Culture of skeletal myoblasts from human donors aged over 40 years: dynamics of cell growth and expression of differentiation markers

BACKGROUND: Local myogenesis, neoangiogenesis and homing of progenitor cells from the bone marrow appear to contribute to repair of the infarcted myocardium. Implantation into heart tissues of autologous skeletal myoblasts has been associated with improved contractile function in animal models and in humans with acute myocardial ischemia. Since heart infarction is most prevalent in individuals of over 40 years of age, we tested whether culture methods available in our laboratory were adequate to obtain sufficient numbers of differentiated skeletal myoblasts from muscle biopsy specimens obtained from patients aged 41 to 91. METHODS AND RESULTS: No matter of donor age, differentiated skeletal muscle cells could be produced in vitro in amounts adequate for cellular therapy (≥300 millions). Using desmin as a cytoplasmic marker, about 50% cultured cells were differentiated along myogenic lineages and expressed proteins proper of skeletal muscle (myosin type I and II, actin, actinin, spectrin and dystrophin). Cytogenetic alterations were not detected in cultured muscle cells that had undergone at least 10 population doublings. Molecular methods employed for the screening of persistent viral infections evidenced that HCV failed to replicate in muscle cells cultured from one patient with chronic HCV infection. CONCLUSION: The proposed culture methods appear to hold promise for aged patients not only in the field of cardiovascular medicine, but also in the urologic and orthopedic fields

Serum Extracellular Vesicle-Derived microRNAs as Potential Biomarkers for Pleural Mesothelioma in a European Prospective Study

Simple Summary Malignant pleural mesothelioma (MPM) is an aggressive and still incurable cancer. There is an urgent need to identify effective and reliable tools for detecting and diagnosing the early onset of MPM. In our study, we investigated the whole miRNAs expression profile from serum extracellular vesicles to identify early changes related to MPM development. miR-11400, miR-148a-3p, and miR-409-3p levels were increased in pre-clinical MPM patients up to five years before their diagnosis. The three-miRNA pattern showed a good discrimination capacity to distinguish pre-clinical MPM from cancer-free controls. The three miRNAs also displayed high diagnostic capabilities for differentiating between MPM patients and controls. This study identified a potential EV miRNA signature in preclinical MPM up to five years before diagnosis and raises the possibility of early intervention. Malignant pleural mesothelioma (MPM) is an aggressive cancer with a dismal prognosis. Early therapeutic interventions could improve patient outcomes. We aimed to identify a pattern of microRNAs (miRNAs) as potential early non-invasive markers of MPM. In a case-control study nested in the European Prospective Investigation into Cancer and Nutrition cohort, we screened the whole miRNome in serum extracellular vesicles (EVs) of preclinical MPM cases. In a subgroup of 20 preclinical samples collected five years prior MPM diagnosis, we observed an upregulation of miR-11400 (fold change (FC) = 2.6, adjusted p-value = 0.01), miR-148a-3p (FC = 1.5, p-value = 0.001), and miR-409-3p (FC = 1.5, p-value = 0.04) relative to matched controls. The 3-miRNA panel showed a good classification capacity with an area under the receiver operating characteristic curve (AUC) of 0.81 (specificity = 0.75, sensitivity = 0.70). The diagnostic ability of the model was also evaluated in an independent retrospective cohort, yielding a higher predictive power (AUC = 0.86). A signature of EV miRNA can be detected up to five years before MPM; moreover, the identified miRNAs could provide functional insights into the molecular changes related to the late carcinogenic process, preceding MPM development

GDNA qPCR is statistically more reliable than mRNA analysis in detecting leukemic cells to monitor CML

Chronic Myeloid Leukemia (CML) is a stem cell cancer that arises when t(9;22) translocation occurs in a hematopoietic stem cells. This event results in the expression of the BCR-ABL1 fusion gene, which codes for a constitutively active tyrosine kinase that is responsible for the transformation of a HSC into a CML stem cell, which then gives rise to a clonal myeloproliferative disease. The introduction of Tyrosine Kinase Inhibitors (TKIs) has revolutionized the management of the disease. However, these drugs do not seem to be able to eradicate the malignancy. Indeed, discontinuation trials (STIM; TWISER; DADI) for those patients who achieved a profound molecular response showed 50% relapsing within 12 months. We performed a comparative analysis on 15 CML patients and one B-ALL patient, between the standard quantitative reverse-transcriptase PCR (qRT-PCR) and our genomic DNA patient-specific quantitative PCR assay (gDNA qPCR). Here we demonstrate that gDNA qPCR is better than standard qRT-PCR in disease monitoring after an average follow-up period of 200 days. Specifically, we statistically demonstrated that DNA negativity is more reliable than RNA negativity in indicating when TKIs therapy can be safely stopped

It is time to change thrombosis risk assessment for PV and ET?

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms to be diagnosed according to the WHO classification. Molecular profiling must include the analysis of JAK2 (looking for the V617F point-mutation in PV and ET, screening exon 12 for mutations only in V617F-negative PV), CALR and MPL mutations (both in V617F-negative ET). The current risk stratification to predict thrombosis requires two parameters: age over 60 years and prior history of thrombosis. On the basis of these two risk factors patients can be stratified in low-risk and high-risk and receive a proper treatment. However, a modern stratification of thrombotic risk might consider "new" low-risk patients: conventional low-risk plus absence of leukocytosis from diagnosis onwards and a hematocrit level below 45% during the course of disease for PV; conventional low-risk plus absence of leukocytosis from diagnosis onwards, JAK2 negativity, CALR positivity, and absence of cardiovascular risk factors for ET