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Tissue regeneration or healing both require efficient vascularization within a tissue-damaged area. Based on this concept, a remarkable number of strategies, aimed at developing new tools to support re-vascularization of damaged tissue have emerged. Among the strategies proposed, the use of pro-angiogenic soluble factors, as a cell-free tool, appears as a promising approach, able to overcome the issues concerning the direct use of cells for regenerative medicine therapy. Here, we compared the effectiveness of adipose mesenchymal stem cells (ASCs), use as cell suspension, ASC protein extract or ASC-conditioned-medium (i.e., soluble factors), combined with collagenic scaffold, in supporting in vivo angiogenesis. We also tested the capability of hypoxia in increasing the efficiency of ASC to promote angiogenesis, via soluble factors, both in vivo and in vitro. In vivo studies were performed using the Integra® Flowable Wound Matrix, and the Ultimatrix in sponge assay. Flow cytometry was used to characterize the scaffold- and sponge-infiltrating cells. Real-time PCR was used to evaluate the expression of pro-angiogenic factors by stimulating Human Umbilical-Vein Endothelial Cells with ASC-conditioned media, obtained in hypoxic and normoxic conditions. We found that, in vivo, ACS-conditioned media can support angiogenesis similar to ASCs and ASC protein extract. Also, we observed that hypoxia increases the pro-angiogenic activities of ASC-conditioned media, compared to normoxia, by generating a secretome enriched in pro-angiogenic soluble factors, with bFGF, Adiponectine, ENA78, GRO, GRO-a, and ICAM1-3, as most regulated factors. Finally, ASC-conditioned media, produced in hypoxic condition, induce the expression of pro-angiogenic molecules in HUVECs. Our results provide evidence that ASC-conditioned-medium can be proposed as a cell-free preparation able to support angiogenesis, thus providing a relevant tool to overcome the issues and restrictions associated with the use of cells.

Currently, more than 30 000 allogeneic hematopoietic stem cell (HSC) transplantations have been performed for the treatment of hematological and nonhematological diseases using HSC from umbilical cord blood (CB). However, the wide utilization of CB as a source of HSC is limited by the low number of cells recovered. One strategy to expand ex vivo CB‐HSC is represented by the use of bone marrow mesenchymal stromal cells (BM‐MSCs) as a feeder to enhance HSC proliferation while maintaining HSC stemness. Indeed, BM‐MSCs have been recognized as one of the most relevant players in the HSC niche. Thus, it has been hypothesized that they can support the ex vivo expansion of HSC by mimicking the physiological microenvironment present in the hematopoietic niche. Due to the role of placenta in supporting fetal hematopoiesis, MSC derived from the amniotic membrane (hAMSC) of human term placenta could represent an interesting alternative to BM‐MSC as a feeder layer to enhance the proliferation and maintain HSC stemness. Therefore, in this study we investigated if hAMSC could support the ex vivo expansion of HSC and progenitor cells. The capacity of hAMSCs to support the ex vivo expansion of CB‐HSC was evaluated in comparison to the control condition represented by the CB‐CD34+ cells without a feeder layer. The coculture was performed at two different CD34+:MSC ratios (1:2 and 1:8) in both cell‐to‐cell contact and transwell setting. After 7 days, the cells were collected and analyzed for phenotype and functionality. Our results suggest that hAMSCs represent a valuable alternative to BM‐MSC to support: (a) the ex vivo expansion of CB‐HSC in both contact and transwell systems, (b) the colony forming unit ability, and (c) long‐term culture initiating cells ability. Overall, these findings may contribute to address the unmet need of high HSC content in CB units available for transplantation., Ex vivo expansion of cord blood hematopoietic stem cell (HSC) and progenitor cell is required to improve efficacy of transplantation in adults. Bone marrow mesenchymal stromal cells are the gold standard for HSC expansion, but they can be difficult to obtain. Amniotic membrane‐derived MSC can be a valid alternative that could help meet the demand of HSC for transplants.

Human amniotic membrane‐derived mesenchymal stromal cells (hAMSCs) are easily obtained in large quantities and free from ethical concerns. Promising therapeutic results for both hAMSCs and their secreted factors (secretome) were described by several in vitro and preclinical studies, often for treatment of orthopedic disorders such as osteoarthritis (OA) and tendinopathy. For clinical translation of the hAMSC secretome as cell‐free therapy, a detailed characterization of hAMSC‐secreted factors is mandatory. Herein, we tested the presence of 200 secreted factors and 754 miRNAs in extracellular vesicles (EVs). Thirty‐seven cytokines/chemokines were identified at varying abundance, some of which involved in both chemotaxis and homeostasis of inflammatory cells and in positive remodeling of extracellular matrix, often damaged in tendinopathy and OA. We also found 336 EV‐miRNAs, 51 of which accounted for more than 95% of the genetic message. A focused analysis based on miRNAs related to OA and tendinopathy showed that most abundant EV‐miRNAs are teno‐ and chondro‐protective, able to induce M2 macrophage polarization, inhibit inflammatory T cells, and promote Treg. Functional analysis on IL‐1β treated tenocytes and chondrocytes resulted in downregulation of inflammation‐associated genes. Overall, presence of key regulatory molecules and miRNAs explain the promising therapeutic results of hAMSCs and their secretome for treatment of musculoskeletal conditions and are a groundwork for similar studies in other pathologies. Furthermore, identified molecules will pave the way for future studies aimed at more sharply predicting disease‐targeted clinical efficacy, as well as setting up potency and release assays to fingerprint clinical‐grade batches of whole secretome or purified components., The secretome from human amniotic membrane‐derived mesenchymal stromal cells offers tremendous potential for treatment of musculoskeletal disorders. Combined analysis of soluble factors and extracellular vesicle‐miRNAs show their teno‐/chondro‐protective features, as well as ability to induce M2 macrophage polarization, inhibit inflammatory T cells, and promote Treg. The fingerprint of key regulatory molecules will define the potency of future off‐the‐shelf and cell‐free regenerative therapies.

