20 results on '"Fadi J. Najm"'
Search Results
2. Integrative dissection of gene regulatory elements at base resolution
- Author
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Zeyu Chen, Nauman Javed, Molly Moore, Jingyi Wu, Michael Vinyard, Luca Pinello, Fadi J. Najm, and Bradley E. Bernstein
- Abstract
SummaryAlthough vast numbers of putative gene regulatory elements have been cataloged, the sequence motifs and individual bases that underlie their functions remain largely unknown. Here we combine epigenetic perturbations, base editing, and deep learning models to dissect regulatory sequences within the exemplar immune locus encoding CD69. Focusing on a differentially accessible and acetylated upstream enhancer, we find that the complementary strategies converge on a ∼170 base interval as critical for CD69 induction in stimulated Jurkat T cells. We pinpoint individual cytosine to thymine base edits that markedly reduce element accessibility and acetylation, with corresponding reduction of CD69 expression. The most potent base edits may be explained by their effect on binding competition between the transcriptional activator GATA3 and the repressor BHLHE40. Systematic analysis of GATA and bHLH/Ebox motifs suggests that interplay between these factors plays a general role in rapid T cell transcriptional responses. Our study provides a framework for parsing gene regulatory elements in their endogenous chromatin contexts and identifying operative artificial variants.HighlightsBase editing screens and deep learning pinpoint sequences and single bases affecting immune gene expressionAn artificial C-to-T variant in a regulatory element suppresses CD69 expression by altering the balance of transcription factor bindingCompetition between GATA3 and BHLHE40 regulates inducible immune genes and T cell states
- Published
- 2022
3. Impact of supraphysiologic MDM2 expression on chromatin networks and therapeutic responses in sarcoma
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Samantha M. Bevill, Salvador Casaní-Galdón, Chadi A. El Farran, Eli G. Cytrynbaum, Kevin A. Macias, Sylvie E. Oldeman, Kayla J. Oliveira, Molly M. Moore, Esmat Hegazi, Carmen Adriaens, Fadi J. Najm, George D. Demetri, Sonia Cohen, John T. Mullen, Nicolò Riggi, Sarah E. Johnstone, and Bradley E. Bernstein
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Genetics ,Biochemistry, Genetics and Molecular Biology (miscellaneous) - Published
- 2023
4. Integrative dissection of gene regulatory elements at base resolution
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Zeyu Chen, Nauman Javed, Molly Moore, Jingyi Wu, Gary Sun, Michael Vinyard, Alejandro Collins, Luca Pinello, Fadi J. Najm, and Bradley E. Bernstein
- Subjects
Genetics ,Biochemistry, Genetics and Molecular Biology (miscellaneous) - Published
- 2023
5. Long-range phasing of dynamic, tissue-specific and allele-specific regulatory elements
- Author
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Sofia Battaglia, Kevin Dong, Jingyi Wu, Zeyu Chen, Fadi J. Najm, Yuanyuan Zhang, Molly M. Moore, Vivian Hecht, Noam Shoresh, and Bradley E. Bernstein
- Subjects
Genomic Imprinting ,Enhancer Elements, Genetic ,Insulin-Like Growth Factor II ,Genetics ,Animals ,Humans ,RNA, Long Noncoding ,DNA ,DNA Methylation ,Alleles ,Chromatin ,Transcription Factors - Abstract
Epigenomic maps identify gene regulatory elements by their chromatin state. However, prevailing short-read sequencing methods cannot effectively distinguish alleles, evaluate the interdependence of elements in a locus or capture single-molecule dynamics. Here, we apply targeted nanopore sequencing to profile chromatin accessibility and DNA methylation on contiguous ~100-kb DNA molecules that span loci relevant to development, immunity and imprinting. We detect promoters, enhancers, insulators and transcription factor footprints on single molecules based on exogenous GpC methylation. We infer relationships among dynamic elements within immune loci, and order successive remodeling events during T cell stimulation. Finally, we phase primary sequence and regulatory elements across the H19/IGF2 locus, uncovering primate-specific features. These include a segmental duplication that stabilizes the imprinting control region and a noncanonical enhancer that drives biallelic IGF2 expression in specific contexts. Our study advances emerging strategies for phasing gene regulatory landscapes and reveals a mechanism that overrides IGF2 imprinting in human cells.
