Identification of a miRNA multi-targeting therapeutic strategy in glioblastoma.

Glioblastoma (GBM) is a deadly and the most common primary brain tumor in adults. Due to their regulation of a high number of mRNA transcripts, microRNAs (miRNAs) are key molecules in the control of biological processes and are thereby promising therapeutic targets for GBM patients. In this regard, we recently reported miRNAs as strong modulators of GBM aggressiveness. Here, using an integrative and comprehensive analysis of the TCGA database and the transcriptome of GBM biopsies, we identified three critical and clinically relevant miRNAs for GBM, miR-17-3p, miR-222, and miR-340. In addition, we showed that the combinatorial modulation of three of these miRNAs efficiently inhibited several biological processes in patient-derived GBM cells of all these three GBM subtypes (Mesenchymal, Proneural, Classical), induced cell death, and delayed tumor growth in a mouse tumor model. Finally, in a doxycycline-inducible model, we observed a significant inhibition of GBM stem cell viability and a significant delay of orthotopic tumor growth. Collectively, our results reveal, for the first time, the potential of miR-17-3p, miR-222 and miR-340 multi-targeting as a promising therapeutic strategy for GBM patients.

Optimization of Thymidine Kinase-Based Safety Switch for Neural Cell Therapy.

Cell therapies based on pluripotent stem cells (PSC), have opened new therapeutic strategies for neurodegenerative diseases. However, insufficiently differentiated PSC can lead to tumor formation. Ideally, safety switch therapies should selectively kill proliferative transplant cells while preserving post-mitotic neurons. In this study, we evaluated the potential of nucleoside analogs and thymidine kinase-based suicide genes. Among tested thymidine kinase variants, the humanized SR39 (SR39h) variant rendered cells most sensitive to suicide induction. Unexpectedly, post-mitotic neurons with ubiquitous SR39h expression were killed by ganciclovir, but were spared when SR39h was expressed under the control of the cell cycle-dependent Ki67 promoter. The efficacy of six different nucleoside analogs to induce cell death was then evaluated. Penciclovir (PCV) showed the most interesting properties with an efficiency comparable to ganciclovir (GCV), but low toxicity. We tested three nucleoside analogs in vivo: at concentrations of 40 mg/kg/day, PCV and GCV prevented tumor formation, while acyclovir (ACV) did not. In summary, SR39h under the control of a cell cycle-dependent promoter appears most efficient and selective as safety switch for neural transplants. In this setting, PCV and GCV are efficient inducers of cell death. Because of its low toxicity, PCV might become a preferred alternative to GCV.

Modeling Klinefelter Syndrome Using Induced Pluripotent Stem Cells Reveals Impaired Germ Cell Differentiation.

Klinefelter syndrome (KS), with an incidence between 1/600 and 1/1,000, is the main genetic cause of male infertility. Due to the lack of an accurate study model, the detailed pathogenic mechanisms by which this X chromosome aneuploidy leads to KS features remain unknown. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from a patient with KS: 47XXY-iPSCs. In order to compare the potentials of both 47XXY-iPSCs and 46XY-iPSCs to differentiate into the germ cell lineage, we developed a directed differentiation protocol by testing different combinations of factors including bone morphogenetic protein 4 (BMP4), glial-derived neurotrophic factor (GDNF), retinoic acid (RA) and stem cell factor (SCF) for 42 days. Importantly, we found a reduced ability of 47XXY-iPSCs to differentiate into germ cells when compared to 46XY-iPSCs. In particular, upon germ cell differentiation of 47XXY-iPSCs, we found a reduced proportion of cells positive for BOLL, a protein required for germ cell development and spermatogenesis, as well as a reduced proportion of cells positive for MAGEA4, a spermatogonia marker. This reduced ability to generate germ cells was not associated with a decrease of proliferation of 47XXY-iPSC-derived cells but rather with an increase of cell death upon germ cell differentiation as revealed by an increase of LDH release and of capase-3 expression in 47XXY-iPSC-derived cells. Our study supports the idea that 47XXY-iPSCs provides an excellent in vitro model to unravel the pathophysiology and to design potential treatments for KS patients.

Parallel derivation of X-monosomy induced pluripotent stem cells (iPSCs) with isogenic control iPSCs.

Turner syndrome, caused by partial or complete loss of one copy of X-chromosome (45,X), is the most common sex chromosome abnormality in women with an incidence of 1 in 2500 female births. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) carrying X-monosomy anomaly, with isogenic control iPSCs. Among the iPSC lines generated from 46XX-fibroblasts, one spontaneously lost a copy of X-chromosome following the reprogramming process, establishing the 45X-iPSC line.

