Giuliana Di Rocco

Diabetes impairs adipose tissue–derived stem cell function and efficiency in promoting wound healing

Adipose tissue–derived stem cells (ASCs) are gaining increasing consideration in tissue repair therapeutic application. Recent evidence indicates that ASCs enhance skin repair in animal models of impaired wound healing. To assess the therapeutic activity of autologous vs. allogeneic ASCs in the treatment of diabetic ulcers, we functionally characterized diabetic ASCs and investigated their potential to promote wound healing with respect to nondiabetic ones. Adipose tissue–derived cells from streptozotocin‐induced type 1 diabetic mice were analyzed either freshly isolated as stromal vascular fraction (SVF), or following a single passage of culture (ASCs). Diabetic ASCs showed decreased proliferative potential and migration. Expression of surface markers was altered in diabetic SVF and cultured ASCs, with a reduction in stem cell marker‐positive cells. ASCs from diabetic mice released lower amounts of hepatocyte growth factor, vascular endothelial growth factor (VEGF)‐A, and insulin‐like growth factor‐1, growth factors playing important roles in skin repair. Accordingly, the supernatant of diabetic ASCs manifested reduced capability to promote keratinocyte and fibroblast proliferation and migration. Therapeutic potential of diabetic SVF administered to wounds of diabetic mice was blunted as compared with cells isolated from nondiabetic mice. Our data indicate that diabetes alters ASC intrinsic properties and impairs their function, thus affecting therapeutic potential in the autologous treatment for diabetic ulcers.

Enhanced Healing of Diabetic Wounds by Topical Administration of Adipose Tissue-Derived Stromal Cells Overexpressing Stromal-Derived Factor-1: Biodistribution and Engraftment Analysis by Bioluminescent Imaging

Chronic ulcers represent a major health problem in diabetic patients resulting in pain and discomfort. Conventional therapy does not guarantee adequate wound repair. In diabetes, impaired healing is partly due to poor endothelial progenitor cells mobilisation and homing, with altered levels of the chemokine stromal-derived factor-1 (SDF-1) at the wound site. Adipose tissue-associated stromal cells (AT-SCs) can provide an accessible source of progenitor cells secreting proangiogenic factors and differentiating into endothelial-like cells. We demonstrated that topical administration of AT-SCs genetically modified ex vivo to overexpress SDF-1, promotes wound healing into diabetic mice. In particular, by in vivo bioluminescent imaging analysis, we monitored biodistribution and survival after transplantation of luciferase-expressing cells. In conclusion, this study indicates the therapeutic potential of AT-SCs administration in wound healing, through cell differentiation, enhanced cellular recruitment at the wound site, and paracrine effects associated with local growth-factors production.

Harvest of superficial layers of fat with a microcannula and isolation of adipose tissue-derived stromal and vascular cells.

BACKGROUND Adipose tissue is a source of stromal and vascular cells suitable for regenerative medical applications. Cell recovery depends on several factors, including the characteristics of the cannula used to harvest tissue. OBJECTIVES The authors assess whether aspiration of superficial layers of adipose tissue performed with a microcannula, rather than a standard cannula, allows for improved isolation of stromal and vascular cells, and they evaluate the angiogenic potential of the isolated cells in vitro and in vivo. METHODS Adipose-derived stromal and stem cells (ADSC) were collected from the lipoaspirate of the abdomen and hip regions of 6 healthy female donors. For adipose tissue harvest, several options were compared: (1) a rounded-tip cannula with a length of 170 mm, a diameter of 3 mm, and a single elliptic suction port on the side near its distal end (port diameter: 3 × 9 mm) or (2) a rounded-tip infiltration cannula with a length of 170 mm, a diameter of 2 mm, and 5 round ports placed spirally along the sides of the distal cannula shaft (each port diameter: 1 mm) (Shipper Medical Technologies Corporation, Centennial, Colorado). Isolated cells were characterized for (1) expression of the endothelial specific marker CD31 by immunohistochemical and cytofluorimetric analyses and (2) tubular-like structure formation using a 3-dimensional angiogenesis assay on Matrigel. Human ADSC were transduced to express firefly luciferase as a marker suitable for bioluminescent tracking and transplantation studies into immunosuppressed mice were performed. RESULTS ADSC yield was determined to be significantly higher in samples collected with the microcannula (P = .04). Moreover, isolated cells acquired typical endothelial-like morphology in vitro, formed capillary-like structures, and expressed the distinctive endothelial cell marker CD31. Cells implanted into immunosuppressed mice persisted for several weeks in areas undergoing neovascularization. CONCLUSIONS These results suggest that aspiration of adipose tissue with a microcannula can be a minimally invasive method to obtain clinically relevant numbers of stromal and vascular cells useful for autologous transplant procedures and for promoting tissue regeneration and neovascularization.