- Published
- 2022
6. Renewing your HBO1 subscription
- Author
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Fadi J. Najm and Peter van Galen
- Subjects
Histones ,Immunology ,Cell Biology ,Hematology ,Biochemistry ,Histone Acetyltransferases - Published
- 2021
7. Cell Type Specificity of Intralocus Interactions Reveals Oligodendrocyte Intrinsic Mechanisms For Multiple Sclerosis
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Paul J. Tesar, Robert T. Karl, Drew J. Adams, Tyler E. Miller, Ranjan Dutta, Gursimran Dhillon, Olivia Corradin, An T. Hoang, Parker A. Hall, Bruna R. Lima, Sagar Nisraiyya, Alexey Kozlenkov, Mayur Madhavan, Anna M. Barbeau, Christina Volsko, Cynthia F. Bartels, Peter C. Scacheri, Stella Dracheva, Fadi J. Najm, Kathryn E.A. Hazel, Daniel C. Factor, and Zachary S. Nevin
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Autoimmune disease ,Cell type ,Myelin ,medicine.anatomical_structure ,Immune system ,T cell ,Multiple sclerosis ,Central nervous system ,medicine ,Biology ,medicine.disease ,Oligodendrocyte ,Cell biology - Abstract
Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients face progressive disability due to failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue and are associated with white matter microstructure. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development.
- Published
- 2019
8. Depletion of Olig2 in oligodendrocyte progenitor cells infected by Theiler’s murine encephalomyelitis virus
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Eric C. Freundt, Anthony J Martorell, Padmanabhan Mahadevan, Paul J. Tesar, Bayleigh Benner, and Fadi J. Najm
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Pluripotent Stem Cells ,0301 basic medicine ,viruses ,Cellular differentiation ,Oligodendrocyte Transcription Factor 2 ,Primary Cell Culture ,Biology ,Cell Line ,OLIG2 ,Transcriptome ,Mice ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Theilovirus ,Cricetinae ,Virology ,Demyelinating disease ,medicine ,Animals ,Remyelination ,Induced pluripotent stem cell ,Oligodendrocyte Precursor Cells ,Gene Expression Profiling ,virus diseases ,Cell Differentiation ,Epithelial Cells ,Molecular Sequence Annotation ,medicine.disease ,nervous system diseases ,stomatognathic diseases ,030104 developmental biology ,medicine.anatomical_structure ,Gene Expression Regulation ,Neurology ,Host-Pathogen Interactions ,Neurology (clinical) ,Demyelinating Diseases - Abstract
Theiler's murine encephalomyelitis virus (TMEV) infects the central nervous system of mice and causes a demyelinating disease that is a model for multiple sclerosis. During the chronic phase of the disease, TMEV persists in oligodendrocytes and macrophages. Lack of remyelination has been attributed to insufficient proliferation and differentiation of oligodendrocyte progenitor cells (OPCs), but the molecular mechanisms remain unknown. Here, we employed pluripotent stem cell technologies to generate pure populations of mouse OPCs to study the temporal and molecular effects of TMEV infection. Global transcriptome analysis of RNA sequencing data revealed that TMEV infection of OPCs caused significant up-regulation of 1926 genes, whereas 1853 genes were significantly down-regulated compared to uninfected cells. Pathway analysis revealed that TMEV disrupted many genes required for OPC growth and maturation. Down-regulation of Olig2, a transcription factor necessary for OPC proliferation, was confirmed by real-time PCR, immunofluorescence microscopy, and western blot analysis. Depletion of Olig2 was not found to be specific to viral strain and did not require expression of the leader (L) protein, which is a multifunctional protein important for persistence, modulation of gene expression, and cell death. These data suggest that direct infection of OPCs by TMEV may inhibit remyelination during the chronic phase of TMEV-induced demyelinating disease.
