27 results on '"Sevlever GE"'
Search Results
2. Generation of a human induced pluripotent stem cell line (INEUi001-A) from an amyotrophic lateral sclerosis/frontotemporal dementia patient with a C9ORF72 G4C2 genotype of <2 (GGGGCCG) and 10 repeats.
- Author
-
Nievas M, Romorini L, Isaja L, Clas GS, Rodríguez-Varela S, Mucci S, Itzcovich T, de Ambrosi B, Scassa ME, Sevlever GE, Surace EI, and Marazita MC
- Subjects
- Female, Humans, Aged, C9orf72 Protein genetics, Leukocytes, Mononuclear metabolism, Genotype, Amyotrophic Lateral Sclerosis genetics, Amyotrophic Lateral Sclerosis metabolism, Frontotemporal Dementia genetics, Induced Pluripotent Stem Cells metabolism
- Abstract
Human induced pluripotent stem cell (hiPSC) line INEUi001-A was reprogrammed from peripheral blood mononuclear cells (PBMC) using the lentiviral-hSTEMCCA-loxP vector. PBMCs were obtained from a 75- year-old female ALS/FTD disease patient carrying a heterozygous deletion within the C9ORF72 hexanucleotide repeat region resulting in a GGGGCCG sequence (∼1.16 repeats). C9ORF72 genotype was maintained and stemness and pluripotency confirmed in INEUi001-A hiPSC line., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
3. Acute severe hypoxia induces apoptosis of human pluripotent stem cells by a HIF-1α and P53 independent mechanism.
- Author
-
Mucci S, Isaja L, Rodríguez-Varela MS, Ferriol-Laffouillere SL, Marazita M, Videla-Richardson GA, Sevlever GE, Scassa ME, and Romorini L
- Subjects
- Humans, Cell Hypoxia, Apoptosis, Hypoxia, Hypoxia-Inducible Factor 1, alpha Subunit genetics, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Tumor Suppressor Protein p53 metabolism, Pluripotent Stem Cells metabolism
- Abstract
Human embryonic and induced pluripotent stem cells are self-renewing pluripotent stem cells (hPSCs) that can differentiate into a wide range of specialized cells. Although moderate hypoxia (5% O
2 ) improves hPSC self-renewal, pluripotency, and cell survival, the effect of acute severe hypoxia (1% O2 ) on hPSC viability is still not fully elucidated. In this sense, we explore the consequences of acute hypoxia on hPSC survival by culturing them under acute (maximum of 24 h) physical severe hypoxia (1% O2 ). After 24 h of hypoxia, we observed HIF-1α stabilization concomitant with a decrease in cell viability. We also observed an increase in the apoptotic rate (western blot analysis revealed activation of CASPASE-9, CASPASE-3, and PARP cleavage after hypoxia induction). Besides, siRNA-mediated downregulation of HIF-1α and P53 did not significantly alter hPSC apoptosis induced by hypoxia. Finally, the analysis of BCL-2 family protein expression levels disclosed a shift in the balance between pro- and anti-apoptotic proteins (evidenced by an increase in BAX/MCL-1 ratio) caused by hypoxia. We demonstrated that acute physical hypoxia reduced hPSC survival and triggered apoptosis by a HIF-1α and P53 independent mechanism., (© 2022. The Author(s).)- Published
- 2022
- Full Text
- View/download PDF
4. Noxa and Mcl-1 expression influence the sensitivity to BH3-mimetics that target Bcl-xL in patient-derived glioma stem cells.
- Author
-
Vera MB, Morris-Hanon O, Nogueiras GI, Ripari LB, Esquivel MI, Perez-Castro C, Romorini L, Sevlever GE, Scassa ME, and Videla-Richardson GA
- Subjects
- Humans, Myeloid Cell Leukemia Sequence 1 Protein metabolism, Proto-Oncogene Proteins c-bcl-2 metabolism, Cell Line, Tumor, Apoptosis Regulatory Proteins pharmacology, Apoptosis, Neoplastic Stem Cells metabolism, bcl-X Protein metabolism, Antineoplastic Agents pharmacology, Glioma drug therapy
- Abstract
The recurrence of Glioblastoma is partly attributed to the highly resistant subpopulation of glioma stem cells. A novel therapeutic approach focuses on restoring apoptotic programs in these cancer stem cells, as they are often deregulated. BH3-mimetics, targeting anti-apoptotic Bcl-2 family members, are emerging as promising compounds to sensitize cancer cells to antineoplastic treatments. Herein, we determined that the most abundantly expressed anti-apoptotic Bcl-2 family members, Bcl-xL and Mcl-1, are the most relevant in regulating patient-derived glioma stem cell survival. We exposed these cells to routinely used chemotherapeutic drugs and BH3-mimetics (ABT-263, WEHI-539, and S63845). We observed that the combination of BH3-mimetics targeting Bcl-xL with chemotherapeutic agents caused a marked increase in cell death and that this sensitivity to Bcl-xL inhibition correlated with Noxa expression levels. Interestingly, whereas co-targeting Bcl-xL and Mcl-1 led to massive cell death in all tested cell lines, down-regulation of Noxa promoted cell survival only in cell lines expressing higher levels of this BH3-only. Therefore, in glioma stem cells, the efficacy of Bcl-xL inhibition is closely associated with Mcl-1 activity and Noxa expression. Hence, a potentially effective strategy would consist of combining Bcl-xL inhibitors with chemotherapeutic agents capable of inducing Noxa, taking advantage of this pro-apoptotic factor., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
5. Protocol for morphometric analysis of neurons derived from human pluripotent stem cells.
- Author
-
Mucci S, Rodriguez-Varela MS, Isaja L, Ferriol-Laffouillere SL, Sevlever GE, Scassa ME, and Romorini L
- Subjects
- Dendrites, Humans, Neurons, Pluripotent Stem Cells
- Abstract
The analysis of morphological features of neurons derived from human pluripotent stem cells (hPSCs) is important to describe neuronal phenotypes and changes observed throughout development. Using free and easily accessible tools, we describe a protocol for the morphometric quantification of hPSCs-derived neurons in two- and three-dimensions in vitro cultures. We detail the analysis of soma area and main and secondary dendrites lengths of GFP-transfected neurons and the measurement of area and perimeter of immunostained neurospheres., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
