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In recent years, exosomes have attracted increasing scientific interest and are no longer considered just as containers for cell waste, but as important mediators of intercellular communication. Among many biomedical research topics, a possible direct role of exosomes in the regenerative medicine field has been underlined in recent studies, including those regarding the so called "paracrine hypothesis". In this perspective, a therapeutic role and/or use of exosomes for tissue regeneration seems to be plausible. However, the majority of the cells isolated and cultured in vitro are exposed to an exogenous exosomes source because of the wide use of foetal bovine serum as cell culture supplement. Bovine serum has been gradually considered as a major biological stimulus, but with still unknown outcome. In this review, we present the state of the art about the role of exosomes in regenerative medicine, particularly for the cardiovascular system. We also analyse the most commonly used exosome isolation techniques that, since their discovery, have undergone continuous development to reach the highest degree of scalability for future clinical translation.

Senescence exerts a great impact on both biological and functional properties of circulating endothelial progenitor cells (EPCs), especially in cardiovascular diseases where the physiological process of aging is accelerated upon clinical administration of certain drugs such as doxorubicin. EPC impairment contributes to doxorubicin-induced cardiotoxicity. Doxorubicin accelerates EPC aging, although mechanisms underlying this phenomenon remain to be fully clarified. Here we investigated if Nox2 activity is able to modulate the premature senescence induced in vitro by doxorubicin in human EPCs. Results showed that in conditioned media obtained from late EPC cultures, the levels of interleukin-6, isoprostanes and nitric oxide bioavailability were increased and reduced respectively after 3h of doxorubicin treatment. These derangements returned to physiological levels when cells were co-treated with apocynin or gp91ds-tat (antioxidant and specific Nox2 inhibitors, respectively). Accordingly, Nox2 activity resulted to be activated by doxorubicin. Importantly, we found that Nox2 inhibition reduced doxorubicin-induced EPC senescence, as indicated by a lower percentage of β-gal positive EPCs. In conclusion, Nox2 activity efficiently contributes to the mechanism of oxidative stress-induced increase in premature aging conferred by doxorubicin. The importance of modulation of Nox2 in human EPCs could reveal a useful tool to restore EPC physiological function and properties.

JAK2 exon 12 mutations are associated with more marked and isolated erythrocytosis than JAK2V617F. We analyzed expression profiles of JAK2 exon 12-mutant and wild-type erythroid colonies from patients with polycythemia vera (PV). Exon 12 mutations were associated with interferon-target gene upregulation, STAT1 activation and additional gene expression changes that were quantitatively and qualitatively similar to those in JAK2V617F-heterozygous cells from essential thrombocythemia (ET) patients. These results demonstrate that JAK2 exon 12-mutated PV does not reflect attenuated STAT1 signaling, and that transcriptional consequences of JAK2 mutations are remarkably similar in JAK2 exon 12-mutated PV and JAK2V617F-positive ET.

Β-blockers (BB) are a primary treatment for chronic heart disease (CHD), resulting in prognostic and symptomatic benefits. Cardiac cell therapy represents a promising regenerative treatment and, for autologous cell therapy, the patients clinical history may correlate with the biology of resident progenitors and the quality of the final cell product. This study aimed at uncovering correlations between clinical records of biopsy-donor CHD patients undergoing cardiac surgery and the corresponding yield and phenotype of cardiospheres (CSs) and CS-derived cells (CDCs), which are a clinically relevant population for cell therapy, containing progenitors. We describe a statistically significant association between BB therapy and improved CSs yield and CDCs phenotype. We show that BB-CDCs have a reduced fibrotic-like CD90 + subpopulation, with reduced expression of collagen-I and increased expression of cardiac genes, compared to CDCs from non-BB donors. Moreover BB-CDCs had a distinctive microRNA expression profile, consistent with reduced fibrotic features (miR-21, miR-29a/b/c downregulation), and enhanced regenerative potential (miR-1, miR-133, miR-101 upregulation) compared to non-BB. In vitro adrenergic pharmacological treatments confirmed cytoprotective and anti-fibrotic effects of β1-blocker on CDCs. This study shows anti-fibrotic and pro-commitment effects of BB treatment on endogenous cardiac reparative cells, and suggests adjuvant roles of β-blockers in cell therapy applications.