Modeling Poliovirus Infection Using Human Engineered Neural Tissue Enriched With Motor Neuron Derived From Embryonic Stem Cells.

Poliomyelitis is caused by poliovirus (PV), a positive strand non-enveloped virus. Since its discovery in the 1950s, several cell culture and molecular methods have been developed to detect and characterize the various strains of PV. Here, we provide an accurate and standardized protocol to differentiate human embryonic stem cells (hESCs) toward engineered neural tissue enriched with motor neurons (MN ENTs). These MN ENTs expressed markers of motor neuron CHAT and Hb-9 as revealed by immunofluorescence staining and quantitative RT-PCR. Interestingly, our results suggest that motor neurons are responsible for the permissiveness of poliovirus within the MN ENTs. Moreover, our study revealed the molecular events occurring upon PV-3 infection in the MN ENTs and highlighted the modulation of a set of genes involved in EGR-EP300 complex. Collectively, we report the development of a reliable in vitro model to investigate the pathophysiology of PV infection, allowing to both design and assess novel therapeutic approaches against PV infection.

DYRK1A Protein, A Promising Therapeutic Target to Improve Cognitive Deficits in Down Syndrome.

Down syndrome (DS) caused by a trisomy of chromosome 21 (HSA21), is the most common genetic developmental disorder, with an incidence of 1 in 800 live births. Its phenotypic characteristics include intellectual impairment, early onset of Alzheimer's disease, congenital heart disease, hypotonia, muscle weakness and several other developmental abnormalities, for the majority of which the pathogenetic mechanisms remain unknown. Among the numerous protein coding genes of HSA21, dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A) encodes a proline-directed serine/threonine and tyrosine kinase that plays pleiotropic roles in neurodevelopment in both physiological and pathological conditions. Numerous studies point to a crucial role of DYRK1A protein for brain defects in patients with DS. Thus, DYRK1A inhibition has shown benefits in several mouse models of DS, including improvement of cognitive behaviour. Lastly, a recent clinical trial has shown that epigallocatechine gallate (EGCG), a DYRK1A inhibitor, given to young patients with DS improved visual recognition memory, working memory performance and adaptive behaviour.

Generation of an alveolar epithelial type II cell line from induced pluripotent stem cells.

Differentiation of primary alveolar type II epithelial cells (AEC II) to AEC type I in culture is a major barrier in the study of the alveolar epithelium in vitro. The establishment of an AEC II cell line derived from induced pluripotent stem cells (iPSC) represents a novel opportunity to study alveolar epithelial cell biology, for instance, in the context of lung injury, fibrosis, and repair. In the present study, we generated long-lasting AEC II from iPSC (LL-iPSC-AEC II). LL-iPSC-AEC II displayed morphological characteristics of AEC II, including growth in a cobblestone monolayer, the presence of lamellar bodies, and microvilli, as shown by electron microscopy. Also, LL-iPSC-AEC II expressed AEC type II proteins, such as cytokeratin, surfactant protein C, and LysoTracker DND 26 (a marker for lamellar bodies). Furthermore, the LL-iPSC-AEC II exhibited functional properties of AEC II by an increase of transepithelial electrical resistance over time, secretion of inflammatory mediators in biologically relevant quantities (IL-6 and IL-8), and efficient in vitro alveolar epithelial wound repair. Consistent with the AEC II phenotype, the cell line showed the ability to uptake and release surfactant protein B, to secrete phospholipids, and to differentiate into AEC type I. In summary, we established a long-lasting, but finite AEC type II cell line derived from iPSC as a novel cellular model to study alveolar epithelial cell biology in lung health and disease.

Gene Network Analysis of Interstitial Macrophages After Treatment with Induced Pluripotent Stem Cells Secretome (iPSC-cm) in the Bleomycin Injured Rat Lung.

Idiopathic pulmonary fibrosis (IPF) is a complex disease involving various cell types. Macrophages are essential in maintenance of physiological homeostasis, wound repair and fibrosis in the lung. Macrophages play a crucial role in repair and remodeling by altering their phenotype and secretory pattern in response to injury. The secretome of induced pluripotent stem cells (iPSC-cm) attenuates injury and fibrosis in bleomycin injured rat lungs. In the current study, we evaluate the effect of iPSC-cm on gene expression and phenotype of interstitial macrophage in bleomycin injured rat lungs in vivo. iPSC-cm was intratracheally instilled 7 days after bleomycin induced lung injury and assessed 7 days later and single cell isolation was performed. Macrophages were FACS sorted and microarray analysis was performed. We characterized changes in the rat lung interstitial macrophages using transcriptional profiling. iPSC-cm reduced the total collagen content of the lung and reduced different macrophage populations. Gene set enrichment analysis revealed involvement of three essential pathways (a) immune modulation, (b) branching morphogenesis and (c) canonical Wnt signaling. This study demonstrates that iPSC-cm reduces fibrosis in bleomycin injured rat lung by partially altering the macrophages and regulating their gene expression.