6. Galectins as Emerging Glyco-Checkpoints and Therapeutic Targets in Glioblastoma.
- Author
-
Videla-Richardson GA, Morris-Hanon O, Torres NI, Esquivel MI, Vera MB, Ripari LB, Croci DO, Sevlever GE, and Rabinovich GA
- Subjects
- Animals, Disease Progression, Drug Resistance, Neoplasm, Glioblastoma immunology, Humans, Galectins metabolism, Glioblastoma drug therapy, Glioblastoma metabolism, Molecular Targeted Therapy, Polysaccharides metabolism
- Abstract
Despite recent advances in diagnosis and treatment, glioblastoma (GBM) represents the most common and aggressive brain tumor in the adult population, urging identification of new rational therapeutic targets. Galectins, a family of glycan-binding proteins, are highly expressed in the tumor microenvironment (TME) and delineate prognosis and clinical outcome in patients with GBM. These endogenous lectins play key roles in different hallmarks of cancer by modulating tumor cell proliferation, oncogenic signaling, migration, vascularization and immunity. Additionally, they have emerged as mediators of resistance to different anticancer treatments, including chemotherapy, radiotherapy, immunotherapy, and antiangiogenic therapy. Particularly in GBM, galectins control tumor cell transformation and proliferation, reprogram tumor cell migration and invasion, promote vascularization, modulate cell death pathways, and shape the tumor-immune landscape by targeting myeloid, natural killer (NK), and CD8
+ T cell compartments. Here, we discuss the role of galectins, particularly galectin-1, -3, -8, and -9, as emerging glyco-checkpoints that control different mechanisms associated with GBM progression, and discuss possible therapeutic opportunities based on inhibition of galectin-driven circuits, either alone or in combination with other treatment modalities.- Published
- 2021
- Full Text
- View/download PDF
7. celldeath: A tool for detection of cell death in transmitted light microscopy images by deep learning-based visual recognition.
- Author
-
La Greca AD, Pérez N, Castañeda S, Milone PM, Scarafía MA, Möbbs AM, Waisman A, Moro LN, Sevlever GE, Luzzani CD, and Miriuka SG
- Subjects
- Cell Death, Humans, MCF-7 Cells, Microscopy, Deep Learning, Image Interpretation, Computer-Assisted, Programming Languages
- Abstract
Cell death experiments are routinely done in many labs around the world, these experiments are the backbone of many assays for drug development. Cell death detection is usually performed in many ways, and requires time and reagents. However, cell death is preceded by slight morphological changes in cell shape and texture. In this paper, we trained a neural network to classify cells undergoing cell death. We found that the network was able to highly predict cell death after one hour of exposure to camptothecin. Moreover, this prediction largely outperforms human ability. Finally, we provide a simple python tool that can broadly be used to detect cell death., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2021
- Full Text
- View/download PDF
8. miR-302 family, miR-145 and miR-296 temporal expression profile along the cell cycle of human pluripotent stem cells.
- Author
-
Rodríguez-Varela MS, Mucci S, Videla-Richardson GA, Isaja L, Sevlever GE, Scassa ME, and Romorini L
- Subjects
- Cell Line, Cytostatic Agents pharmacology, Humans, Induced Pluripotent Stem Cells drug effects, Induced Pluripotent Stem Cells physiology, MicroRNAs metabolism, Cell Cycle Checkpoints, Induced Pluripotent Stem Cells metabolism, MicroRNAs genetics
- Abstract
Human pluripotent stem cells (hPSCs), like embryonic (hESCs) and induced pluripotent stem cells (hiPSCs), exhibit an unusual cell cycle structure characterized by a short G1 phase and cells being most of time in S phase. hPSCs are receptive to differentiation cues during their transition through G1 phase when lineage determination is decided. Although several MicroRNAs (miRNAs) have been shown to target transcripts that directly or indirectly coordinate the cell cycle of pluripotent cells, its temporal expression profile along hPSCs cell cycle remains poorly characterized. miR-145 and miR-296 are induced during differentiation and silence the self-renewal and pluripotency program. miR-302 family is essential for hPSCs stemness and its expression decreases during differentiation. We aimed to study how the aforementioned miRNAs are regulated along the cell cycle of hPSCs. We demonstrated by pharmacological synchronization and block and release experiments that miR-145, miR-296 and miR-302 family are periodically expressed in hPSCs. Importantly, miR-302 family expression is induced at G1/S boundary and remained high at S phase, presumably to impede differentiation onset. Besides, we confirmed by a gene ontology analysis that many validated miR-302 family target genes are involved in cell cycle regulation., (Copyright © 2021 Elsevier B.V. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
9. Generation of a human induced pluripotent stem cell line from a familial Alzheimer's disease PSEN1 T119I patient.
- Author
-
Isaja L, Rodríguez-Varela MS, Marazita M, Mucci S, Itzcovich T, Chrem-Méndez P, Niikado M, Ferriol-Laffouillere SL, Allegri R, Martinetto H, Sevlever GE, Scassa ME, Surace EI, and Romorini L
- Subjects
- Aged, Cell Differentiation, Fibroblasts, Humans, Male, Presenilin-1 genetics, Alzheimer Disease genetics, Induced Pluripotent Stem Cells
- Abstract
Human induced pluripotent stem cells (hiPSC) line FLENIi001-A was reprogrammed from dermal fibroblasts using the lentiviral-hSTEMCCA-loxP vector. Fibroblasts were obtained from a skin biopsy of a 72-year-old Caucasian male familial Alzheimer's disease patient carrying the T119I mutation in the PSEN1 gene. PSEN1 genotype was maintained and stemness and pluripotency confirmed in the FLENIi001-A hiPSC line., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
10. Chemical hypoxia induces apoptosis of human pluripotent stem cells by a NOXA-mediated HIF-1α and HIF-2α independent mechanism.
- Author
-
Isaja L, Mucci S, Vera J, Rodríguez-Varela MS, Marazita M, Morris-Hanon O, Videla-Richardson GA, Sevlever GE, Scassa ME, and Romorini L
- Subjects
- Caspase 3 genetics, Caspase 9 genetics, Cell Death genetics, Cell Survival genetics, DNA Fragmentation, Down-Regulation genetics, Humans, Membrane Proteins genetics, Mitochondria genetics, Signal Transduction genetics, Tumor Suppressor Protein p53 genetics, Up-Regulation genetics, Apoptosis genetics, Basic Helix-Loop-Helix Transcription Factors genetics, Cell Hypoxia genetics, Hypoxia-Inducible Factor 1, alpha Subunit genetics, Pluripotent Stem Cells metabolism, Proto-Oncogene Proteins c-bcl-2 genetics
- Abstract
Human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are self-renewing human pluripotent stem cells (hPSCs) that can differentiate to a wide range of specialized cells. Notably, hPSCs enhance their undifferentiated state and self-renewal properties in hypoxia (5% O
2 ). Although thoroughly analyzed, hypoxia implication in hPSCs death is not fully determined. In order to evaluate the effect of chemically mimicked hypoxia on hPSCs cell survival, we analyzed changes in cell viability and several aspects of apoptosis triggered by CoCl2 and dimethyloxalylglycine (DMOG). Mitochondrial function assays revealed a decrease in cell viability at 24 h post-treatments. Moreover, we detected chromatin condensation, DNA fragmentation and CASPASE-9 and 3 cleavages. In this context, we observed that P53, BNIP-3, and NOXA protein expression levels were significantly up-regulated at different time points upon chemical hypoxia induction. However, only siRNA-mediated downregulation of NOXA but not HIF-1α, HIF-2α, BNIP-3, and P53 did significantly affect the extent of cell death triggered by CoCl2 and DMOG in hPSCs. In conclusion, chemically mimicked hypoxia induces hPSCs cell death by a NOXA-mediated HIF-1α and HIF-2α independent mechanism.- Published
