Elena De Falco

The Potential of GMP-Compliant Platelet Lysate to Induce a Permissive State for Cardiovascular Transdifferentiation in Human Mediastinal Adipose Tissue-Derived Mesenchymal Stem Cells

Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs.

Circulating tumor cells detection has independent prognostic impact in high-risk non-muscle invasive bladder cancer

High‐risk non‐muscle invasive bladder cancer (NMIBC) progresses to metastatic disease in 10–15% of cases, suggesting that micrometastases may be present at first diagnosis. The prediction of risks of progression relies upon EORTC scoring systems, based on clinical and pathological parameters, which do not accurately identify which patients will progress. Aim of the study was to investigate whether the presence of CTC may improve prognostication in a large population of patients with Stage I bladder cancer who were all candidate to conservative surgery. A prospective single center trial was designed to correlate the presence of CTC to local recurrence and progression of disease in high‐risk T1G3 bladder cancer. One hundred two patients were found eligible, all candidate to transurethral resection of the tumor followed by endovesical adjuvant immunotherapy with BCG. Median follow‐up was 24.3 months (minimum–maximum: 4–36). The FDA‐approved CellSearch System was used to enumerate CTC. Kaplan–Meier methods, log‐rank test and multivariable Cox proportional hazard analysis was applied to establish the association of circulating tumor cells with time to first recurrence (TFR) and progression‐free survival. CTC were detected in 20% of patients and predicted both decreased TFR (log‐rank p < 0.001; multivariable adjusted hazard ratio [HR] 2.92 [95% confidence interval: 1.38–6.18], p = 0.005), and time to progression (log‐rank p < 0.001; HR 7.17 [1.89–27.21], p = 0.004). The present findings provide evidence that CTC analyses can identify patients with Stage I bladder cancer who have already a systemic disease at diagnosis and might, therefore, potentially benefit from systemic treatment.

Bridging regenerative medicine based therapies into the 21st Century: solo or symphony?

Clinical translation in the field of regenerative medicine means manufacturing a safe, reproducible and effective clinical product for the benefit of patients. This represents the ultimate goal of applied research, but beyond researchers and clinicians, multiple intermediate players are involved, including other researchers, reviewers, funding agencies, scientific societies, guideline authors, and policy regulators. Consequently, bridging translational research and regenerative medicine therapies into the 21 st Century requires a resolute effort. We envisage that strategic and synergistic efforts in seven key areas will facilitate the mainstream adoption and implementation of regenerative medicine based therapies.

Dark Chocolate Acutely Improves Walking Autonomy in Patients With Peripheral Artery Disease

Background NOX‐2, the catalytic subunit of NADPH oxidase, has a key role in the formation of reactive oxidant species and is implicated in impairing flow‐mediated dilation (FMD). Dark chocolate exerts artery dilatation via down‐regulating NOX2‐mediated oxidative stress. The aim of this study was to investigate whether dark chocolate improves walking autonomy in peripheral artery disease (PAD) patients via an oxidative stress‐mediated mechanism. Methods and Results FMD, serum levels of isoprostanes, nitrite/nitrate (NOx) and sNOX2‐dp, a marker of blood NOX2 activity, maximal walking distance (MWD) and maximal walking time (MWT) were studied in 20 PAD patients (14 males and 6 females, mean age: 69±9 years) randomly allocated to 40 g of dark chocolate (>85% cocoa) or 40 g of milk chocolate (≤35% cocoa) in a single blind, cross‐over design. The above variables were assessed at baseline and 2 hours after chocolate ingestion. Dark chocolate intake significantly increased MWD (+11%; P<0.001), MWT (+15%; P<0.001), serum NOx (+57%; P<0.001) and decreased serum isoprostanes (−23%; P=0.01) and sNOX2‐dp (−37%; P<0.001); no changes of the above variables were observed after milk chocolate intake. Serum epicatechin and its methylated metabolite significantly increased only after dark chocolate ingestion. Multiple linear regression analysis showed that Δ of MWD was independently associated with Δ of MWT (P<0.001) and Δ of NOx (P=0.018). In vitro study demonstrated that HUVEC incubated with a mixture of polyphenols significantly increased nitric oxide (P<0.001) and decreased E‐selectin (P<0.001) and VCAM1 (P<0.001). Conclusion In PAD patients dark but not milk chocolate acutely improves walking autonomy with a mechanism possibly related to an oxidative stress‐mediated mechanism involving NOX2 regulation. Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT01947712.