- Published
- 2015
9. Cell Type-Specific Intralocus Interactions Reveal Oligodendrocyte Mechanisms in MS
- Author
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Gursimran Dhillon, Cynthia F. Bartels, Ranjan Dutta, Peter C. Scacheri, Stella Dracheva, Lucille R. Hu, Kevin C. Allan, Fadi J. Najm, Michael D. Gallagher, Anna M. Barbeau, Christina Volsko, Mayur Madhavan, Parker A. Hall, Bruna R. Lima, Kathryn E.A. Hazel, Daniel C. Factor, William Phu, Alexandra G. Sibert, Yanwei Song, Robert T. Karl, Olivia Corradin, An T. Hoang, Drew J. Adams, Sagar Nisraiyya, Alexey Kozlenkov, Zachary S. Nevin, Tyler E. Miller, and Paul J. Tesar
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Central Nervous System ,Cell type ,Multiple Sclerosis ,Central nervous system ,Cell Communication ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Myelin ,0302 clinical medicine ,Immune system ,Risk Factors ,medicine ,Animals ,Humans ,Disease ,Remyelination ,Myelin Sheath ,030304 developmental biology ,Epigenomics ,Neurons ,0303 health sciences ,Multiple sclerosis ,Brain ,medicine.disease ,Oligodendrocyte ,Cell biology ,Oligodendroglia ,medicine.anatomical_structure ,030217 neurology & neurosurgery ,Demyelinating Diseases - Abstract
Summary Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. Video Abstract
- Published
- 2020
10. Reprogramming of Mouse Fibroblasts to Induced Oligodendrocyte Progenitor Cells
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Robert T. Karl, Paul J. Tesar, Fadi J. Najm, and Angela M. Lager
- Subjects
Real-time polymerase chain reaction ,biology ,Lentivirus ,Oligodendrocyte progenitor ,RNA-Seq ,biology.organism_classification ,Reprogramming ,Molecular biology ,Immunostaining - Published
- 2017
11. Nucleolar asymmetry and the importance of septin integrity upon cell cycle arrest
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Alan M. Tartakoff, Fadi J. Najm, and Urvashi Rai
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0301 basic medicine ,Nucleolus ,Gene Expression ,lcsh:Medicine ,Septin ,Biochemistry ,Spindle pole body ,Animal Cells ,Medicine and Health Sciences ,Cell Cycle and Cell Division ,lcsh:Science ,Anaphase ,Centromeres ,Multidisciplinary ,Chromosome Biology ,Cell cycle ,Chromatin ,Cell biology ,medicine.anatomical_structure ,Cell Processes ,Epigenetics ,Cellular Structures and Organelles ,Cellular Types ,Cell Nucleolus ,Research Article ,Chromosome Structure and Function ,Saccharomyces cerevisiae Proteins ,Immune Cells ,Immunology ,Antigen-Presenting Cells ,CDC20 ,Saccharomyces cerevisiae ,Biology ,Chromosomes ,Anaphase-Promoting Complex-Cyclosome ,03 medical and health sciences ,medicine ,Genetics ,Cell Nucleus ,030102 biochemistry & molecular biology ,lcsh:R ,Biology and Life Sciences ,Proteins ,Cell Biology ,Cell Cycle Checkpoints ,Cell nucleus ,030104 developmental biology ,Genetic Loci ,lcsh:Q ,Anaphase-promoting complex ,Septins - Abstract
Cell cycle arrest can be imposed by inactivating the anaphase promoting complex (APC). In S. cerevisiae this arrest has been reported to stabilize a metaphase-like intermediate in which the nuclear envelope spans the bud neck, while chromatin repeatedly translocates between the mother and bud domains. The present investigation was undertaken to learn how other features of nuclear organization are affected upon depletion of the APC activator, Cdc20. We observe that the spindle pole bodies and the spindle repeatedly translocate across the narrow orifice at the level of the neck. Nevertheless, we find that the nucleolus (organized around rDNA repeats on the long right arm of chromosome XII) remains in the mother domain, marking the polarity of the nucleus. Accordingly, chromosome XII is polarized: TelXIIR remains in the mother domain and its centromere is predominantly located in the bud domain. In order to learn why the nucleolus remains in the mother domain, we studied the impact of inhibiting rRNA synthesis in arrested cells. We observed that this fragments the nucleolus and that these fragments entered the bud domain. Taken together with earlier observations, the restriction of the nucleolus to the mother domain therefore can be attributed to its massive structure. We also observed that inactivation of septins allowed arrested cells to complete the cell cycle, that the alternative APC activator, Cdh1, was required for completion of the cell cycle and that induction of Cdh1 itself caused arrested cells to progress to the end of the cell cycle.