Summary Enhancer RNAs (eRNAs) are a subset of long noncoding RNA generated from genomic enhancers: they are thought to act as potent promoters of the expression of nearby genes through interaction with the transcriptional and epigenomic machineries. In the present work, we describe two eRNAs transcribed from the enhancer of Nkx2-5—a gene specifying a master cardiomyogenic lineage transcription factor (TF)—which we call Intergenic Regulatory Element Nkx2-5 Enhancers (IRENEs). The IRENEs are encoded, respectively, on the same strand (SS) and in the divergent direction (div) respect to the nearby gene. Of note, these two eRNAs have opposing roles in the regulation of Nkx2-5: IRENE-SS acts as a canonical promoter of transcription, whereas IRENE-div represses the activity of the enhancer through recruitment of the histone deacetylase sirtuin 1. Thus, we have identified an autoregulatory loop controlling expression of the master cardiac TF NKX2-5, in which one eRNA represses transcription., Graphical Abstract, Highlights • Two eRNAs (IRENE-SS, IRENE-div) with opposing functions are found upstream of Nkx2-5 • IRENE-SS works as a classical eRNA, acting as a transcriptional activator • IRENE-div acts unconventionally, functioning as a transcriptional repressor • IRENEs epigenetically control enhancer status and, subsequently, locus architecture, Biological Sciences; Molecular Biology; Molecular Mechanism of Gene Regulation

Background It is now well established that factors (free or in extracellular vesicles) secreted by mesenchymal stromal cells (MSC) are important mediators of MSC regenerative actions. Herein we produced the secretome (conditioned medium, CM) from MSC isolated from the amniotic membrane (hAMSC) and CM from the intact amniotic membrane (hAM, no manipulation or enzymatic digestion) in order to potentially identify an effective, easy and less expensive secretome to produce for potential applications in regenerative medicine. Given that immunomodulation is a key mechanism of action through which hAMSC contributes to tissue regeneration, we used a comprehensive panel of in vitro immunomodulatory tests to compare the CMs. Methods Amniotic membranes were either cut into fragments or used for hAMSC isolation. CMs from hAMSC at passages 0 and 2 were collected after a standard 5-day culture while CM from hAM was collected after a 2- and 5-day culture. Immunomodulation was assessed in terms of PBMC and T-cell proliferation, T-cell subset polarization, T-regulatory cell induction, cell cytotoxicity and monocyte differentiation toward antigen-presenting cells. Furthermore, we performed a comparison between CM obtained from single donors and pooled CM. We also assessed the impact of lyophilization on the immunomodulatory properties of CM. Results We demonstrate that CM from hAM has comparable immunomodulatory properties to CM from hAMSC at passages 0 and 2. Furthermore, we demonstrate that pooled CMs have similar effects when compared to CM from single donors used separately. Finally, we demonstrate that lyophilization does not alter the in vitro immunomodulatory properties of CM from hAM and hAMSC. Conclusions The results presented herein support the possibility to produce secretome from intact hAM and open the prospect to highly improve the scalability of the GMP production process while reducing the costs and time related to the process of cell isolation and expansion. Moreover, the possibility of having a lyophilized secretome that maintains its original properties would allow for a ready-to-use product with easier handling, shipping and storage. The use of a lyophilized product will also facilitate clinicians by permitting customized reconstitution volumes and methods according to the most suitable formula required by the clinical application.