Concise Review: Methods and Cell Types Used to Generate Down Syndrome Induced Pluripotent Stem Cells.

Down syndrome (DS, trisomy 21), is the most common viable chromosomal disorder, with an incidence of 1 in 800 live births. Its phenotypic characteristics include intellectual impairment and several other developmental abnormalities, for the majority of which the pathogenetic mechanisms remain unknown. Several models have been used to investigate the mechanisms by which the extra copy of chromosome 21 leads to the DS phenotype. In the last five years, several laboratories have been successful in reprogramming patient cells carrying the trisomy 21 anomaly into induced pluripotent stem cells, i.e., T21-iPSCs. In this review, we summarize the different T21-iPSCs that have been generated with a particular interest in the technical procedures and the somatic cell types used for the reprogramming.

DNA-Methylation Patterns in Trisomy 21 Using Cells from Monozygotic Twins.

DNA methylation is essential in mammalian development. We have hypothesized that methylation differences induced by trisomy 21 (T21) contribute to the phenotypic characteristics and heterogeneity in Down syndrome (DS). In order to determine the methylation differences in T21 without interference of the interindividual genomic variation, we have used fetal skin fibroblasts from monozygotic (MZ) twins discordant for T21. We also used skin fibroblasts from MZ twins concordant for T21, normal MZ twins without T21, and unrelated normal and T21 individuals. Reduced Representation Bisulfite Sequencing (RRBS) revealed 35 differentially methylated promoter regions (DMRs) (Absolute methylation differences = 25%, FDR < 0.001) in MZ twins discordant for T21 that have also been observed in comparison between unrelated normal and T21 individuals. The identified DMRs are enriched for genes involved in embryonic organ morphogenesis (FDR = 1.60 e -03) and include genes of the HOXB and HOXD clusters. These DMRs are maintained in iPS cells generated from this twin pair and are correlated with the gene expression changes. We have also observed an increase in DNA methylation level in the T21 methylome compared to the normal euploid methylome. This observation is concordant with the up regulation of DNA methyltransferase enzymes (DNMT3B and DNMT3L) and down regulation of DNA demethylation enzymes (TET2 and TET3) observed in the iPSC of the T21 versus normal twin. Altogether, the results of this study highlight the epigenetic effects of the extra chromosome 21 in T21 on loci outside of this chromosome that are relevant to DS associated phenotypes.

Domains of genome-wide gene expression dysregulation in Down's syndrome.

Trisomy 21 is the most frequent genetic cause of cognitive impairment. To assess the perturbations of gene expression in trisomy 21, and to eliminate the noise of genomic variability, we studied the transcriptome of fetal fibroblasts from a pair of monozygotic twins discordant for trisomy 21. Here we show that the differential expression between the twins is organized in domains along all chromosomes that are either upregulated or downregulated. These gene expression dysregulation domains (GEDDs) can be defined by the expression level of their gene content, and are well conserved in induced pluripotent stem cells derived from the twins' fibroblasts. Comparison of the transcriptome of the Ts65Dn mouse model of Down's syndrome and normal littermate mouse fibroblasts also showed GEDDs along the mouse chromosomes that were syntenic in human. The GEDDs correlate with the lamina-associated (LADs) and replication domains of mammalian cells. The overall position of LADs was not altered in trisomic cells; however, the H3K4me3 profile of the trisomic fibroblasts was modified and accurately followed the GEDD pattern. These results indicate that the nuclear compartments of trisomic cells undergo modifications of the chromatin environment influencing the overall transcriptome, and that GEDDs may therefore contribute to some trisomy 21 phenotypes.

Modelling and rescuing neurodevelopmental defect of Down syndrome using induced pluripotent stem cells from monozygotic twins discordant for trisomy 21.