Towards Therapeutic Delivery of Extracellular Vesicles: Strategies for In Vivo Tracking and Biodistribution Analysis

Extracellular vesicles (EVs), such as microvesicles and exosomes, are membranous structures containing bioactive material released by several cells types, including mesenchymal stem/stromal cells (MSCs). Increasing lines of evidences point to EVs as paracrine mediators of the beneficial effects on tissue remodeling associated with cell therapy. Administration of MSCs-derived EVs has therefore the potential to open new and safer therapeutic avenues, alternative to cell-based approaches, for degenerative diseases. However, an enhanced knowledge about in vivo EVs trafficking upon delivery is required before effective clinical translation. Only a few studies have focused on the biodistribution analysis of exogenously administered MSCs-derived EVs. Nevertheless, current strategies for in vivo tracking in animal models have provided valuable insights on the biodistribution upon systemic delivery of EVs isolated from several cellular sources, indicating in liver, spleen, and lungs the preferential target organs. Different strategies for targeting EVs to specific tissues to enhance their therapeutic efficacy and reduce possible off-target effects have been investigated. Here, in the context of a possible clinical application of MSC-derived EVs for tissue regeneration, we review the existing strategies for in vivo tracking and targeting of EVs isolated from different cellular sources and the studies elucidating the biodistribution of exogenously administered EVs.

p53 centrosomal localization diagnoses ataxia-telangiectasia homozygotes and heterozygotes

Ataxia-telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder characterized by radiosensitivity, genomic instability, and predisposition to cancer. A-T is caused by biallelic mutations in the ataxia-telangiectasia mutated (ATM) gene, but heterozygous carriers, though apparently healthy, are believed to be at increased risk for cancer and more sensitive to ionizing radiation than the general population. Despite progress in functional and sequencing-based assays, no straightforward, rapid, and inexpensive test is available for the identification of A-T homozygotes and heterozygotes, which is essential for diagnosis, genetic counseling, and carrier prediction. The oncosuppressor p53 prevents genomic instability and centrosomal amplification. During mitosis, p53 localizes at the centrosome in an ATM-dependent manner. We capitalized on the latter finding and established a simple, fast, minimally invasive, reliable, and inexpensive test to determine mutant ATM zygosity. The percentage of mitotic lymphoblasts or PBMCs bearing p53 centrosomal localization clearly discriminated among healthy donors (>75%), A-T heterozygotes (40%-56%), and A-T homozygotes (<30%). The test is specific for A-T, independent of the type of ATM mutations, and recognized tumor-associated ATM polymorphisms. In a preliminary study, our test confirmed that ATM is a breast cancer susceptibility gene. These data open the possibility of cost-effective, early diagnosis of A-T homozygotes and large-scale screenings for heterozygotes.

Human epicardium-derived cells fuse with high efficiency with skeletal myotubes and differentiate toward the skeletal muscle phenotype: a comparison study with stromal and endothelial cells

EPDCs fuse with skeletal myotubes with higher efficiency when compared to MSCs and endothelial cells. Independently from the cell origin, all nuclei recruited inside myotubes express muscle-specific genes. VCAM1 expression in nonmuscle cells is induced by soluble factors secreted by myotubes, and its function is required for fusion to occur.

Challenges and Strategies for Improving the Regenerative Effects of Mesenchymal Stromal Cell-Based Therapies

Cell-based therapies have the potential to revolutionize current treatments for diseases with high prevalence and related economic and social burden. Unfortunately, clinical trials have made only modest improvements in restoring normal function to degenerating tissues. This limitation is due, at least in part, to the death of transplanted cells within a few hours after transplant due to a combination of mechanical, cellular, and host factors. In particular, mechanical stress during implantation, extracellular matrix loss upon delivery, nutrient and oxygen deprivation at the recipient site, and host inflammatory response are detrimental factors limiting long-term transplanted cell survival. The beneficial effect of cell therapy for regenerative medicine ultimately depends on the number of administered cells reaching the target tissue, their viability, and their promotion of tissue regeneration. Therefore, strategies aiming at improving viable cell engraftment are crucial for regenerative medicine. Here we review the major factors that hamper successful cell engraftment and the strategies that have been studied to enhance the beneficial effects of cell therapy. Moreover, we provide a perspective on whether mesenchymal stromal cell-derived extracellular vesicle delivery, as a cell-free regenerative approach, may circumvent current cell therapy limitations.