Perinatal cells, including cells from placenta, fetal annexes (amniotic and chorionic membranes), umbilical cord, and amniotic fluid display intrinsic immunological properties which very likely contribute to the development and growth of a semiallogeneic fetus during pregnancy. Many studies have shown that perinatal cells can inhibit the activation and modulate the functions of various inflammatory cells of the innate and adaptive immune systems, including macrophages, neutrophils, natural killer cells, dendritic cells, and T and B lymphocytes. These immunological properties, along with their easy availability and lack of ethical concerns, make perinatal cells very useful/promising in regenerative medicine. In recent years, extracellular vesicles (EVs) have gained great interest as a new therapeutic tool in regenerative medicine being a cell-free product potentially capable, thanks to the growth factors, miRNA and other bioactive molecules they convey, of modulating the inflammatory microenvironment thus favoring tissue regeneration. The immunomodulatory actions of perinatal cells have been suggested to be mediated by still not fully identified factors (secretoma) secreted either as soluble proteins/cytokines or entrapped in EVs. In this review, we will discuss how perinatal derived EVs may contribute toward the modulation of the immune response in various inflammatory pathologies (acute and chronic) by directly targeting different elements of the inflammatory microenvironment, ultimately leading to the repair and regeneration of damaged tissues.

This work is addressed to provide, by in vitro experiments, results on the repercussion that a nanostructured scaffold could have on viability, differentiation and secretion of bioactive factors of human adipose-derived stem cells (hASCs) when used in association to promote angiogenesis, a crucial condition to favour tissue regeneration. To achieve this aim, we evaluated cell viability and morphology by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and microscopy analysis, respectively. We also investigated the expression of some of those genes involved in angiogenesis and differentiation processes utilizing quantitative polymerase chain reaction (qPCR), whereas the amounts of Vascular Endothelial Growth Factor A, Interleukin 6 and Fatty Acid-Binding Protein 4 secreted in the culture medium, were quantified by enzyme-linked immunosorbent assay (ELISA). Results suggested that, in the presence of the scaffold, cell proliferation and the exocytosis of factors involved in the angiogenesis process are reduced, by contrast, the expression of those genes involved in hASC differentiation appeared enhanced. To guarantee cell survival, the construct dimensions are, generally, smaller than clinically required. Furthermore, being the paracrine event the primary mechanism exerting the beneficial effects on injured tissues, the use of conditioned culture medium instead of cells may be convenient.

Adult vascular smooth muscle cells (VSMCs) dedifferentiate in response to extracellular cues such as vascular damage and inflammation. Dedifferentiated VSMCs are proliferative, migratory, less contractile, and can contribute to vascular repair as well as to cardiovascular pathologies such as intimal hyperplasia/restenosis in coronary artery and arterial aneurysm. We here demonstrate the role of ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) as an epigenetic master regulator of VSMC plasticity. UHRF1 expression correlated with the development of vascular pathologies associated with modulation of noncoding RNAs, such as microRNAs. miR-145 — pivotal in regulating VSMC plasticity, which is reduced in vascular diseases — was found to control Uhrf1 mRNA translation. In turn, UHRF1 triggered VSMC proliferation, directly repressing promoters of cell-cycle inhibitor genes (including p21 and p27) and key prodifferentiation genes via the methylation of DNA and histones. Local vascular viral delivery of Uhrf1 shRNAs or Uhrf1 VSMC-specific deletion prevented intimal hyperplasia in mouse carotid artery and decreased vessel damage in a mouse model of aortic aneurysm. Our study demonstrates the fundamental role of Uhrf1 in regulating VSMC phenotype by promoting proliferation and dedifferentiation. UHRF1 targeting may hold therapeutic potential in vascular pathologies.

Autologous platelet rich plasma (PRP) is clinically used to induce repair of different tissues through the release of bioactive molecules. In some patients, the production of an efficient autologous PRP is unfeasible due to their compromised health. We developed an allogeneic PRP mismatched for AB0 and Rh antigens. To broadcast its clinical applications avoiding side effects the outcome of allogeneic PRP on immune response should be defined. Thus, we investigated whether PRP affected the differentiation of peripheral blood monocytes to dendritic cells upon stimulation with granulocyte monocyte colony stimulating factor and interleukin-4. Indeed, these cells are the main players of immune response and tissue repair. PRP inhibited the differentiation of monocytes to CD1a+ dendritic cells and favored the expansion of phagocytic CD163+CD206+ fibrocyte-like cells. These cells produced inteleukin-10 and prostaglandin-E2, but not interferon-γ, upon stimulation with lipopolysaccharides. Moreover, they promoted the expansion of regulatory CD4+CD25+FoxP3+ T cells upon allostimulation or antigen specific priming. Finally, the conditioned medium harvested from monocytes differentiated with PRP triggered a strong chemotactic effect on mesenchymal cells in both scratch and transwell migration assays. These results strongly suggest that allogeneic PRP can foster the differentiation of monocytes to a regulatory anti-inflammatory population possibly favoring wound healing.

The tumor microenvironment (TME) plays a critical role in tumorigenesis and is composed of different cellular components, including immune cells and mesenchymal stromal cells (MSCs). In this review, we will discuss MSCs in the TME setting and more specifically their interactions with immune cells and how they can both inhibit (immunosurveillance) and favor (immunoediting) tumor growth. We will also discuss how MSCs are used as a therapeutic strategy in cancer. Due to their unique immunomodulatory properties, MSCs isolated from perinatal tissues are intensely explored as therapeutic interventions in various inflammatory-based disorders with promising results. However, their therapeutic applications in cancer remain for the most part controversial and, importantly, the interactions between administered perinatal MSC and immune cells in the TME remain to be clearly defined.