Subclones homozygous for JAK2V617F are more common in polycythemia vera (PV) than essential thrombocythemia (ET), but their prevalence and significance remain unclear. The JAK2 mutation status of 6495 BFU-E, grown in low erythropoietin conditions, was determined in 77 patients with PV or ET. Homozygous-mutant colonies were common in patients with JAK2V617F-positive PV and were surprisingly prevalent in JAK2V617F-positive ET and JAK2 exon 12-mutated PV. Using microsatellite PCR to map loss-of-heterozygosity breakpoints within individual colonies, we demonstrate that recurrent acquisition of JAK2V617F homozygosity occurs frequently in both PV and ET. PV was distinguished from ET by expansion of a dominant homozygous subclone, the selective advantage of which is likely to reflect additional genetic or epigenetic lesions. Our results suggest a model in which development of a dominant JAK2V617F-homzygous subclone drives erythrocytosis in many PV patients, with alternative mechanisms operating in those with small or undetectable homozygous-mutant clones.

We welcome the paper by Wever-Pinzon et al. [(1)][1] who investigated the longitudinal effects of left ventricular assist device (LVAD) unloading on cardiac function. Notably, subjects who experienced cardiac recovery more frequently took beta-blockers (BBs), which have an established

Retinoic acid (RA), the major bioactive metabolite of retinol or vitamin A, induces a spectrum of pleiotropic effects in cell growth and differentiation that are relevant for embryonic development and adult physiology. The RA activity is mediated primarily by members of the retinoic acid receptor (RAR) subfamily, namely RARα, RARβ and RARγ, which belong to the nuclear receptor (NR) superfamily of transcription factors. RARs form heterodimers with members of the retinoid X receptor (RXR) subfamily and act as ligand-regulated transcription factors through binding specific RA response elements (RAREs) located in target genes promoters. RARs also have non-genomic effects and activate kinase signaling pathways, which fine-tune the transcription of the RA target genes. The disruption of RA signaling pathways is thought to underlie the etiology of a number of hematological and non-hematological malignancies, including leukemias, skin cancer, head/neck cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, liver cancer, glioblastoma and neuroblastoma. Of note, RA and its derivatives (retinoids) are employed as potential chemotherapeutic or chemopreventive agents because of their differentiation, anti-proliferative, pro-apoptotic, and anti-oxidant effects. In humans, retinoids reverse premalignant epithelial lesions, induce the differentiation of myeloid normal and leukemic cells, and prevent lung, liver, and breast cancer. Here, we provide an overview of the biochemical and molecular mechanisms that regulate the RA and retinoid signaling pathways. Moreover, mechanisms through which deregulation of RA signaling pathways ultimately impact on cancer are examined. Finally, the therapeutic effects of retinoids are reported.

The epithelial-to-mesenchymal transition (EMT) is an essential trans-differentiation process, which plays a critical role in embryonic development, wound healing, tissue regeneration, organ fibrosis, and cancer progression. It is the fundamental mechanism by which epithelial cells lose many of their characteristics while acquiring features typical of mesenchymal cells, such as migratory capacity and invasiveness. Depending on the contest, EMT is complemented and balanced by the reverse process, the mesenchymal-to-epithelial transition (MET). In the saving economy of the living organisms, the same (Ying-Yang) tool is integrated as a physiological strategy in embryonic development, as well as in the course of reparative or disease processes, prominently fibrosis, tumor invasion and metastasis. These mechanisms and their related signaling (e.g., TGF-β and BMPs) have been effectively studied in vitro by tissue-derived cell spheroids models. These three-dimensional (3D) cell culture systems, whose phenotype has been shown to be strongly dependent on TGF-β-regulated EMT/MET processes, present the advantage of recapitulating in vitro the hypoxic in vivo micro-environment of tissue stem cell niches and their formation. These spheroids, therefore, nicely reproduce the finely regulated Ying-Yang equilibrium, which, together with other mechanisms, can be determinant in cell fate decisions in many pathophysiological scenarios, such as differentiation, fibrosis, regeneration, and oncogenesis. In this review, current progress in the knowledge of signaling pathways affecting EMT/MET and stemness regulation will be outlined by comparing data obtained from cellular spheroids systems, as ex vivo niches of stem cells derived from normal and tumoral tissues. The mechanistic correspondence in vivo and the possible pharmacological perspective will be also explored, focusing especially on the TGF-β-related networks, as well as others, such as SNAI1, PTEN, and EGR1. This latter, in particular, for its ability to convey multiple types of stimuli into relevant changes of the cell transcriptional program, can be regarded as a heterogeneous "stress-sensor" for EMT-related inducers (growth factor, hypoxia, mechano-stress), and thus as a therapeutic target.

