cells are exposed to extrinsic hostile factors and prevent a fibroblast senescence phenotype including paracrine functions associated with skin aging.ConclusionsCollectively, our findings propose natural systems carrying the physiological balance of in-vivo produced secretome that could improve cutaneous wound healing and tissue repair. This approach, representing an innovative perspective and therapeutic strategy in regenerative medicine, could also be combined with autologous stem cell grafts to treat chronic nonhealing wounds, stable vitiligo, severe burns, and post-oncological scarring.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0956-4) contains supplementary material, which is available to authorized users.
BackgroundCombination therapy with BRAF and MEK inhibitors significantly improves survival in BRAF mutated melanoma patients but is unable to prevent disease recurrence due to the emergence of drug resistance. Cancer stem cells (CSCs) have been involved in these long-term treatment failures. We previously reported in lung cancer that CSCs maintenance is due to altered lipid metabolism and dependent upon Stearoyl-CoA-desaturase (SCD1)-mediated upregulation of YAP and TAZ. On this ground, we investigated the role of SCD1 in melanoma CSCs.MethodsSCD1 gene expression data of melanoma patients were downloaded from TCGA and correlated with disease progression by bioinformatics analysis and confirmed on patient’s tissues by qRT-PCR and IHC analyses. The effects of combination of BRAF/MEKi and the SCD1 inhibitor MF-438 were monitored by spheroid-forming and proliferation assays on a panel of BRAF-mutated melanoma cell lines grown in 3D and 2D conditions, respectively. SCD1, YAP/TAZ and stemness markers were evaluated in melanoma cells and tissues by qRT-PCR, WB and Immunofluorescence.ResultsWe first observed that SCD1 expression increases during melanoma progression. BRAF-mutated melanoma 3D cultures enriched for CSCs overexpressed SCD1 and were more resistant than 2D differentiated cultures to BRAF and MEK inhibitors. We next showed that exposure of BRAF-mutated melanoma cells to MAPK pathway inhibitors enhanced stemness features by upregulating the expression of YAP/TAZ and downstream genes but surprisingly not SCD1. However, SCD1 pharmacological inhibition was able to downregulate YAP/TAZ and to revert at the same time CSC enrichment and resistance to MAPK inhibitors.ConclusionsOur data underscore the role of SCD1 as prognostic marker in melanoma and promote the use of SCD1 inhibitors in combination with MAPK inhibitors for the control of drug resistance.Electronic supplementary materialThe online version of this article (10.1186/s13046-018-0989-7) contains supplementary material, which is available to authorized users.
There is an increasing interest in the apparently normal skin in vitiligo. Altered expression of the adhesion molecule E-cadherin and persistent deregulated intracellular redox status that promotes the acquisition of a stress-induced senescent phenotype in melanocytes of normally pigmented skin from patients with vitiligo have been described. Growing evidence has shown that such cellular and functional alterations are not necessarily restricted to melanocytes but may be extended to other cutaneous cell populations in both lesional and nonlesional areas. However, whether dermal fibroblasts exhibit related alterations that may contribute to the defects associated with melanocytes in vitiligo is not known. Here we reveal within the dermal compartment cells a myofibroblast phenotype and a predisposition to premature senescence, indicating the existence of altered cross-talk between dermal and epidermal components that may affect melanocyte functionality even in the apparently normal skin of patients with vitiligo.
Vitiligo is characterized by death or functional defects of epidermal melanocytes through still controversial pathogenic process. Previously, we showed that mitochondria-driven pre-senescent phenotype diminishes the capability of vitiligo melanocytes to cope with stressful stimuli. In the current study, we investigated markers of mitochondrial energy metabolism including the PGC1a axis, and then we determined the index of mitochondrial impairment using a cytomic approach. We found in cultured epidermal vitiligo melanocytes, compared to healthy ones, low ATP, increased proton leakage, and altered expression of several glycolytic enzymes (hexokinase II, pyruvic dehydrogenase kinase 1 and pyruvic kinase M2), We suggest that the low ATP production may be sufficient in steady-state conditions but it is unable to cover further needs. We also found in vitiligo melanocyrtes hyper-activation of the PGC1α axis, finalized to counteract the energy defect. Cytomic analysis, supported by MitoTracker Red pattern and ex-vivo immunohistochemistry, suggested an increased mitochondrial mass, possibly useful to ensure the essential ATP level. Finally, pharmacological cardiolipin stabilization reverted the energetic impairment, confirming the initial mitochondrial role. In conclusion, we report new insight in the pathogenetic mechanism of viitligo and indicate that the mitochondrial failure rescue by cardiolipin manipulation may be a new intriguing target in treatment development.