Methylation at 5-cytosine (5-mC) is a fundamental epigenetic DNA modification associated recently with cardiac disease. In contrast, the role of 5-hydroxymethylcytosine (5-hmC)—5-mC's oxidation product—in cardiac biology and disease is unknown. Here we assess the hydroxymethylome in embryonic, neonatal, adult and hypertrophic mouse cardiomyocytes, showing that dynamic modulation of hydroxymethylated DNA is associated with specific transcriptional networks during heart development and failure. DNA hydroxymethylation marks the body of highly expressed genes as well as distal regulatory regions with enhanced activity. Moreover, pathological hypertrophy is characterized by a shift towards a neonatal 5-hmC distribution pattern. We also show that the ten-eleven translocation 2 (TET2) enzyme regulates the expression of key cardiac genes, such as Myh7, through 5-hmC deposition on the gene body and at enhancers. Thus, we provide a genome-wide analysis of 5-hmC in the cardiomyocyte and suggest a role for this epigenetic modification in heart development and disease., 5-hydroxymethylation of cysteine (5-hmC) plays a role in epigenetic regulation. Here the authors analyse the hydroxymethylome in embryonic, neonatal, adult and hypertrophic mouse cardiomyocytes and show that the dynamic modulation of hydroxymethylated DNA is important for cardiomyocyte gene expression programming in heart development and failure.

Background. The tumor microenvironment is typically an immunosuppressive niche often characterized by low oxygen tension, representing an intrinsic limitation to the success of immunotherapeutic approaches. Several lines of evidence indicate hypoxia is a master regulator of the adenosinergic axis, up-regulating on one side expression of CD39 and CD73, the two enzymes that generate adenosine starting from ATP/ADP and on the other side the adenosine receptors, which are powerful inhibitors of immune responses. Our previous studies using primary samples indicate that CLL cells can produce extracellular adenosine and that the CLL microenvironment is rich in expression of the A2A receptor, which potently inhibits T cell responses and skews macrophages towards type 2 responses(1, 2). Aim of the work. The main aims of this work are i) to dynamically study expression and activity of the adenosinergic axis using an in vivo model of CLL, and ii) to investigate whether its targeting may restore immune responses. Results. We exploited the TCL1 mouse model of CLL by adoptively transferring different leukemias into immunocompetent wild-type C57BL/6 mice and following the re-organization of the microenvironment and the reshaping of immune responses during disease progression. The first observation is that the appearance of large leukemic nodules that subvert normal splenic architecture is accompanied by increased staining with the hypoxia marker pimonidazole and by lactic acidosis, as witnessed by progressive increase in LDH activity within the tissue. The second observation concerns the onset of progressive immunosuppression with appearance of terminally differentiated and dysfunctional T lymphocytes and skewing of classical inflammatory monocytes to anti-inflammatory patrolling monocytes. Leukemia development is also accompanied by increased adenosine bio-synthetic potential due to the up-regulation of CD39 and CD73 and to the marked and generalized up-regulation of the A2A adenosine receptor, which is evident in all cells of the microenvironment, including leukemic cells, T lymphocytes and macrophages, as documented by real-time PCR studies on purified populations and by flow cytometry and immunohistochemistry analyses. This finding suggests that A2A may be a common mediator of immune suppression and that it may be a suitable therapeutic target. To test this hypothesis, we adoptively transferred different TCL1 leukemias in naïve C57BL/6 mice, allowed engraftment for 10 days and then treated with the commercially available SCH58621 A2A inhibitor every other day for two weeks (1mg/kg, intra-peritoneally). At the end of treatment, mice were euthanized and immune features examined as above. Results very consistently showed that A2A inhibition increased the naïve component of both CD4+ and CD8+ subsets, with a concomitant partial reduction in effector T lymphocytes and in regulatory T cells. Moreover, both CD4+ and CD8+ subsets recovered their cytotoxic functions as indicated by the production of IFN-γ and IL-2. When examining the macrophage compartment, SCH58261 repolarized monocytes, by increasing the inflammatory subset at the expense of patrolling monocytes. Conclusions. Taken together, these results highlight the relevance of the adenosinergic axis in the creation and maintenance of a tumor microenvironment that favors immune escape. This could be due, at least in part, to the presence of a highly hypoxic leukemic niche. Interrupting this network using an A2A receptor antagonist restores the functions of the different immune cell subsets. Therefore, these data suggest that the adenosinergic axis may represent a good target in treatment strategies that combine anti-leukemic drugs with agents able to repolarize the CLL environment towards immune competence.Serra S, et al. (2016) Adenosine signaling mediates hypoxic responses in the chronic lymphocytic leukemia microenvironment. 1(1).doi:10.1182/bloodadvances.2016000984.The.Serra S, et al. (2011) CD73-generated extracellular adenosine in chronic lymphocytic leukemia creates local conditions counteracting drug-induced cell death. 118(23):6141-6153. Disclosures Deaglio: iTeos therapeutics: Research Funding; Verastem: Research Funding; VelosBio inc: Research Funding.