- Published
- 2017
12. Diverse Chemical Scaffolds Enhance Oligodendrocyte Formation by Inhibiting CYP51, TM7SF2, or EBP
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Zita Hubler, Drew J. Adams, William L. Seibel, Ilya Bederman, Fadi J. Najm, Paul J. Tesar, Dharmaraja Allimuthu, and Hong Tang
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Phenotypic screening ,Clinical Biochemistry ,Steroid Isomerases ,Biology ,01 natural sciences ,Biochemistry ,Small Molecule Libraries ,Mice ,Myelin ,Drug Discovery ,medicine ,Animals ,Remyelination ,Enhancer ,Molecular Biology ,Cells, Cultured ,Pharmacology ,chemistry.chemical_classification ,010405 organic chemistry ,Cell Differentiation ,Small molecule ,Oligodendrocyte ,0104 chemical sciences ,Cell biology ,Oligodendroglia ,Drug repositioning ,medicine.anatomical_structure ,Enzyme ,chemistry ,14-alpha Demethylase Inhibitors ,Molecular Medicine ,Oxidoreductases - Abstract
Small molecules that promote oligodendrocyte formation have been identified in "drug repurposing" screens to nominate candidate therapeutics for diseases in which myelin is lost, including multiple sclerosis. We recently reported that many such molecules enhance oligodendrocyte formation not by their canonical targets but by inhibiting a narrow range of enzymes in cholesterol biosynthesis. Here we identify enhancers of oligodendrocyte formation obtained by screening a structurally diverse library of 10,000 small molecules. Identification of the cellular targets of these validated hits revealed a majority inhibited the cholesterol biosynthesis enzymes CYP51, TM7SF2, or EBP. In addition, evaluation of analogs led to identification of CW3388, a potent EBP-inhibiting enhancer of oligodendrocyte formation poised for further optimization.
- Published
- 2019
13. Transcription factor–mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells
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Daniel C. Factor, Robert H. Miller, Anita Zaremba, Angela M. Lager, Fadi J. Najm, Andrew V. Caprariello, Robert T. Karl, Tadao Maeda, Krysta Wyatt, and Paul J. Tesar
- Subjects
Biomedical Engineering ,Bioengineering ,Biology ,Applied Microbiology and Biotechnology ,Article ,03 medical and health sciences ,0302 clinical medicine ,Animals ,Myelin Sheath ,030304 developmental biology ,0303 health sciences ,Induced stem cells ,Stem Cells ,Transdifferentiation ,Fibroblasts ,Embryonic stem cell ,Neural stem cell ,Cell biology ,Endothelial stem cell ,Oligodendroglia ,Immunology ,Molecular Medicine ,Stem cell ,Reprogramming ,030217 neurology & neurosurgery ,Transcription Factors ,Biotechnology ,Adult stem cell - Abstract
Cell-based therapies for myelin disorders, such as multiple sclerosis and leukodystrophies, require technologies to generate functional oligodendrocyte progenitor cells. Here we describe direct conversion of mouse embryonic and lung fibroblasts to “induced” oligodendrocyte progenitor cells (iOPCs) using sets of either eight or three defined transcription factors. iOPCs exhibit a bipolar morphology and global gene expression profile consistent with bona fide OPCs. They can be expanded in vitro for at least five passages while retaining the ability to differentiate into multiprocessed oligodendrocytes. When transplanted to hypomyelinated mice, iOPCs are capable of ensheathing host axons and generating compact myelin. Lineage conversion of somatic cells to expandable iOPCs provides a strategy to study the molecular control of oligodendrocyte lineage identity and may facilitate neurological disease modeling and autologous remyelinating therapies.