Altered SDF-1-mediated differentiation of bone marrow-derived endothelial progenitor cells in diabetes mellitus

In diabetic patients and animal models of diabetes mellitus (DM), circulating endothelial progenitor cell (EPC) number is lower than in normoglycaemic conditions and EPC angiogenic properties are inhibited. Stromal cell derived factor‐1 (SDF‐1) plays a key role in bone marrow (BM) c‐kit+ stem cell mobilization into peripheral blood (PB), recruitment from PB into ischemic tissues and differentiation into endothelial cells. The aim of the present study was to examine the effect of DM in vivo and in vitro, on murine BM‐derived c‐kit+ cells and on their response to SDF‐1. Acute hindlimb ischemia was induced in streptozotocin‐treated DM and control mice; circulating c‐kit+ cells exhibited a rapid increase followed by a return to control levels which was significantly faster in DM than in control mice. CXCR4 expression by BM c‐kit+ cells as well as SDF‐1 protein levels in the plasma and in the skeletal muscle, both before and after the induction of ischemia, were similar between normoglycaemic and DM mice. However, BM‐derived c‐kit+ cells from DM mice exhibited an impaired differentiation towards the endothelial phenotype in response to SDF‐1; this effect was associated with diminished protein kinase phosphorylation. Interestingly, SDF‐1 ability to induce differentiation of c‐kit+ cells from DM mice was restored when cells were cultured under normoglycaemic conditions whereas c‐kit+ cells from normoglycaemic mice failed to differentiate in response to SDF‐1 when they were cultured in hyperglycaemic conditions. These results show that DM diminishes circulating c‐kit+ cell number following hindlimb ischemia and inhibits SDF‐1‐mediated AKT phosphorylation and differentiation towards the endothelial phenotype of BM‐derived c‐kit+ cells.

Circulating tumor cells: Exploring intratumor heterogeneity of colorectal cancer

The hypothesis of the “liquid biopsy” using circulating tumor cells (CTCs) emerged as a minimally invasive alternative to traditional tissue biopsy to determine cancer therapy. Discordance for biomarkers expression between primary tumor tissue and circulating tumor cells (CTCs) has been widely reported, thus rendering the biological characterization of CTCs an attractive tool for biomarkers assessment and treatment selection. Studies performed in metastatic colorectal cancer (mCRC) patients using CellSearch, the only FDA-cleared test for CTCs assessment, demonstrated a much lower yield of CTCs in this tumor type compared with breast and prostate cancer, both at baseline and during the course of treatment. Thus, although attractive, the possibility to use CTCs as therapy-related biomarker for colorectal cancer patients is still limited by a number of technical issues mainly due to the low sensitivity of the CellSearch method. In the present study we found a significant discordance between CellSearch and AdnaTest in the detection of CTCs from mCRC patients. We then investigated KRAS pathway activating mutations in CTCs and determined the degree of heterogeneity for KRAS oncogenic mutations between CTCs and tumor tissues. Whether KRAS gene amplification may represent an alternative pathway responsible for KRAS activation was further explored. KRAS gene amplification emerged as a functionally equivalent and mutually exclusive mechanism of KRAS pathway activation in CTCs, possibly related to transcriptional activation. The serial assessment of CTCs may represent an early biomarker of treatment response, able to overcome the intrinsic limit of current molecular biomarkers represented by intratumor heterogeneity.

A Review of the Molecular Mechanisms Underlying the Development and Progression of Cardiac Remodeling

Pathological molecular mechanisms involved in myocardial remodeling contribute to alter the existing structure of the heart, leading to cardiac dysfunction. Among the complex signaling network that characterizes myocardial remodeling, the distinct processes are myocyte loss, cardiac hypertrophy, alteration of extracellular matrix homeostasis, fibrosis, defective autophagy, metabolic abnormalities, and mitochondrial dysfunction. Several pathophysiological stimuli, such as pressure and volume overload, trigger the remodeling cascade, a process that initially confers protection to the heart as a compensatory mechanism. Yet chronic inflammation after myocardial infarction also leads to cardiac remodeling that, when prolonged, leads to heart failure progression. Here, we review the molecular pathways involved in cardiac remodeling, with particular emphasis on those associated with myocardial infarction. A better understanding of cell signaling involved in cardiac remodeling may support the development of new therapeutic strategies towards the treatment of heart failure and reduction of cardiac complications. We will also discuss data derived from gene therapy approaches for modulating key mediators of cardiac remodeling.