Background: Correct gene expression programming of the cardiomyocyte underlies the normal functioning of the heart. Alterations to this can lead to the loss of cardiac homeostasis, triggering heart dysfunction. Although the role of some histone methyltransferases in establishing the transcriptional program of postnatal cardiomyocytes during heart development has been shown, the function of this class of epigenetic enzymes is largely unexplored in the adult heart. In this study, we investigated the role of G9a/Ehmt2, a histone methyltransferase that defines a repressive epigenetic signature, in defining the transcriptional program for cardiomyocyte homeostasis and cardiac hypertrophy. Methods: We investigated the function of G9a in normal and stressed cardiomyocytes with the use of a conditional, cardiac-specific G9a knockout mouse, a specific G9a inhibitor, and high-throughput approaches for the study of the epigenome (chromatin immunoprecipitation sequencing) and transcriptome (RNA sequencing); traditional methods were used to assess cardiac function and cardiovascular disease. Results: We found that G9a is required for cardiomyocyte homeostasis in the adult heart by mediating the repression of key genes regulating cardiomyocyte function via dimethylation of H3 lysine 9 and interaction with enhancer of zeste homolog 2, the catalytic subunit of polycomb repressive complex 2, and MEF2C-dependent gene expression by forming a complex with this transcription factor. The G9a-MEF2C complex was found to be required also for the maintenance of heterochromatin needed for the silencing of developmental genes in the adult heart. Moreover, G9a promoted cardiac hypertrophy by repressing antihypertrophic genes. Conclusions: Taken together, our findings demonstrate that G9a orchestrates critical epigenetic changes in cardiomyocytes in physiological and pathological conditions, thereby providing novel therapeutic avenues for cardiac pathologies associated with dysregulation of these mechanisms.

Cardiac hypertrophy, initially an adaptive response of the myocardium to stress, can progress to heart failure. The epigenetic signature underlying this phenomenon is poorly understood. Here, we report on the genome-wide distribution of seven histone modifications in adult mouse cardiomyocytes subjected to a prohypertrophy stimulus in vivo. We found a set of promoters with an epigenetic pattern that distinguishes specific functional classes of genes regulated in hypertrophy and identified 9,207 candidate active enhancers whose activity was modulated. We also analyzed the transcriptional network within which these genetic elements act to orchestrate hypertrophy gene expression, finding a role for myocyte enhancer factor (MEF)2C and MEF2A in regulating enhancers. We propose that the epigenetic landscape is a key determinant of gene expression reprogramming in cardiac hypertrophy and provide a basis for understanding the role of chromatin in regulating this phenomenon.

Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c-KIT+ hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O2 versus 1% O2 preconditioning). The capacity of the c-KIT+ hAFS-derived EV (hAFS-EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS-EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA-Cre, SmnF7/F7 mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS-EV. In conclusion, this is the first study showing that c-KIT+ hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy.

Temozolomide (TMZ) and carmustine (BCNU), cancer-drugs usually used in the treatment of gliomas, are DNA-methylating agents producing O6-methylguanine. It has been shown that 06-methylguanine triggers DNA mismatch repair and in turn induce apoptosis and senescence, respectively, over a 4 and 6 days period [Y. Hirose, M.S. Berger, R.O. Pieper, p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells, Cancer Res. 61 (2001) 1957–1963; W. Roos, M. Baumgartner, B. Kaina, Apoptosis triggered by DNA damage O6-methylguanine in human lymphocytes requires DNA replication and is mediated by p53 and Fas/CD95/Apo-1, Oncogene 23 (2004) 359–367]. Here we show that TMZ and BCNU have an earlier effect on nuclear organization and chromatin structure. In particular, we report that TMZ and BCNU induce clustering of pericentromeric heterochromatin regions and increase the amount of heterochromatic proteins MeCP2 and HP1α bound to chromatin. These drugs also decrease global levels of histone H3 acetylation and increase levels of histone H3 trimethylated on lysine 9 (H3-triMeK9). These events precede the senescence status. We conclude that TMZ and BCNU efficacy in glioma treatment may implicate a first event characterized by changes in heterochromatin organization and its silencing which is then followed by apoptosis and senescence.

Heterochromatin plays an important role in transcriptional repression, for the correct segregation of chromosomes and in the maintenance of genome stability. Pericentric heterochromatin (PH) replication and formation have been proposed to occur in the pericentric heterochromatin duplication body (pHDB). A central question is how the underacetylated state of heterochromatic histone H4 tail is established and controlled, because it is a key event during PH replication and is essential to maintain the compacted and silenced state of these regions. Np95 is a cell cycle regulated and is a nuclear histone-binding protein that also recruits HDAC-1 to target promoters. It is essential for S phase and for embryonic formation and is implicated in chromosome stability. Here we show that Np95 is part of the pHDB, and its functional ablation causes a strong reduction in PH replication. Depletion of Np95 also causes a hyperacetylation of lysines 8, 12, and 16 of heterochromatin histone H4 and an increase of pericentromeric major satellite transcription, whose RNAs are key players for heterochromatin formation. We propose that Np95 is a new relevant protein involved in heterochromatin replication and formation.