- 2020
- Full Text
- View/download PDF
11. Integrin alpha-5 subunit is critical for the early stages of human pluripotent stem cell cardiac differentiation.
- Author
-
Neiman G, Scarafía MA, La Greca A, Santín Velazque NL, Garate X, Waisman A, Möbbs AM, Kasai-Brunswick TH, Mesquita F, Martire-Greco D, Moro LN, Luzzani C, Bastos Carvalho A, Sevlever GE, Campos de Carvalho A, Guberman AS, and Miriuka SG
- Subjects
- CRISPR-Cas Systems, Cell Differentiation, Cell Line, Down-Regulation, Gene Expression Regulation, Developmental, HEK293 Cells, Human Embryonic Stem Cells metabolism, Humans, Myocytes, Cardiac metabolism, Pluripotent Stem Cells cytology, Pluripotent Stem Cells metabolism, Stem Cell Niche, Human Embryonic Stem Cells cytology, Integrin alpha5 genetics, Myocytes, Cardiac cytology
- Abstract
The stem cell niche has a strong influence in the differentiation potential of human pluripotent stem cells with integrins playing a major role in communicating cells with the extracellular environment. However, it is not well understood how interactions between integrins and the extracellular matrix are involved in cardiac stem cell differentiation. To evaluate this, we performed a profile of integrins expression in two stages of cardiac differentiation: mesodermal progenitors and cardiomyocytes. We found an active regulation of the expression of different integrins during cardiac differentiation. In particular, integrin α5 subunit showed an increased expression in mesodermal progenitors, and a significant downregulation in cardiomyocytes. To analyze the effect of α5 subunit, we modified its expression by using a CRISPRi technique. After its downregulation, a significant impairment in the process of epithelial-to-mesenchymal transition was seen. Early mesoderm development was significantly affected due to a downregulation of key genes such as T Brachyury and TBX6. Furthermore, we observed that repression of integrin α5 during early stages led to a reduction in cardiomyocyte differentiation and impaired contractility. In summary, our results showed the link between changes in cell identity with the regulation of integrin α5 expression through the alteration of early stages of mesoderm commitment.
- Published
- 2019
- Full Text
- View/download PDF
12. Human embryonic stem cells display a pronounced sensitivity to the cyclin dependent kinase inhibitor Roscovitine.
- Author
-
Videla-Richardson GA, Furmento VA, Garcia CP, Morris-Hanon O, Sevlever GE, Romorini L, and Scassa ME
- Subjects
- Apoptosis drug effects, Cell Cycle drug effects, Cell Cycle Checkpoints drug effects, Cell Line, Down-Regulation drug effects, Human Embryonic Stem Cells drug effects, Humans, Myeloid Cell Leukemia Sequence 1 Protein metabolism, Phosphorylation drug effects, Protein Domains, RNA Polymerase II chemistry, RNA Polymerase II metabolism, Signal Transduction drug effects, Small Molecule Libraries pharmacology, Tumor Suppressor Protein p53 metabolism, Cyclin-Dependent Kinases pharmacology, Human Embryonic Stem Cells cytology, Protein Kinase Inhibitors pharmacology, Roscovitine pharmacology
- Abstract
Background: The essentially unlimited expansion potential and the pluripotency of human embryonic stem cells (hESCs) make them attractive for cell-based therapeutic purposes. Although hESCs can indefinitely proliferate in culture, unlike transformed cancer cells, they are endowed with a cell-intrinsic property termed mitochondrial priming that renders them highly sensitive to apoptotic stimuli. Thus, all attempts to broaden the insights into hESCs apoptosis may be helpful for establishing pro-survival strategies valuable for its in vitro culture and further use in clinical applications. Cyclin-dependent kinases (CDKs), a family of serine/threonine protein kinases originally identified as regulators of the eukaryotic cell cycle, can also regulate transcription and differentiation. Moreover, there are compelling data suggesting that its activities are involved in certain apoptotic programs in different cell types. Currently, it is not completely determined whether CDKs regulate apoptotic processes in rapidly proliferating and apoptosis-prone hESCs. In this study, to elucidate the effect of CDKs inhibition in hESCs we used Roscovitine (ROSC), a purine analogue that selectively inhibits the activities of these kinases., Results: Inhibition of CDKs by ROSC triggers programmed cell death in hESCs but not in proliferating somatic cells (human fibroblasts). The apoptotic process encompasses caspase-9 and -3 activation followed by PARP cleavage. ROSC treatment also leads to p53 stabilization, which coincides with site-specific phosphorylation at serine 46 and decreased levels of Mdm2. Additionally, we observed a transcriptional induction of p53AIP1, a repression of pro-survival factor Mcl-1 and an up-regulation of pro-apoptotic BH3-only proteins NOXA and PUMA. Importantly, we found that the role of CDK2 inhibition appears to be at best accessory as an active CDK2 is not required to ensure hESCs survival., Conclusion: Our experimental data reveal that hESCs, contrary to fibroblasts, exhibit a pronounced sensitivity to ROSC.