Sirtuins 1–7 expression in human adipose-derived stem cells from subcutaneous and visceral fat depots: influence of obesity and hypoxia

The sirtuin family comprises seven NAD+-dependent deacetylases which control the overall health of organisms through the regulation of pleiotropic metabolic pathways. Sirtuins are important modulators of adipose tissue metabolism and their expression is higher in lean than obese subjects. At present, the role of sirtuins in adipose-derived stem cells has not been investigated yet. Therefore, in this study, we evaluated the expression of the complete panel of sirtuins in adipose-derived stem cells isolated from both subcutaneous and visceral fat of non-obese and obese subjects. We aimed at investigating the influence of obesity on sirtuins’ levels, their role in obesity-associated inflammation, and the relationship with the peroxisome proliferator-activated receptor delta, which also plays functions in adipose tissue metabolism. The mRNA levels in the four types of adipose-derived stem cells were evaluated by quantitative polymerase chain reaction, in untreated cells and also after 8 h of hypoxia exposure. Correlations among sirtuins’ expression and clinical and molecular parameters were also analyzed. We found that sirtuin1–6 exhibited significant higher mRNA expression in visceral adipose-derived stem cells compared to subcutaneous adipose-derived stem cells of non-obese subjects. Sirtuin1–6 levels were markedly reduced in visceral adipose-derived stem cells of obese patients. Sirtuins’ expression in visceral adipose-derived stem cells correlated negatively with body mass index and C-reactive protein and positively with peroxisome proliferator-activated receptor delta. Finally, only in the visceral adipose-derived stem cells of obese patients hypoxia-induced mRNA expression of all of the sirtuins. Our results highlight that sirtuins’ levels in adipose-derived stem cells are consistent with protective effects against visceral obesity and inflammation, and suggest a transcriptional mechanism through which acute hypoxia up-regulates sirtuins in the visceral adipose-derived stem cells of obese patients.

Promotion of Survival and Engraftment of Transplanted Adipose Tissue-Derived Stromal and Vascular Cells by Overexpression of Manganese Superoxide Dismutase

Short-term persistence of transplanted cells during early post-implant period limits clinical efficacy of cell therapy. Poor cell survival is mainly due to the harsh hypoxic microenvironment transplanted cells face at the site of implantation and to anoikis, driven by cell adhesion loss. We evaluated the hypothesis that viral-mediated expression of a gene conferring hypoxia resistance to cells before transplant could enhance survival of grafted cells in early stages after implant. We used adipose tissue as cell source because it consistently provides high yields of adipose-tissue-derived stromal and vascular cells (ASCs), suitable for regenerative purposes. Luciferase positive cells were transduced with lentiviral vectors expressing either green fluorescent protein as control or human manganese superoxide dismutase (SOD2). Cells were then exposed in vitro to hypoxic conditions, mimicking cell transplantation into an ischemic site. Cells overexpressing SOD2 displayed survival rates significantly greater compared to mock transduced cells. Similar results were also obtained in vivo after implantation into syngeneic mice and assessment of cell engraftment by in vivo bioluminescent imaging. Taken together, these findings suggest that ex vivo gene transfer of SOD2 into ASCs before implantation confers a cytoprotective effect leading to improved survival and engraftment rates, therefore enhancing cell therapy regenerative potential.

Exosomes and other extracellular vesicles-mediated microRNA delivery for cancer therapy

Abstract: Extracellular vesicles (EVs), including exosomes and microvesicles, are critical mediators of cellto- cell communication in tissue homeostasis and repair, both in physiological and pathological conditions. Recently, progress has been achieved in their use in regenerative medicine as transfer agents for active biomolecules. Specifically, EVs are natural carriers of microRNAs (miRNAs), protecting their cargo from plasma ribonucleases and delivering their content to recipient cells. Expression of miRNAs is dysregulated in virtually all forms of cancer. Therefore, EVs-mediated miRNA delivery may represent a valuable tool for cancer therapeutic intervention, aiming at restoring cancer miRNAs expression to normal levels. MicroRNAs may act both as tumor suppressors or oncogenes, consequently different alternative approaches for regulating miRNA expression in tumor tissues have been developed. Here, we review the various strategies for miRNA loading into EVs and highlight studies of EVs-mediated miRNA delivery which have been employed for cancer treatment, both in vitro and in vivo . Collectively, these data support the use of EVs in miRNAs/miRNAs antagonist transfer for cancer therapy, but challenges related to EV biology have yet to be further addressed before safe clinical translation.