The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kit pos cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kitpos eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage. © 2013 Elsevier Inc.

Adult mammalian cells can be reprogrammed to a pluripotent state by forcing the expression of a few embryonic transcription factors. The resulting induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers. It is well known that post-natal cardiomyocytes (CMs) lack the capacity to proliferate. Here, we report that neonatal CMs can be reprogrammed to generate iPS cells that express embryonic-specific markers and feature gene-expression profiles similar to those of mouse embryonic stem (mES) cell and cardiac fibroblast (CF)-derived iPS cell populations. CM-derived iPS cells are able to generate chimeric mice and, moreover, re-differentiate toward CMs more efficiently then either CF-derived iPS cells or mES cells. The increased differentiation capacity is possibly related to CM-derived iPS cells retaining an epigenetic memory of the phenotype of their founder cell. CM-derived iPS cells may thus lead to new information on differentiation processes underlying cardiac differentiation and proliferation.

Np95 is an important determinant in cell cycle progression. Its expression is tightly regulated and becomes detectable shortly before the entry of cells into S phase. Accordingly, Np95 is absolutely required for the G1/S transition. Its continued expression throughout the S/G2/M phases further suggests additional roles. Indeed, Np95 has been implicated in DNA damage response. Here, we show that Np95 is tightly bound to chromatin in vivo and that it binds to histones in vivo and in vitro. The binding to histones is direct and shows a remarkable preference for histone H3 and its N-terminal tail. A novel protein domain, the SRA-YDG domain, contained in Np95 is indispensable both for the interaction with histones and for chromatin binding in vivo. Np95 contains a RING finger. We show that this domain confers E3 ubiquitin ligase activity on Np95, which is specific for core histones, in vitro. Finally, Np95 shows specific E3 activity for histone H3 when the endogenous core octamer, coimmunoprecipitating with Np95, is used as a substrate. Histone ubiquitination is an important determinant in the regulation of chromatin structure and gene transcription. Thus, the demonstration that Np95 is a chromatin-associated ubiquitin ligase suggests possible molecular mechanisms for its action as a cell cycle regulator.

Background aims Platelet derivatives have been proposed as alternatives to animal sera given that for cell therapy applications, the use of fetal bovine/calf serum (FBS/FCS) is subjected to severe limitations for safety and ethical concerns. We developed a cell culture medium additive obtained by the combination of two blood-derived standardized components. Methods A platelet lysate (PL) and a platelet-poor plasma (PPP) were produced in a lyophilized form. Each component was characterized for its growth factor content (platelet-derived growth factor-BB/vascular endothelial growth factor). PL and PPP were used as single components or in combination in different ratio at cumulative 5% final concentration in the culture medium. Results The single components were less effective than the component combination. In primary cell cultures (bone marrow stromal cells, adipose derived adult stem cells, osteoblasts, chondrocytes, umbilical cord–derived mesenchymal stromal cells, lymphocytes), the PL/PPP supplement promoted an increased cell proliferation in respect to the standard FCS culture in a dose-dependent manner, maintaining the cell functionality, clonogenicity, phenotype and differentiative properties throughout the culture. At a different component ratio, the supplement was also used to support proliferation of a cell line (U-937). Conclusions The PL/PPP supplement is an efficient cell culture medium additive that can replace FCS to promote cell proliferation. It can outdo FCS, especially when adopted in primary cultures from tissue biopsies. Moreover, the dual component nature of the supplement allows the researcher to determine the more appropriate ratio of the two components for the nutritional and functional requirements of the cell type of interest.

Background: There is an urgent need of neuronal cell models to be applied to high-throughput screening settings while recapitulating physiological and/or pathological events occurring in the Central Nervous System (CNS). Stem cells offer a great opportunity in this direction since their self renewal capacity allows for large scale expansion. Protocols for directed differentiation also promise to generate populations of biochemically homogenous neuronal progenies. NS (Neural Stem) cells are a novel population of stem cells that undergo symmetric cell division in monolayer and chemically defined media, while remaining highly neurogenic. Results: We report the full adaptation of the NS cell systems for their growth and neuronal differentiation to 96and 384-well microplates. This optimized system has also been exploited in homogeneous and high-content assays. Conclusions: Our results show that these mouse NS cells may be suitable for a series of applications in highthroughput format. Background Highly-informative automated assays that monitor cell morphology, proliferation, death, motility and differentiation offer, nowadays, a great opportunity for the discovery of new gene functions, the study of molecular networks, the identification and validation of disease-relevant drug targets as well as the selection of pharmacologically active compounds. These assays are crucial for the obtainment of rapid, reliable and reproducible results. However, biological systems inevitably bring along some disadvantages with respect to a pure biochemical assay. Therefore one aim is to optimize the number and quality of the information gained from the selected cell model. Generally, the closest the cells employed recapitulate their in vivo counterparts, the more reliable the obtained data are for a given situation or pathology. Cells for drug discovery research are typically obtained from primary tissues, genetically transformed or immortalized tumor cells [1]. More recent stem cell