State of the Art on the Evidence Base in Cardiac Regenerative Therapy: Overview of 41 Systematic Reviews.

Objectives. To provide a comprehensive appraisal of the evidence from secondary research on cardiac regenerative therapy. Study Design and Setting. Overview of systematic reviews of controlled clinical trials concerning stem cell administration or mobilization in patients with cardiovascular disease. Results. After a systematic database search, we short-listed 41 reviews (660 patients). Twenty-two (54%) reviews focused on acute myocardial infarction (AMI), 19 (46%) on chronic ischemic heart disease (IHD) or heart failure (HF), 29 (71%) on bone marrow-derived stem-cells (BMSC), and 36 (88%) to randomized trials only. Substantial variability among reviews was found for validity (AMSTAR score: median 9 [minimum 3]; 1st quartile 9; 3rd quartile 10; maximum 11), effect estimates (change in ejection fraction from baseline to follow-up: 3.47% [0.02%; 2.90%; 4.22%; 6.11%]), and citations (Web of Science yearly citations: 4.1 [0; 2.2; 6.5; 68.9]). No significant association was found between these three features. However, reviews focusing on BMSC therapy had higher validity scores (P = 0.008) and showed more pronounced effect estimates (P = 0.002). Higher citations were associated with journal impact factor (P = 0.007), corresponding author from North America/Europe (P = 0.022), and inclusion of nonrandomized trials (P = 0.046). Conclusions. Substantial heterogeneity is apparent among these reviews in terms of quality and effect estimates.

Optimization of the isolation and expansion method of human mediastinal–adipose tissue derived mesenchymal stem cells with virally inactivated GMP-grade platelet lysate

Mesenchymal stem cells (MSCs) are adult multipotent cells currently employed in several clinical trials due to their immunomodulating, angiogenic and repairing features. The adipose tissue is certainly considered an eligible source of MSCs. Recently, putative adipose tissue derived MSCs (ADMSCs) have been isolated from the mediastinal depots. However, very little is known about the properties, the function and the potential of human mediastinal ADMSCs (hmADMSCs). However, the lack of standardized methodologies to culture ADMSCs prevents comparison across. Herein for the first time, we report a detailed step by step description to optimize the isolation and the expansion methodology of hmADMSCs using a virally inactivated good manufacturing practice (GMP)-grade platelet lysate, highlighting the critical aspects of the procedure and providing useful troubleshooting suggestions. Our approach offers a reproducible system which could provide standardization across laboratories. Moreover, our system is time and cost effective, and it can provide a reproducible source of adipose stem cells to enable future studies to unravel new insights regard this promising stem cell population.

Serum and supplement optimization for EU GMP‐compliance in cardiospheres cell culture

Cardiac progenitor cells (CPCs) isolated as cardiospheres (CSs) and CS‐derived cells (CDCs) are a promising tool for cardiac cell therapy in heart failure patients, having CDCs already been used in a phase I/II clinical trial. Culture standardization according to Good Manufacturing Practices (GMPs) is a mandatory step for clinical translation. One of the main issues raised is the use of xenogenic additives (e.g. FBS, foetal bovine serum) in cell culture media, which carries the risk of contamination with infectious viral/prion agents, and the possible induction of immunizing effects in the final recipient. In this study, B27 supplement and sera requirements to comply with European GMPs were investigated in CSs and CDCs cultures, in terms of process yield/efficiency and final cell product gene expression levels, as well as phenotype. B27− free CS cultures produced a significantly reduced yield and a 10‐fold drop in c‐kit expression levels versus B27+ media. Moreover, autologous human serum (aHS) and two different commercially available GMP AB HSs were compared with standard research‐grade FBS. CPCs from all HSs explants had reduced growth rate, assumed a senescent‐like morphology with time in culture, and/or displayed a significant shift towards the endothelial phenotype. Among three different GMP gamma‐irradiated FBSs (giFBSs) tested, two provided unsatisfactory cell yields, while one performed optimally, in terms of CPCs yield/phenotype. In conclusion, the use of HSs for the isolation and expansion of CSs/CDCs has to be excluded because of altered proliferation and/or commitment, while media supplemented with B27 and the selected giFBS allows successful EU GMP‐complying CPCs culture.