Down syndrome (trisomy 21) is the most common viable chromosomal disorder with intellectual impairment and several other developmental abnormalities. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from monozygotic twins discordant for trisomy 21 in order to eliminate the effects of the variability of genomic background. The alterations observed by genetic analysis at the iPSC level and at first approximation in early development illustrate the developmental disease transcriptional signature of Down syndrome. Moreover, we observed an abnormal neural differentiation of Down syndrome iPSCs in vivo when formed teratoma in NOD-SCID mice, and in vitro when differentiated into neuroprogenitors and neurons. These defects were associated with changes in the architecture and density of neurons, astroglial and oligodendroglial cells together with misexpression of genes involved in neurogenesis, lineage specification and differentiation. Furthermore, we provide novel evidence that dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) on chromosome 21 likely contributes to these defects. Importantly, we found that targeting DYRK1A pharmacologically or by shRNA results in a considerable correction of these defects.

Data in brief: Transcriptome analysis of induced pluripotent stem cells from monozygotic twins discordant for trisomy 21.

Down syndrome (DS, trisomy 21), is the most common viable chromosomal disorder, with an incidence of 1 in 800 live births. Its phenotypic characteristics include intellectual impairment and several other developmental abnormalities, for the majority of which the pathogenetic mechanisms remain unknown. In this "Data in Brief" paper, we sum up the whole genome analysis by mRNA sequencing of normal and DS induced pluripotent stem cells that was recently published by Hibaoui et al. in EMBO molecular medicine.

Human pluripotent stem cells: applications and challenges in neurological diseases.

The ability to generate human pluripotent stem cells (hPSCs) holds great promise for the understanding and the treatment of human neurological diseases in modern medicine. The hPSCs are considered for their in vitro use as research tools to provide relevant cellular model for human diseases, drug discovery, and toxicity assays and for their in vivo use in regenerative medicine applications. In this review, we highlight recent progress, promises, and challenges of hPSC applications in human neurological disease modeling and therapies.

Melatonin improves muscle function of the dystrophic mdx5Cv mouse, a model for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe X-linked muscle-wasting disease caused by the absence of the cytoskeletal protein dystrophin. In addition to abnormal calcium handling, numerous studies point to a crucial role of oxidative stress in the pathogenesis of the disease. Considering the impressive results provided by antioxidants on dystrophic muscle structure and function, we investigated whether melatonin can protect the mdx(5Cv) mouse, an animal model for DMD. Male mdx(5Cv) mouse pups were treated with melatonin by daily intraperitoneal (i.p.) injection (30 mg/kg body weight) or by subcutaneous (s.c.) implant(s) (18 or 54 mg melatonin as Melovine® implants) from 17/18 to 28/29 days of age. Isometric force of the triceps surae was recorded at the end of the treatment. The i.p. treatment increased the phasic twitch tension of mdx(5Cv) mice. The maximal tetanic tension was ameliorated by 18 mg s.c. and 30 mg/kg i.p. treatments. Melatonin caused the dystrophic muscle to contract and relax faster. The force-frequency relationship of melatonin-treated dystrophic mice was shifted to the right. In accordance with improved muscle function, melatonin decreased plasma creatine kinase activity, a marker for muscle injury. Melatonin treatment increased total glutathione content and lowered the oxidized/reduced glutathione ratio, indicating a better redox status of the muscle. In light of the present investigation, the therapeutic potential of melatonin should be further considered for patients with DMD.

Melatonin prevents oxidative stress-mediated mitochondrial permeability transition and death in skeletal muscle cells.

Oxidative stress-induced mitochondrial dysfunction plays a crucial role in the pathogenesis of a wide range of diseases including muscle disorders. In this study, we demonstrate that melatonin readily rescued mitochondria from oxidative stress-induced dysfunction and effectively prevented subsequent apoptosis of primary muscle cultures prepared from C57BL/6J mice. In particular, melatonin (10(-4)-10(-6) m) fully prevented myotube death induced by tert-butylhydroperoxide (t-BHP; 10 microm-24 hr) as assessed by acid phosphatase, caspase-3 activities and cellular morphological changes. Using fluorescence imaging, we showed that the mitochondrial protection provided by melatonin was associated with an inhibition of t-BHP-induced reactive oxygen species generation. In line with this observation, melatonin prevented t-BHP-induced mitochondrial depolarization and mitochondrial permeability transition pore (PTP) opening. This was associated with a highly reduced environment as reflected by an increased glutathione content and an increased ability to maintain mitochondrial pyridine nucleotides and glutathione in a reduced state. Using isolated mitochondria, in a similar manner as cyclosporin A, melatonin (10(-8)-10(-6) m) desensitized the PTP to Ca(2+) and prevented t-BHP-induced mitochondrial swelling, pyridine nucleotide and glutathione oxidation. In conclusion, our findings suggest that inhibition of the PTP essentially contributes to the protective effect of melatonin against oxidative stress in myotubes.