Monitoring Editor: Yixian Zheng Heterochromatic chromosomal regions undergo large-scale reorganization and progressively aggregate, forming chromocenters. These are dynamic structures that rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and differentiation. Np95-ICBP90 (m- and h-UHRF1) is a histone binding protein expressed only in proliferating cells. During pericentromeric heterochromatin (PH) replication, Np95 specifically relocalizes to chromocenters where it highly concentrates in the replication factories that correspond to less compacted DNA. Np95 recruits HDAC and DNMT1 to PH and depletion of Np95 impairs PH replication. Here we show that Np95 causes large-scale modifications of chromocenters independently from the H3:K9 and H4:K20 trimethylation pathways, from the expression levels of HP1, from DNA methylation and from the cell cycle. The PHD domain is essential to induce this effect. The PHD domain is also required in vitro to increase access of a restriction enzyme to DNA packaged into nucleosomal arrays. We propose that the PHD domain of Np95-ICBP90 contributes to the opening and/or stabilization of dense chromocenter structures to support the recruitment of modifying enzymes - like HDAC and DNMT1 - required for the replication and formation of PH.

Terminal differentiation exerts a remarkably tight control on cell proliferation. However, the oncogenic products of DNA tumor viruses, such as adenovirus E1A, can force postmitotic cells to proliferate, thus representing a powerful tool to study progression into S phase. In this study, we identified the gene encoding Np95, a murine nuclear phosphoprotein, as an early target of E1A-induced transcriptional events. In terminally differentiated (TD) cells, the activation of Np95 was specifically induced by E1A, but not by overexpression of E2F-1 or of the cyclin E (cycE)–cyclin-dependent kinase 2 (cdk2) complex. In addition, the concomitant expression of Np95 and of cycE–cdk2 was alone sufficient to induce S phase in TD cells. In NIH-3T3 cells, the expression of Np95 was tightly regulated during the cell cycle, and its functional ablation resulted in abrogation of DNA synthesis. Thus, expression of Np95 is essential for S phase entry. Previous evidence suggested that E1A, in addition to its well characterized effects on the pRb/E2F-1 pathway, activates a parallel and complementary pathway that is also required for the reentry in S phase of TD cells (Tiainen, M., D. Spitkousky, P. Jansen-Dürr, A. Sacchi, and M. Crescenzi. 1996. Mol. Cell. Biol. 16:5302–5312). From our results, Np95 appears to possess all the characteristics to represent the first molecular determinant identified in this pathway.

INTRODUCTION: Lenalidomide monotherapy exerts clinical activity in relapsed/refractory Diffuse Large B-cell Lymphoma (DLBCL) with better response rate and progression-free survival being recorded in activated B-cell-like (ABC) rather than germinal center B-cell-like (GCB)-DLBCL. Reasons for such a difference are likely due to different expression of key molecules involved in mediating activity of Lenalidomide, such as Interferon regulatory factor 4(IRF4) and cereblon (CRBN). Evidences supporting the key role of DNA methylation and histone modifications in regulating genome stability and gene expression in DLBCL prompted us to investigate the capacity of Azacytidine in modulating Lenalidomide activity, thereby sensitizing GCB-DLBCL to Lenalidomide and enhancing Lenalidomide efficacy in ABC-DLBCL. METHODS: DLBCL cell lines with ABC (U-2932, RIVA) or GCB (SU-DHL4, SU-DHL6) genotype were used to investigate the effects of Lenalidomide and Azacytidine on cell growth and cell death. Western blotting (WB) and immunofluorescence analysis were used to assess modulating effects of the two-drug combination on molecular determinants of Lenalidomide activity. Additionally, we studied CRBN, IRF4 and CRBN binding proteins expression, such as Ikaros and Aiolos (IKZF1 and IKZF3) by real time polymerase chain reaction (RT-PCR) in response to drug treatment. RESULTS: Graded concentrations of Lenalidomide (0.1-100 µM) inhibited cell proliferation by 20% to 40% and increased cell death up to 30% to 40% in ABC-DLBCL cell lines, whereas had minimal effects on GCB-DLBCL cell lines. Untreated ABC-DLBCL but not GCB-DLBCL consistently showed a high expression of CRBN and IRF4. Upon Lenalidomide treatment (3 days) CRBN was significantly upregulated and IRF4 downregulated in ABC-DLBCL, but not GCB-DLBCL cells. Since DNA methylation regulates gene expression in DLBCL cell lines, we next examined whether Azacytdine could modulate CRBN and IRF4 expression and in turn enhance responsiveness to Lenalidomide. Exposure of both ABC- and GCB-DLBCL cell lines to Azacytidine (up to 72 hours) induced a marked increase of CRBN and IRF4 transcripts; addition of Lenalidomide strongly increased Azacytidine-induced increase of CRBN and significantly downregulated IRF4 expression; the combined treatment induced a marked downregulation of Ikaros and Aiolos protein levels. At the cellular level, the concomitant Azacytidine (10 μM)/Lenalidomide (10 μM) treatment inhibited in a synergistic manner the mean (±SEM) cell growth of both ABC-DLBCL (Lena: -16 ± 4%; AZA: -22 ± 2%; AZA/Lena: -70 ± 1%, P CONCLUSIONS: Our results indicate that Azacytidine sensitizes GCB-DLBCL to the cytotoxic effects of Lenalidomide and enhances Lenalidomide efficacy against ABC-DLBCL resulting in synergistic anti-proliferative and pro-apoptotic effects in both ABC- and GCB-DLBCL cell lines. Cytotoxicity of the two drug combination is mediated by signaling events involving CRBN upregulation and IRF4 downregulation leading to CRBN-binding proteins downregulation. Azacytidine-dependent activation of CRBN and IRF4 expression allow to hypothesize a methylation-driven regulation of these genes. These results might provide a rationale for clinical studies using Azacytidine and Lenalidomide combination in ABC- and GCB-DLBCL. Disclosures No relevant conflicts of interest to declare.