In the past decade, adipose tissue has become a highly interesting source of adult stem cells for plastic surgery and regenerative medicine. The adipose source offers two options for the isolation of regenerative cells: the enzymatic digestion an expensive time-consuming procedure lacking a common standard operating protocol, or the non-enzymatic dissociation methods based on mechanical forces to break the processed adipose tissue. Here, we propose innovative inexpensive non-enzymatic protocols to collect and concentrate clinically useful regenerative cells from adipose tissue by centrifugation of the infranatant fraction of lipoaspirate as first step, usually discarded as a byproduct of the surgical procedure, and by fat shaking and wash as second enrichment step. The isolated cells were characterized according to the criteria proposed by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) to define human mesenchymal stem cells, and the results were compared with matched lipoaspirate samples processed with collagenase. The results demonstrated the usability of these new procedures as an alternative to fat grafting for treating stem cell-depleted tissues and for specific application requiring minimal or null soft tissue augmentation, such as skin diseases including severe burn and post-oncological scaring, chronic non-healing wounds, and vitiligo.
Deregulations or mutations of WNT/β-catenin signaling have been associated to both tumour formation and progression. However, contradictory results concerning the role of β-catenin in human melanoma address an open question on its oncogenic nature and prognostic value in this tumour. Changes in WNT signaling pathways have been linked to phenotype switching of melanoma cells between a highly proliferative/non-invasive and a slow proliferative/metastatic condition. We used a novel panel of cell lines isolated from melanoma specimens, at initial passages, to investigate phenotype differences related to the levels and activity of WNT/β-catenin signaling pathway. This in vitro cell system revealed a marked heterogeneity that comprises, in some cases, two distinct tumour-derived subpopulations of cells presenting a different activation level and cellular distribution of β-catenin. In cells derived from the same tumor, we demonstrated that the prevalence of LEF1 (high β-catenin expressing cells) or TCF4 (low β-catenin expressing cells) as β-catenin partner for DNA binding, is associated to the expression of two distinct profiles of WNT-responsive genes. Interestingly, melanoma cells expressing relative low level of β-catenin and an invasive markers signature were associated to the TNF-α-induced pro-inflammatory pathway and to the chemotherapy resistance, suggesting that the co-existence of melanoma subpopulations with distinct biological properties could influence the impact of chemo- and immunotherapy.
Solid tumors are complex systems characterized by dynamic interactions between neoplastic cells, non-tumoral cells, and extracellular components. Among all the stromal cells that populate tumor microenvironment, fibroblasts are the most abundant elements and are critically involved in disease progression. Cancer-associated fibroblasts (CAFs) have pleiotropic functions in tumor growth and extracellular matrix remodeling implicated in local invasion and distant metastasis. CAFs additionally participate in the inflammatory response of the tumor site by releasing a variety of chemokines and cytokines. It is becoming clear that understanding the dynamic, mutual melanoma–fibroblast relationship would enable treatment options to be amplified. To better characterize melanoma-associated fibroblasts, here we analyzed low-passage primary CAFs derived from advanced-stage primary skin melanomas, focusing on the immuno-phenotype. Furthermore, we assessed the expression of several CAF markers and the production of growth factors. To deepen the study of CAF–melanoma cell crosstalk, we employed CAF-derived supernatants and trans-well co-culture systems to evaluate the influences of CAFs on (i) the motogenic ability of melanoma cells, (ii) the chemotherapy-induced cytotoxicity, and (iii) the release of mediators active in modulating tumor growth and spread.
Techniques for medical tissue regeneration require an abundant source of human adult stem cells. There is increasing evidence that adipose stem cells contribute to restoration of tissue vascularization and organ function. The object of our study was to isolate and characterize adult adipose-derived stem cells from patients undergoing on lipoaspirate transplant with the aim to improve tissue regeneration. Adipose-derived stem cells were isolated and purified from the lipoaspirate of 15 patients and characterized for CD markers and the ability to differentiate toward the adipogenic lineage. We found that purified adipose stem cells express high level of CD49d, CD44, CD90, CD105, CD13, and CD71 and these markers of staminality were maintained at high level for at least 3 months and seven passages of in vitro culture. As expected, these cells resulted negative for the endothelial and hematopoietic-specific markers CD31, CD106, CD34, and CD45. Differentiation towards adipogenic lineage demonstrated that purified adipose-derived stem cells are still able to become adipocytes at least 3 months after in vitro culture. The analysis of Akt and MAPK phosphorylation confirmed a modulation of their activity during differentiation. Interestingly, we established for the first time that, among the p53 family members, a strong upregulation of p63 expression occurs in adipocytic differentiation, indicating a role for this transcription factor in adipocytic differentiation. Taken together, these data indicate that purified lipoaspirate-derived stem cells maintain their characteristic of staminality for a long period of in vitro culture, suggesting that they could be applied for cell-based therapy to improve autologous lipoaspirate transplant.
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