- Published
- 2013
14. Adaptive Chromatin Remodeling Drives Glioblastoma Stem Cell Plasticity and Drug Tolerance
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Laura Donohue, Daniel P. Cahill, Mario L. Suvà, Jon C. Aster, Hiroaki Wakimoto, Sarah J. Shareef, Jeremy N. Rich, Christopher D. Carey, Brian B. Liau, Peter van Galen, Fadi J. Najm, William A. Flavahan, Christine Hebert, Shawn M. Gillespie, Tyler E. Miller, Scott J. Rodig, Cem Sievers, Anoop P. Patel, Andrew S. Venteicher, and Bradley E. Bernstein
- Subjects
0301 basic medicine ,Jumonji Domain-Containing Histone Demethylases ,Transcription, Genetic ,Notch signaling pathway ,Biology ,Methylation ,Chromatin remodeling ,Article ,Histones ,03 medical and health sciences ,Cell Line, Tumor ,Histone methylation ,Genetics ,Biomarkers, Tumor ,Humans ,Epigenetics ,Protein Kinase Inhibitors ,Cell Proliferation ,Histone Demethylases ,Base Sequence ,Receptors, Notch ,Lysine ,Cell Cycle ,Brain ,Nuclear Proteins ,Acetylation ,Cell Biology ,Chromatin Assembly and Disassembly ,Chromatin ,030104 developmental biology ,Enhancer Elements, Genetic ,Drug Resistance, Neoplasm ,Cancer cell ,Cancer research ,biology.protein ,Neoplastic Stem Cells ,Molecular Medicine ,Demethylase ,Stem cell ,Glioblastoma ,Protein Kinases ,Protein Binding ,Signal Transduction - Abstract
Glioblastoma, the most common and aggressive malignant brain tumor, is propagated by stem-like cancer cells refractory to existing therapies. Understanding the molecular mechanisms that control glioblastoma stem cell (GSC) proliferation and drug resistance may reveal opportunities for therapeutic interventions. Here we show GSCs can reversibly transition to a slow-cycling, persistent state in response to targeted kinase inhibitors. In this state, GSCs upregulate primitive developmental programs and are dependent upon Notch signaling. This transition is accompanied by widespread redistribution of repressive histone methylation. Accordingly, persister GSCs upregulate, and are dependent on, the histone demethylases KDM6A/B. Importantly, slow-cycling cells with high Notch activity and histone demethylase expression are present in primary glioblastomas before treatment, potentially contributing to relapse. Our findings illustrate how cancer cells may hijack aspects of native developmental programs for deranged proliferation, adaptation, and tolerance. They also suggest strategies for eliminating refractory tumor cells by targeting epigenetic and developmental pathways.
- Published
- 2016
15. Rapid and robust generation of functional oligodendrocyte progenitor cells from epiblast stem cells
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Anita Zaremba, Shreya Nayak, Eric C. Freundt, Andrew V. Caprariello, Peter C. Scacheri, Fadi J. Najm, Paul J. Tesar, and Robert H. Miller
- Subjects
Biology ,Biochemistry ,Article ,03 medical and health sciences ,0302 clinical medicine ,Directed differentiation ,medicine ,Animals ,Humans ,Progenitor cell ,Induced pluripotent stem cell ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Induced stem cells ,Stem Cells ,Cell Differentiation ,Cell Biology ,Oligodendrocyte ,Neural stem cell ,Cell biology ,Oligodendroglia ,medicine.anatomical_structure ,nervous system ,Immunology ,Stem cell ,030217 neurology & neurosurgery ,Biotechnology ,Adult stem cell - Abstract
Reported is the robust directed differentiation of mouse epiblast stem cells to oligodendrocyte precursor cells, which then differentiate into myelinating oligodendrocytes in vitro and in vivo. The system should prove useful for basic research and for drug screens. Myelin-related disorders such as multiple sclerosis and leukodystrophies, for which restoration of oligodendrocyte function would be an effective treatment, are poised to benefit greatly from stem cell biology. Progress in myelin repair has been constrained by difficulties in generating pure populations of oligodendrocyte progenitor cells (OPCs) in sufficient quantities. Pluripotent stem cells theoretically provide an unlimited source of OPCs, but current differentiation strategies are poorly reproducible and generate heterogenous populations of cells. Here we provide a platform for the directed differentiation of pluripotent mouse epiblast stem cells (EpiSCs) through defined developmental transitions into a pure population of highly expandable OPCs in 10 d. These OPCs robustly differentiate into myelinating oligodendrocytes in vitro and in vivo. Our results demonstrate that mouse pluripotent stem cells provide a pure population of myelinogenic oligodendrocytes and offer a tractable platform for defining the molecular regulation of oligodendrocyte development and drug screening.