The epigenetic signature for transcription regulation is poorly understood in cardiac hypertrophy. Here, we investigate the genome-wide distribution of several histone modifications and the correlated transcriptome in adult mouse cardiomyocytes subjected to a pro-hypertrophy stimulus in vivo. We demonstrate that the epigenetic profile dynamic of cardiac genes changes in response to the stimulus, and that distinct epigenetic signatures at promoters regulate the gene re-programming occurring with cardiac hypertrophy. Moreover, we identify 9,207 enhancers that impact transcription in normal and hypertrophic cardiomyocytes and whose activity is governed by unique epigenetic signatures. In fact, combinations of H3K27ac, H3K9ac, and H3K4me3 marks define new subclasses of highly active and dynamic enhancers in cardiac myocytes. We also analyzed the transcriptional network within which these genetic elements act to orchestrate hypertrophy gene expression, finding a role for myocyte enhancer factor (MEF)2C and MEF2A in regulating enhancers. The association of cardiac hypertrophy with a specific epigenetic profile provides a new basis for understanding the molecular mechanisms underlying hear failure, and opens up the possibility of developing new therapies based on the control of the epigenetic profile of cardiomyocytes.

Epigenetic changes on DNA and chromatin are implicated in cell differentiation and organogenesis. For the heart, distinct histone methylation profiles were recently linked to stage-specific gene expression programs during cardiac differentiation in vitro. However, the enzymes catalyzing these modifications and the genes regulated by them remain poorly defined. We therefore decided to identify the epigenetic enzymes that are potentially involved in cardiomyogenesis by analyzing the expression profile of the 85 genes encoding the epigenetic-related proteins in mouse cardiomyocytes (CMs), and then study how they affect gene expression during differentiation and maturation of this cell type. We show here with gene expression screening of epigenetic enzymes that the highly expressed H3 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) drives a transitional pattern of di-methylation on H3 lysine 79 (H3K79) in CMs at different stages of differentiation in vitro and in vivo. Through a genome-wide chromatin-immunoprecipitation DNA-sequencing approach, we found H3K79me2 enriched at genes expressed during cardiac differentiation. Moreover, knockdown of Dot1L affected the expression of H3K79me2-enriched genes. Our results demonstrate that histone methylation, and in particular DOT1L-mediated H3K79me2 modification, drives cardiomyogenesis through the definition of a specific transcriptional landscape.

Placenta-derived mesenchymal stromal cells (MSC) have attracted more attention for their immune modulatory properties and poor immunogenicity, which makes them suitable for allogeneic transplantation. Although MSC isolated from different areas of the placenta share several features, they also present significant biological differences, which might point to distinct clinical applications. Hence, we compared cells from full term placenta distinguishing them on the basis of their origin, either maternal or fetal. We used cells developed by Pluristem LTD: PLacenta expanded mesenchymal-like adherent stromal cells (PLX), maternal-derived cells (PLX-PAD), fetal-derived cells (PLX-R18), and amniotic membrane-derived MSC (hAMSC). We compared immune modulatory properties evaluating effects on T-lymphocyte proliferation, expression of cytotoxicity markers, T-helper and T-regulatory cell polarization, and monocyte differentiation toward antigen presenting cells (APC). Furthermore, we investigated cell immunogenicity. We show that MSCs and MSC-like cells from both fetal and maternal sources present immune modulatory properties versus lymphoid (T cells) and myeloid (APC) cells, whereby fetal-derived cells (PLX-R18 and hAMSC) have a stronger capacity to modulate immune cell proliferation and differentiation. Our results emphasize the importance of understanding the cell origin and characteristics in order to obtain a desired result, such as modulation of the inflammatory response that is critical in fostering regenerative processes.