- Published
- 2019
- Full Text
- View/download PDF
13. Deep Learning Neural Networks Highly Predict Very Early Onset of Pluripotent Stem Cell Differentiation.
- Author
-
Waisman A, La Greca A, Möbbs AM, Scarafía MA, Santín Velazque NL, Neiman G, Moro LN, Luzzani C, Sevlever GE, Guberman AS, and Miriuka SG
- Subjects
- Cells, Cultured, Humans, Image Processing, Computer-Assisted, Machine Learning, Microscopy, Cell Differentiation, Deep Learning, Neural Networks, Computer, Pluripotent Stem Cells cytology, Pluripotent Stem Cells metabolism
- Abstract
Deep learning is a significant step forward for developing autonomous tasks. One of its branches, computer vision, allows image recognition with high accuracy thanks to the use of convolutional neural networks (CNNs). Our goal was to train a CNN with transmitted light microscopy images to distinguish pluripotent stem cells from early differentiating cells. We induced differentiation of mouse embryonic stem cells to epiblast-like cells and took images at several time points from the initial stimulus. We found that the networks can be trained to recognize undifferentiated cells from differentiating cells with an accuracy higher than 99%. Successful prediction started just 20 min after the onset of differentiation. Furthermore, CNNs displayed great performance in several similar pluripotent stem cell (PSC) settings, including mesoderm differentiation in human induced PSCs. Accurate cellular morphology recognition in a simple microscopic set up may have a significant impact on how cell assays are performed in the near future., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
14. MicroRNA characterization in equine induced pluripotent stem cells.
- Author
-
Moro LN, Amin G, Furmento V, Waisman A, Garate X, Neiman G, La Greca A, Santín Velazque NL, Luzzani C, Sevlever GE, Vichera G, and Miriuka SG
- Subjects
- Animals, Cell Differentiation genetics, Fibroblasts cytology, Gene Expression Profiling, Induced Pluripotent Stem Cells cytology, Kruppel-Like Factor 4, Nuclear Transfer Techniques, Horses, Induced Pluripotent Stem Cells metabolism, MicroRNAs genetics
- Abstract
Cell reprogramming has been well described in mouse and human cells. The expression of specific microRNAs has demonstrated to be essential for pluripotent maintenance and cell differentiation, but not much information is available in domestic species. We aim to generate horse iPSCs, characterize them and evaluate the expression of different microRNAs (miR-302a,b,c,d, miR-205, miR-145, miR-9, miR-96, miR-125b and miR-296). Two equine iPSC lines (L2 and L3) were characterized after the reprogramming of equine fibroblasts with the four human Yamanaka's factors (OCT-4/SOX-2/c-MYC/KLF4). The pluripotency of both lines was assessed by phosphatase alkaline activity, expression of OCT-4, NANOG and REX1 by RT-PCR, and by immunofluorescence of OCT-4, SOX-2 and c-MYC. In vitro differentiation to embryo bodies (EBs) showed the capacity of the iPSCs to differentiate into ectodermal, endodermal and mesodermal phenotypes. MicroRNA analyses resulted in higher expression of the miR-302 family, miR-9 and miR-96 in L2 and L3 vs. fibroblasts (p<0.05), as previously shown in human pluripotent cells. Moreover, downregulation of miR-145 and miR-205 was observed. After differentiation to EBs, higher expression of miR-96 was observed in the EBs respect to the iPSCs, and also the expression of miR-205 was induced but only in the EB-L2. In addition, in silico alignments of the equine microRNAs with mRNA targets suggested the ability of miR-302 family to regulate cell cycle and epithelial mesenchymal transition genes, miR-9 and miR-96 to regulate neural determinant genes and miR-145 to regulate pluripotent genes, similarly as in humans. In conclusion, we could obtain equine iPSCs, characterize them and determine for the first time the expression level of microRNAs in equine pluripotent cells., Competing Interests: We have the following interests: Author Gabriel Vichera is employed by Kheiron Biotech. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.
- Published
- 2018
- Full Text
- View/download PDF
15. Extracellular vesicles from pluripotent stem cell-derived mesenchymal stem cells acquire a stromal modulatory proteomic pattern during differentiation.
- Author
-
La Greca A, Solari C, Furmento V, Lombardi A, Biani MC, Aban C, Moro L, García M, Guberman AS, Sevlever GE, Miriuka SG, and Luzzani C
- Subjects
- Cell Line, Extracellular Vesicles ultrastructure, Humans, Principal Component Analysis, Stromal Cells metabolism, Tandem Mass Spectrometry, Wharton Jelly cytology, Cell Differentiation, Extracellular Vesicles metabolism, Mesenchymal Stem Cells metabolism, Pluripotent Stem Cells cytology, Pluripotent Stem Cells metabolism, Proteomics
- Abstract
Mesenchymal stem/stromal cells (MSCs) obtained from pluripotent stem cells (PSCs) constitute an interesting alternative to classical MSCs in regenerative medicine. Among their many mechanisms of action, MSC extracellular vesicles (EVs) are a potential suitable substitute for MSCs in future cell-free-based therapeutic approaches. Unlike cells, EVs do not elicit acute immune rejection, and they can be produced in large quantities and stored until ready to use. Although the therapeutic potential of MSC EVs has already been proven, a thorough characterization of MSC EVs is lacking. In this work, we used a label-free liquid chromatography tandem mass spectrometry proteomic approach to identify the most abundant proteins in EVs that are secreted from MSCs derived from PSCs (PD-MSCs) and from their parental induced PSCs (iPSCs). Next, we compared both datasets and found that while iPSC EVs enclose proteins that modulate RNA and microRNA stability and protein sorting, PD-MSC EVs are rich in proteins that organize extracellular matrix, regulate locomotion, and influence cell-substrate adhesion. Moreover, compared to their respective cells, iPSCs and iPSC EVs share a greater proportion of proteins, while the PD-MSC proteome appears to be more specific. Correlation and principal component analysis consistently aggregate iPSCs and iPSC EVs but segregate PD-MSC and their EVs. Altogether, these findings suggest that during differentiation, compared with their parental iPSC EVs, PD-MSC EVs acquire a more specific set of proteins; arguably, this difference might confer their therapeutic properties.
- Published
- 2018
- Full Text
- View/download PDF
16. Identification of the miRNAome of early mesoderm progenitor cells and cardiomyocytes derived from human pluripotent stem cells.
- Author
-
Garate X, La Greca A, Neiman G, Blüguermann C, Santín Velazque NL, Moro LN, Luzzani C, Scassa ME, Sevlever GE, Romorini L, and Miriuka SG
- Subjects
- Cell Differentiation, Cells, Cultured, Genome, Human, High-Throughput Nucleotide Sequencing, Humans, Mesoderm cytology, Myocytes, Cardiac cytology, Pluripotent Stem Cells cytology, Biomarkers metabolism, Cell Lineage genetics, Gene Expression Regulation, Mesoderm metabolism, MicroRNAs genetics, Myocytes, Cardiac metabolism, Pluripotent Stem Cells metabolism
- Abstract
MicroRNAs are small non-coding RNAs involved in post-transcriptional regulation of gene expression related to many cellular functions. We performed a small-RNAseq analysis of cardiac differentiation from pluripotent stem cells. Our analyses identified some new aspects about microRNA expression in this differentiation process. First, we described a dynamic expression profile of microRNAs where some of them are clustered according to their expression level. Second, we described the extensive network of isomiRs and ADAR modifications. Third, we identified the microRNAs families and clusters involved in the establishment of cardiac lineage and define the mirRNAome based on these groups. Finally, we were able to determine a more accurate miRNAome associated with cardiomyocytes by comparing the expressed microRNAs with other mature cells. MicroRNAs exert their effect in a complex and interconnected way, making necessary a global analysis to better understand their role. Our data expands the knowledge of microRNAs and their implications in cardiomyogenesis.