- Published
- 2011
16. Isolation of Epiblast Stem Cells from Preimplantation Mouse Embryos
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Josh G. Chenoweth, Fadi J. Najm, Paul J. Tesar, Joseph H. Nadeau, Ronald D.G. McKay, Philip D. Anderson, and Raymond W. Redline
- Subjects
Pluripotent Stem Cells ,Cell type ,Cellular differentiation ,Molecular Sequence Data ,Germ layer ,Cell Separation ,Biology ,03 medical and health sciences ,Mice ,0302 clinical medicine ,medicine ,Genetics ,Animals ,Humans ,Blastocyst ,Induced pluripotent stem cell ,030304 developmental biology ,0303 health sciences ,Base Sequence ,Gene Expression Profiling ,Stem Cells ,Gene Expression Regulation, Developmental ,Cell Differentiation ,Cell Biology ,DNA Methylation ,Embryonic stem cell ,Cell biology ,medicine.anatomical_structure ,Epiblast ,Molecular Medicine ,CpG Islands ,Stem cell ,030217 neurology & neurosurgery ,Germ Layers - Abstract
SummaryPluripotent stem cells provide a platform to interrogate control elements that function to generate all cell types of the body. Despite their utility for modeling development and disease, the relationship of mouse and human pluripotent stem cell states to one another remains largely undefined. We have shown that mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) are distinct, pluripotent states isolated from pre- and post-implantation embryos respectively. Human ES cells are different than mouse ES cells and share defining features with EpiSCs, yet are derived from pre-implantation human embryos. Here we show that EpiSCs can be routinely derived from pre-implantation mouse embryos. The preimplantation-derived EpiSCs exhibit molecular features and functional properties consistent with bona fide EpiSCs. These results provide a simple method for isolating EpiSCs and offer direct insight into the intrinsic and extrinsic mechanisms that regulate the acquisition of distinct pluripotent states.
- Published
- 2011
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17. Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo
- Author
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Daniel C. Factor, Joseph R. Podojil, Andrew P. Robinson, Robert T. Karl, Mayur Madhavan, Ruben Papoian, Min Shen, Christopher Kantor, Tyler E. Miller, Elizabeth Shick, Robert H. Miller, Zachary S. Nevin, Matthew B. Boxer, Anita Zaremba, Daniela Schlatzer, Fadi J. Najm, Kevin L. Quick, Stephen D. Miller, Paul J. Tesar, Alex Sargent, Ajit Jadhav, Kyle R. Brimacombe, and Hong Tang
- Subjects
Male ,Pluripotent Stem Cells ,Encephalomyelitis, Autoimmune, Experimental ,Multiple Sclerosis ,Miconazole ,MAP Kinase Signaling System ,Cellular differentiation ,Neural degeneration ,Biology ,Tissue Culture Techniques ,03 medical and health sciences ,Myelin ,Mice ,0302 clinical medicine ,Receptors, Glucocorticoid ,Cerebellum ,medicine ,Animals ,Humans ,Regeneration ,Remyelination ,Progenitor cell ,Myelin Sheath ,030304 developmental biology ,0303 health sciences ,Clobetasol ,Multidisciplinary ,Experimental autoimmune encephalomyelitis ,Lysophosphatidylcholines ,Cell Differentiation ,medicine.disease ,Oligodendrocyte ,3. Good health ,Cell biology ,Disease Models, Animal ,Oligodendroglia ,medicine.anatomical_structure ,Phenotype ,Immunology ,Female ,Stem cell ,Mitogen-Activated Protein Kinases ,030217 neurology & neurosurgery ,Germ Layers ,Demyelinating Diseases - Abstract
Two drugs, miconazole and clobetasol, have functions that modulate differentiation of oligodendrocyte progenitor cells directly, enhance remyelination, and significantly reduce disease severity in mouse models of multiple sclerosis. Multiple sclerosis is characterized by an autoimmune response and failure of remyelination in the brain due to defects in differentiation of myelin-producing cells from oligodendrocyte progenitor cells. Most current treatments target the immune system. Paul Tesar and colleagues screened for compounds that can enhance oligodendrocyte maturation from mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitors. They found two drugs — miconazole (an antifungal) and clobetasol (a steroid) — that enhance myelin production in vivo in mouse models of multiple sclerosis and enhanced the differentiation of human oligodendrocytes progenitors in vitro. Mechanistically, these compounds appear to target both the immune response and oligodendrocyte progenitor cells. Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes1. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention2. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells3,4,5. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.