Several studies have shown that adaptation of various viruses to grow in certain cell lines of vertebrates, leads to the selection of virus variants that bind heparan sulfate (HS) with high affinity. In this study we investigated the susceptibility of strains of dengue virus (DENV) to oversulfated heparin an analogue of HS after passages in BHK-21 cells. Field isolates of the four serotypes of DENV with a limited number of passes in mosquito cells C6/36HT were serially passaged eight times in BHK-21 cells. The adaptation of the DENV to the cell culture selected viral variants with an increased replicative capacity in BHK-21 cells and an increased susceptibility to heparin compared with the original not adapted strains, with a more significant inhibition of the infectivity in DENV-2 and DENV-4.The E protein of the adapted strains showed changes in the amino acid sequence, particularly at the position K204R to DENV-1, N67K to DENV-2, K308R and V452A for DENV-3 and E327G to DENV-4. These substitutions implicated a gain of basic residues that increased the net positive charge of the protein. These results suggest that adaptation of DENV strains to BHK-21 cells implies changes in the envelope protein, changes associated to the protein reactivity with heparin, the inhibitory effectiveness of this compound varying depending on the viral strain. In addition, these results suggest that the HS can play an important role in the infectivity of the DENV strains adapted to vertebrate cell culture, but not in the infectivity of non-adapted DENV isolates.

Hematopoiesis is a regulated multistep process, whereby transcriptional and epigenetic events contribute to progenitor fate determination. miRNAs have emerged as key players in hematopoietic cell development, differentiation and malignant transformation. From embryonic development through to adult life, miRNAs cooperate with, or are regulated, by epigenetic factors. Moreover, recent findings suggest that they contribute to chromatin structural modification, and the functional relevance of this ‘epigenetic–miRNA axis‘ will be discussed in this article. Finally, emerging evidence has highlighted that miRNAs have functional control in human hematopoietic cells, involving targeted recruitment of epigenetic complexes to evolutionarily conserved complementary genomic loci. We propose the existence of epigenetic–miRNA loops that are able to organize the whole gene expression profile in hematopoietic cells.

Subclones homozygous for JAK2V617F are more common and larger in patients with polycythemia vera compared to essential thrombocythemia, but their role in determining phenotype remains unclear. We genotyped 4564 erythroid colonies from 59 patients with polycythemia vera or essential thrombocythemia to investigate whether the proportion of JAK2V617F -homozygous precursors, compared to heterozygous precursors, is associated with clinical or demographic features. In polycythemia vera, a higher proportion of homozygous-mutant precursors was associated with more extreme blood counts at diagnosis, consistent with a causal role for homozygosity in polycythemia vera pathogenesis. Larger numbers of homozygous-mutant colonies were associated with older age, and with male gender in polycythemia vera but female gender in essential thrombocythemia. These results suggest that age promotes development or expansion of homozygous-mutant clones and that gender modulates the phenotypic consequences of JAK2V617F homozygosity, thus providing a potential explanation for the long-standing observations of a preponderance of men with polycythemia vera but of women with essential thrombocythemia.