- Published
- 2018
- Full Text
- View/download PDF
17. Regulation of cyclin E1 expression in human pluripotent stem cells and derived neural progeny.
- Author
-
Rodríguez Varela MS, Mucci S, Videla Richardson GA, Morris Hanon O, Furmento VA, Miriuka SG, Sevlever GE, Scassa ME, and Romorini L
- Subjects
- Cell Proliferation, Cells, Cultured, Cyclin E metabolism, E2F Transcription Factors metabolism, Extracellular Signal-Regulated MAP Kinases metabolism, Fibroblasts metabolism, G1 Phase Cell Cycle Checkpoints, G2 Phase, Humans, Mitogen-Activated Protein Kinase Kinases metabolism, Mitosis, Neural Stem Cells metabolism, Oncogene Proteins metabolism, Pluripotent Stem Cells metabolism, Proto-Oncogene Proteins c-myc metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Cyclin E genetics, Gene Expression Regulation, Neurons cytology, Neurons metabolism, Oncogene Proteins genetics, Pluripotent Stem Cells cytology
- Abstract
Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells (hESCs and hiPSCs) show unique cell cycle characteristics, such as a short doubling time due to an abbreviated G1 phase. Whether or not the core cell cycle machinery directly regulates the stemness and/or the differentiation potential of hPSCs remains to be determined. To date, several scenarios describing the atypical cell cycle of hPSCs have been suggested, and therefore there is still controversy over how cyclins, master regulators of the cell cycle, are expressed and regulated. Furthermore, the cell cycle profile and the expression pattern of major cyclins in hESCs-derived neuroprogenitors (NP) have not been studied yet. Therefore, herein we characterized the expression pattern of major cyclins in hPSCs and NP. We determined that all studied cyclins mRNA expression levels fluctuate along cell cycle. Particularly, after a thorough analysis of synchronized cell populations, we observed that cyclin E1 mRNA levels increased sharply in G1/S concomitantly with cyclin E1 protein accumulation in hPSCs and NP. Additionally, we demonstrated that cyclin E1 mRNA expression levels involves the activation of MEK/ERK pathway and the transcription factors c-Myc and E2Fs in hPSCs. Lastly, our results reveal that proteasome mediates the marked down-regulation (degradation) of cyclin E1 protein observed in G2/M by a mechanism that requires a functional CDK2 but not GSK3β activity., Abbreviations: hPSCs: human pluripotent stem cells; hESCs: human embryonic stem cells; hiPSCs: human induced pluripotent stem cells; NP: neuroprogenitors; HF: human foreskin fibroblasts; MEFs: mouse embryonic fibroblasts; iMEFs: irradiated mouse embryonic fibroblasts; CDKs: cyclindependent kinases; CKIs: CDK inhibitors; CNS: central nervous system; Oct-4: Octamer-4; EB: embryoid body; AFP: Alpha-fetoprotein; cTnT: Cardiac Troponin T; MAP-2: microtubule-associated protein; TUJ-1: neuron-specific class III β-tubulin; bFGF: basic fibroblastic growth factor; PI3K: Phosphoinositide 3-kinase; KSR: knock out serum replacement; CM: iMEF conditioned medium; E8: Essential E8 medium.
- Published
- 2018
- Full Text
- View/download PDF
18. The Cell Cycle Inhibitors p21 Cip1 and p27 Kip1 Control Proliferation but Enhance DNA Damage Resistance of Glioma Stem Cells.
- Author
-
Morris-Hanon O, Furmento VA, Rodríguez-Varela MS, Mucci S, Fernandez-Espinosa DD, Romorini L, Sevlever GE, Scassa ME, and Videla-Richardson GA
- Subjects
- Cell Cycle Checkpoints genetics, Cell Line, Tumor, Cell Proliferation, Cyclin-Dependent Kinase Inhibitor p21 metabolism, Cyclin-Dependent Kinase Inhibitor p27 metabolism, Gene Expression Regulation, Neoplastic, Gene Silencing, Glioma metabolism, Glioma pathology, Humans, Protein Transport, RNA, Small Interfering genetics, Stress, Physiological genetics, Cyclin-Dependent Kinase Inhibitor p21 genetics, Cyclin-Dependent Kinase Inhibitor p27 genetics, DNA Damage, Drug Resistance, Neoplasm genetics, Glioma genetics, Neoplastic Stem Cells metabolism
- Abstract
High-grade gliomas are the most prevalent and lethal primary brain tumors. They display a hierarchical arrangement with a population of self-renewing and highly tumorigenic cells called cancer stem cells. These cells are thought to be responsible for tumor recurrence, which make them main candidates for targeted therapies. Unbridled cell cycle progression may explain the selective sensitivity of some cancer cells to treatments. The members of the Cip/Kip family p21
Cip1 and p27Kip1 were initially considered as tumor suppressors based on their ability to block proliferation. However, they are currently looked at as proteins with dual roles in cancer: one as tumor suppressor and the other as oncogene. Therefore, the aim of this study was to determine the functions of these cell cycle inhibitors in five patient-derived glioma stem cell-enriched cell lines. We found that these proteins are functional in glioma stem cells. They negatively regulate cell cycle progression both in unstressed conditions and in response to genotoxic stress. In addition, p27Kip1 is upregulated in nutrient-restricted and differentiating cells, suggesting that this Cip/Kip is a mediator of antimitogenic signals in glioma cells. Importantly, the lack of these proteins impairs cell cycle halt in response to genotoxic agents, rendering cells more vulnerable to DNA damage. For these reasons, these proteins may operate both as tumor suppressors, limiting cell proliferation, and as oncogenes, conferring cell resistance to DNA damage. Thus, deepening our knowledge on the biological functions of these Cip/Kips may shed light on how some cancer cells develop drug resistance., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2017