- Published
- 2014
18. Generation and characterization of epiblast stem cells from blastocyst-stage mouse embryos
- Author
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Daniel C, Factor, Fadi J, Najm, and Paul J, Tesar
- Subjects
Mice ,Blastocyst ,Germ Cells ,Gastrulation ,Cell Culture Techniques ,Animals ,Cell Differentiation ,Embryonic Stem Cells ,Germ Layers ,Cell Line - Abstract
Mouse epiblast stem cells (EpiSCs) are pluripotent embryonic cells that can be used to interrogate developmental transitions that occur during gastrulation. EpiSCs can also be robustly differentiated into functional somatic and germ cell derivatives making them a useful tool for studying development and regenerative medicine. Typically, mouse EpiSCs are isolated from the early postimplantation epiblast around 5.5 days post coitum (dpc). This chapter describes the methods for isolation of mouse EpiSCs from preimplantation blastocyst-stage mouse embryos (3.5 dpc). This technique enables the routine ability to derive EpiSC lines as it is much less labor intensive than isolation of EpiSCs from the postimplantation epiblast. We also detail relevant assays used to characterize new EpiSC lines and distinguish them from mouse embryonic stem cells.
- Published
- 2013
19. Generation and Characterization of Epiblast Stem Cells from Blastocyst-Stage Mouse Embryos
- Author
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Paul J. Tesar, Daniel C. Factor, and Fadi J. Najm
- Subjects
Rex1 ,Days post coitum ,Biology ,Embryonic stem cell ,Cell biology ,Gastrulation ,medicine.anatomical_structure ,Epiblast ,embryonic structures ,medicine ,Blastocyst ,Stem cell ,reproductive and urinary physiology ,Germ cell - Abstract
Mouse epiblast stem cells (EpiSCs) are pluripotent embryonic cells that can be used to interrogate developmental transitions that occur during gastrulation. EpiSCs can also be robustly differentiated into functional somatic and germ cell derivatives making them a useful tool for studying development and regenerative medicine. Typically, mouse EpiSCs are isolated from the early postimplantation epiblast around 5.5 days post coitum (dpc). This chapter describes the methods for isolation of mouse EpiSCs from preimplantation blastocyst-stage mouse embryos (3.5 dpc). This technique enables the routine ability to derive EpiSC lines as it is much less labor intensive than isolation of EpiSCs from the postimplantation epiblast. We also detail relevant assays used to characterize new EpiSC lines and distinguish them from mouse embryonic stem cells.
- Published
- 2013
20. NPTX1 Regulates Neural Lineage Specification from Human Pluripotent Stem Cells
- Author
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Barbara Corneo, Sheila Le, Steven Lotz, Sarah E. Hirsch, Fadi J. Najm, Christopher A. Fasano, Andrew P. Minotti, Nathan C. Boles, Qingjie Wang, and Paul J. Tesar
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Nervous system ,Neurogenesis ,Induced Pluripotent Stem Cells ,Nerve Tissue Proteins ,Biology ,Bioinformatics ,GPI-Linked Proteins ,General Biochemistry, Genetics and Molecular Biology ,Transcriptome ,Downregulation and upregulation ,Neural Stem Cells ,medicine ,Humans ,Cell Lineage ,Induced pluripotent stem cell ,Receptor ,Gene ,lcsh:QH301-705.5 ,Embryonic Stem Cells ,Cell biology ,Neoplasm Proteins ,Up-Regulation ,medicine.anatomical_structure ,C-Reactive Protein ,lcsh:Biology (General) ,Bone Morphogenetic Proteins ,Intercellular Signaling Peptides and Proteins ,NODAL ,Neural development ,Protein Binding - Abstract
SummaryNeural induction is the first fundamental step in nervous system formation. During development, a tightly regulated niche modulates transient extracellular signals to influence neural lineage commitment. To date, however, the cascade of molecular events that sustain these signals in humans is not well understood. Here we show that NPTX1, a secreted protein, is rapidly upregulated during neural induction from human pluripotent stem cells (hPSCs). By manipulating its expression, we were able to reduce or initiate neural lineage commitment. A time-course transcriptome analysis and functional assays show that NPTX1 acts in part by binding the Nodal receptor cofactor TDGF1, reducing both Nodal and BMP signaling. Our findings identify one of the earliest genes expressed upon neural induction and provide insight into human neural lineage specification.
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