- Full Text
- View/download PDF
19. AKT/GSK3β signaling pathway is critically involved in human pluripotent stem cell survival.
- Author
-
Romorini L, Garate X, Neiman G, Luzzani C, Furmento VA, Guberman AS, Sevlever GE, Scassa ME, and Miriuka SG
- Subjects
- Cell Survival, Cells, Cultured, Gene Expression Regulation, Humans, Glycogen Synthase Kinase 3 beta metabolism, Oncogene Protein v-akt metabolism, Pluripotent Stem Cells physiology, Signal Transduction
- Abstract
Human embryonic and induced pluripotent stem cells are self-renewing pluripotent stem cells (PSC) that can differentiate into a wide range of specialized cells. Basic fibroblast growth factor is essential for PSC survival, stemness and self-renewal. PI3K/AKT pathway regulates cell viability and apoptosis in many cell types. Although it has been demonstrated that PI3K/AKT activation by bFGF is relevant for PSC stemness maintenance its role on PSC survival remains elusive. In this study we explored the molecular mechanisms involved in the regulation of PSC survival by AKT. We found that inhibition of AKT with three non-structurally related inhibitors (GSK690693, AKT inhibitor VIII and AKT inhibitor IV) decreased cell viability and induced apoptosis. We observed a rapid increase in phosphatidylserine translocation and in the extent of DNA fragmentation after inhibitors addition. Moreover, abrogation of AKT activity led to Caspase-9, Caspase-3, and PARP cleavage. Importantly, we demonstrated by pharmacological inhibition and siRNA knockdown that GSK3β signaling is responsible, at least in part, of the apoptosis triggered by AKT inhibition. Moreover, GSK3β inhibition decreases basal apoptosis rate and promotes PSC proliferation. In conclusion, we demonstrated that AKT activation prevents apoptosis, partly through inhibition of GSK3β, and thus results relevant for PSC survival.
- Published
- 2016
- Full Text
- View/download PDF
20. Human Pluripotent Stem Cells and Derived Neuroprogenitors Display Differential Degrees of Susceptibility to BH3 Mimetics ABT-263, WEHI-539 and ABT-199.
- Author
-
García CP, Videla Richardson GA, Dimopoulos NA, Fernandez Espinosa DD, Miriuka SG, Sevlever GE, Romorini L, and Scassa ME
- Subjects
- Ataxia Telangiectasia Mutated Proteins metabolism, Camptothecin pharmacology, Cell Line, Human Embryonic Stem Cells cytology, Humans, Induced Pluripotent Stem Cells cytology, Neural Stem Cells cytology, Proto-Oncogene Proteins c-bcl-2 metabolism, Tumor Suppressor Protein p53 metabolism, bcl-X Protein metabolism, Aniline Compounds pharmacology, Bridged Bicyclo Compounds, Heterocyclic pharmacology, Human Embryonic Stem Cells metabolism, Induced Pluripotent Stem Cells metabolism, Neural Stem Cells metabolism, Sulfonamides pharmacology
- Abstract
Human embryonic stem cells (hESCs) are hypersensitive to genotoxic stress and display lower survival ability relative to their differentiated progeny. Herein, we attempted to investigate the source of this difference by comparing the DNA damage responses triggered by the topoisomerase I inhibitor camptothecin, in hESCs, human induced pluripotent stem cells (hiPSCs) and hESCs-derived neuroprogenitors (NP). We observed that upon camptothecin exposure pluripotent stem cells underwent apoptosis more swiftly and at a higher rate than differentiated cells. However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types. Importantly, we observed that hESCs and hiPSCs express lower levels of the anti-apoptotic protein Bcl-2 than NP. To assess whether Bcl-2 abundance could account for this differential response we treated cells with ABT-263, WEHI-539 and ABT-199, small molecules that preferentially target the BH3-binding pocket of Bcl-xL and/or Bcl-2 and reduce their ability to sequester pro-apoptotic factors. We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs. Conversely, all tested cell types appeared to be highly resistant to the Bcl-2 specific inhibitor, ABT-199. However, in all cases we determined that ABT-263 or WEHI-539 treatment exacerbated camptothecin-induced apoptosis. Importantly, similar responses were observed after siRNA-mediated down-regulation of Bcl-xL or Bcl-2. Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells. The emerging knowledge of the relative dependence of pluripotent and progenitor cells on Bcl-2 and Bcl-xL activities may help to predict cellular responses and potentially manipulate these cells for therapeutic purposes in the near future.
- Published
- 2016
- Full Text
- View/download PDF
21. Generation of iPSC line iPSC-FH2.1 in hypoxic conditions from human foreskin fibroblasts.
- Author
-
Questa M, Romorini L, Blüguermann C, Solari CM, Neiman G, Luzzani C, Scassa MÉ, Sevlever GE, Guberman AS, and Miriuka SG
- Subjects
- Cell Differentiation, Cells, Cultured, Cellular Reprogramming, Comparative Genomic Hybridization, DNA Methylation, Humans, Induced Pluripotent Stem Cells metabolism, Karyotype, Kruppel-Like Factor 4, Male, Microscopy, Fluorescence, Octamer Transcription Factor-3 genetics, Promoter Regions, Genetic, Real-Time Polymerase Chain Reaction, Transcription Factors genetics, Transcription Factors metabolism, Fibroblasts cytology, Foreskin cytology, Induced Pluripotent Stem Cells cytology
- Abstract
Human foreskin fibroblasts were used to generate the iPSC line iPSC-FH2.1 using the EF1a-hSTEMCCA-loxP vector expressing OCT4, SOX2, c-MYC and KLF4, in 5% O2 culture conditions. Stemness was confirmed, as was pluripotency both in vivo and in vitro, in normoxia and hypoxia. Human Embryonic Stem Cell (hESC) line WA-09 and reprogrammed fibroblast primary culture HFF-FM were used as controls., (Copyright © 2015 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
22. Specific Preferences in Lineage Choice and Phenotypic Plasticity of Glioma Stem Cells Under BMP4 and Noggin Influence.
- Author
-
Videla Richardson GA, Garcia CP, Roisman A, Slavutsky I, Fernandez Espinosa DD, Romorini L, Miriuka SG, Arakaki N, Martinetto H, Scassa ME, and Sevlever GE
- Subjects
- Aged, Antigens, CD metabolism, Bone Morphogenetic Protein 4 pharmacology, Carrier Proteins pharmacology, Cell Differentiation drug effects, Cell Differentiation genetics, Female, Gene Expression Regulation, Neoplastic genetics, Humans, Intercellular Signaling Peptides and Proteins pharmacology, Male, Middle Aged, Nerve Tissue Proteins metabolism, Phenotype, Signal Transduction drug effects, Signal Transduction physiology, Tumor Cells, Cultured pathology, Tumor Cells, Cultured physiology, Bone Morphogenetic Protein 4 metabolism, Brain Neoplasms pathology, Carrier Proteins metabolism, Glioma pathology, Neoplastic Stem Cells metabolism, Neoplastic Stem Cells pathology
- Abstract
Although BMP4-induced differentiation of glioma stem cells (GSCs) is well recognized, details of the cellular responses triggered by this morphogen are still poorly defined. In this study, we established several GSC-enriched cell lines (GSC-ECLs) from high-grade gliomas. The expansion of these cells as adherent monolayers, and not as floating neurospheres, enabled a thorough study of the phenotypic changes that occurred during their differentiation. Herein, we evaluated GSC-ECLs' behavior toward differentiating conditions by depriving them of growth factors and/or by adding BMP4 at different concentrations. After analyzing cellular morphology, proliferation and lineage marker expression, we determined that GSC-ECLs have distinct preferences in lineage choice, where some of them showed an astrocyte fate commitment and others a neuronal one. We found that this election seems to be dictated by the expression pattern of BMP signaling components present in each GSC-ECL. Additionally, treatment of GSC-ECLs with the BMP antagonist, Noggin, also led to evident phenotypic changes. Interestingly, under certain conditions, some GSC-ECLs adopted an unexpected smooth muscle-like phenotype. As a whole, our findings illustrate the wide differentiation potential of GSCs, highlighting their molecular complexity and paving a way to facilitate personalized differentiating therapies., (© 2015 International Society of Neuropathology.)
- Published
- 2016
- Full Text
- View/download PDF
23. A therapy-grade protocol for differentiation of pluripotent stem cells into mesenchymal stem cells using platelet lysate as supplement.
- Author
-
Luzzani C, Neiman G, Garate X, Questa M, Solari C, Fernandez Espinosa D, García M, Errecalde AL, Guberman A, Scassa ME, Sevlever GE, Romorini L, and Miriuka SG
- Subjects
- Antigens, Surface metabolism, Cell Differentiation drug effects, Cells, Cultured, DNA Methylation, Human Embryonic Stem Cells cytology, Humans, Intercellular Signaling Peptides and Proteins pharmacology, Mesenchymal Stem Cells metabolism, Microscopy, Fluorescence, Phenotype, Pluripotent Stem Cells drug effects, Pluripotent Stem Cells metabolism, Promoter Regions, Genetic, Blood Platelets metabolism, Mesenchymal Stem Cells cytology, Pluripotent Stem Cells cytology
- Abstract
Introduction: Mesenchymal stem cells (MSCs) are a promising source of cells for regenerative therapies. Although they can be isolated easily from several tissues, cell expansion is limited since their properties are lost with successive passages. Hence, pluripotent derived MSCs (PD-MSCs) arise as a suitable alternative for MSC production. Nevertheless, at present, PD-MSC derivation protocols are either expensive or not suitable for clinical purposes., Methods: In this work we present a therapy-grade, inexpensive and simple protocol to derive MSCs from pluripotent stem cells (PSCs) based on the use of platelet lysate (PL) as medium supplement., Results: We showed that the PD-MSCPL expressed multiple MSC markers, including CD90, CD73, CD105, CD166, and CD271, among others. These cells also show multilineage differentiation ability and immunomodulatory effects on pre-stimulated lymphocytes. Thorough characterization of these cells showed that a PD-MSCPL resembles an umbilical cord (UC) MSC and differs from a PSC in surface marker and extracellular matrix proteins and integrin expression. Moreover, the OCT-4 promoter is re-methylated with mesenchymal differentiation comparable with the methylation levels of UC-MSCs and fibroblasts. Lastly, the use of PL-supplemented medium generates significantly more MSCs than the use of fetal bovine serum., Conclusions: This protocol can be used to generate a large amount of PD-MSCs with low cost and is compatible with clinical therapies.
- Published
- 2015
- Full Text
- View/download PDF
24. Topoisomerase I inhibitor, camptothecin, induces apoptogenic signaling in human embryonic stem cells.
- Author
-
García CP, Videla Richardson GA, Romorini L, Miriuka SG, Sevlever GE, and Scassa ME
- Subjects
- Animals, Apoptosis drug effects, Cell Cycle drug effects, Cell Survival drug effects, Cells, Cultured, Cyclin-Dependent Kinase Inhibitor p21 metabolism, DNA Damage, DNA Fragmentation, Embryonic Stem Cells cytology, Embryonic Stem Cells metabolism, Flow Cytometry, Humans, Mice, Phosphorylation, Signal Transduction drug effects, Tumor Suppressor Protein p53 metabolism, Antineoplastic Agents, Phytogenic pharmacology, Camptothecin pharmacology, Embryonic Stem Cells drug effects, Topoisomerase I Inhibitors pharmacology
- Abstract
Embryonic stem cells (ESCs) need to maintain their genomic integrity in response to DNA damage to safeguard the integrity of the organism. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and, if not repaired correctly, they can lead to cell death, genomic instability and cancer. How human ESCs (hESCs) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. In the present study we aim to determine the hESC response to the DSB inducing agent camptothecin (CPT). We find that hESCs are hypersensitive to CPT, as evidenced by high levels of apoptosis. CPT treatment leads to DNA-damage sensor kinase (ATM and DNA-PKcs) phosphorylation on serine 1981 and serine 2056, respectively. Activation of ATM and DNA-PKcs was followed by histone H2AX phosphorylation on Ser 139, a sensitive reporter of DNA damage. Nuclear accumulation and ATM-dependent phosphorylation of p53 on serine 15 were also observed. Remarkably, hESC viability was further decreased when ATM or DNA-PKcs kinase activity was impaired by the use of specific inhibitors. The hypersensitivity to CPT treatment was markedly reduced by blocking p53 translocation to mitochondria with pifithrin-μ. Importantly, programmed cell death was achieved in the absence of the cyclin dependent kinase inhibitor, p21(Waf1), a bona fide p53 target gene. Conversely, differentiated hESCs were no longer highly sensitive to CPT. This attenuated apoptotic response was accompanied by changes in cell cycle profile and by the presence of p21(Waf1). The results presented here suggest that p53 has a key involvement in preventing the propagation of damaged hESCs when genome is threatened. As a whole, our findings support the concept that the phenomenon of apoptosis is a prominent player in normal embryonic development., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
25. Effect of antibiotics against Mycoplasma sp. on human embryonic stem cells undifferentiated status, pluripotency, cell viability and growth.
- Author
-
Romorini L, Riva DA, Blüguermann C, Videla Richardson GA, Scassa ME, Sevlever GE, and Miriuka SG
- Subjects
- Anti-Bacterial Agents toxicity, Apoptosis drug effects, Biomarkers metabolism, Cell Differentiation drug effects, Cell Line, Cell Proliferation drug effects, Cell Survival drug effects, Ciprofloxacin pharmacology, Ciprofloxacin toxicity, Embryonic Stem Cells cytology, Embryonic Stem Cells microbiology, Humans, Karyotype, Macrolides pharmacology, Macrolides toxicity, Mycoplasma drug effects, Anti-Bacterial Agents pharmacology, Embryonic Stem Cells drug effects, Embryonic Stem Cells metabolism
- Abstract
Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate into specialized cells and hold great promise as models for human development and disease studies, cell-replacement therapies, drug discovery and in vitro cytotoxicity tests. The culture and differentiation of these cells are both complex and expensive, so it is essential to extreme aseptic conditions. hESCs are susceptible to Mycoplasma sp. infection, which is hard to detect and alters stem cell-associated properties. The purpose of this work was to evaluate the efficacy and cytotoxic effect of Plasmocin(TM) and ciprofloxacin (specific antibiotics used for Mycoplasma sp. eradication) on hESCs. Mycoplasma sp. infected HUES-5 884 (H5 884, stable hESCs H5-brachyury promoter-GFP line) cells were effectively cured with a 14 days Plasmocin(TM) 25 µg/ml treatment (curative treatment) while maintaining stemness characteristic features. Furthermore, cured H5 884 cells exhibit the same karyotype as the parental H5 line and expressed GFP, through up-regulation of brachyury promoter, at day 4 of differentiation onset. Moreover, H5 cells treated with ciprofloxacin 10 µg/ml for 14 days (mimic of curative treatment) and H5 and WA09 (H9) hESCs treated with Plasmocin(TM) 5 µg/ml (prophylactic treatment) for 5 passages retained hESCs features, as judged by the expression of stemness-related genes (TRA1-60, TRA1-81, SSEA-4, Oct-4, Nanog) at mRNA and protein levels. In addition, the presence of specific markers of the three germ layers (brachyury, Nkx2.5 and cTnT: mesoderm; AFP: endoderm; nestin and Pax-6: ectoderm) was verified in in vitro differentiated antibiotic-treated hESCs. In conclusion, we found that Plasmocin(TM) and ciprofloxacin do not affect hESCs stemness and pluripotency nor cell viability. However, curative treatments slightly diminished cell growth rate. This cytotoxic effect was reversible as cells regained normal growth rate upon antibiotic withdrawal.
- Published
- 2013
- Full Text
- View/download PDF
26. Human embryonic stem cells and derived contractile embryoid bodies are susceptible to Coxsakievirus B infection and respond to interferon Iβ treatment.
- Author
-
Scassa ME, Jaquenod de Giusti C, Questa M, Pretre G, Richardson GA, Bluguermann C, Romorini L, Ferrer MF, Sevlever GE, Miriuka SG, and Gómez RM
- Subjects
- Cell Line, Coxsackievirus Infections genetics, Coxsackievirus Infections metabolism, Embryoid Bodies drug effects, Embryonic Stem Cells drug effects, Enterovirus B, Human drug effects, Humans, Receptors, Virus genetics, Receptors, Virus metabolism, Virus Replication drug effects, Coxsackievirus Infections virology, Embryoid Bodies virology, Embryonic Stem Cells virology, Enterovirus B, Human physiology, Interferon-beta pharmacology
- Abstract
We studied the susceptibility of human embryonic stem cells and derived contractile embryoid bodies from WAO9, HUES-5 and HUES-16 cell lines to Coxsackievirus B infection. After validating stem cell-like properties and cardiac phenotype, Coxsackievirus B receptors CAR and DAF, as well as type I interferon receptors were detected in all cell lines and differentiation stages studied. Real-time PCR analysis showed that CAR mRNA levels were 3.4-fold higher in undifferentiated cells, while DAF transcript levels were 2.78-fold more abundant in differentiated cultures (P<0.05). All cell lines were susceptible to Coxsackievirus serotypes B1-5 infection as shown by RT-PCR detection of viral RNA, immunofluorescence detection of viral protein and infectivity titration of cell culture supernatants resulting in cell death. Supernatants infectivity titers 24-48 h post-infection ranged from 10⁵-10⁶ plaque forming units (PFU)/ml, the highest titers were detected in undifferentiated cells. Cell viability detected by a colorimetric assay, showed inverse correlation with infectivity titers of cell culture supernatants. Treatment with 100 U of interferon Iβ significantly reduced viral replication and associated cell death during a 24-48 h observation period, as detected by reduced infectivity titers in the supernatants and increased cell viability by a colorimetric assay, respectively. We propose human embryonic stem cell and derived contractile embryoid bodies as a valid model to study cardiac Coxsackievirus B infection., (Copyright © 2010 Elsevier B.V. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
27. Detection of Helicobacter pylori in human carotid atherosclerotic plaques.
- Author
-
Ameriso SF, Fridman EA, Leiguarda RC, and Sevlever GE
- Subjects
- Age Factors, Aged, Carotid Arteries metabolism, Carotid Stenosis complications, Carotid Stenosis metabolism, DNA, Bacterial isolation & purification, Female, Helicobacter Infections complications, Helicobacter Infections diagnosis, Helicobacter Infections metabolism, Helicobacter Infections microbiology, Helicobacter pylori genetics, Humans, Immunohistochemistry, Intercellular Adhesion Molecule-1 metabolism, Logistic Models, Male, Odds Ratio, Risk Factors, Sex Factors, Carotid Arteries microbiology, Carotid Arteries pathology, Carotid Stenosis microbiology, Carotid Stenosis pathology, Helicobacter pylori isolation & purification
- Abstract
Background and Purpose: Several lines of evidence point toward a relationship between infection and atherosclerotic vascular disease. Thus, infection and inflammation often precede ischemic neurological events. Transient alterations in coagulation and direct arterial invasion by certain microorganisms have been reported. Helicobacter pylori infection is the major cause of peptic ulcer disease and appears to be a risk factor for ischemic cerebrovascular disease. However, in contrast to other chronic infectious agents, H pylori has not been consistently isolated from atherosclerotic lesions., Methods: We investigated the presence of H pylori in 38 atherosclerotic plaques obtained at carotid endarterectomy by using morphological and immunohistochemical techniques and a highly sensitive polymerase chain reaction method. We performed immunohistochemical detection of intercellular adhesion molecule-1, a marker related to inflammatory cell response. We also examined 7 carotid arteries obtained at autopsy from subjects without carotid atherosclerosis., Results: H pylori DNA was found in 20 of 38 atherosclerotic plaques. Ten of the H pylori DNA-positive plaques also showed morphological and immunohistochemical evidence of H pylori infection. None of 7 normal carotid arteries was positive for H pylori. Intercellular adhesion molecule-1 was expressed in 75% of H pylori-positive plaques and in 22% of H pylori-negative plaques. The presence of the microorganism was associated with male sex but was independent of age, vascular risk factor profile, and prior neurological symptoms., Conclusions: H pylori is present in a substantial number of carotid atherosclerotic lesions and is associated with features of inflammatory cell response. This study provides additional evidence of the relationship between H pylori infection and atherosclerotic disease.
- Published
- 2001
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.