138 results on '"Stanton LW"'
Search Results
2. SARS-CoV transmission epidemiology revealed by MALDI-TOF mass spectrometry-based viral genotyping
- Author
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Liu, J, Lan, LS, Yijun, R, EE, LA, Drosten, C, Liu, ET, Stanton, LW, Hibberd, ML, Liu, J, Lan, LS, Yijun, R, EE, LA, Drosten, C, Liu, ET, Stanton, LW, and Hibberd, ML
- Published
- 2004
3. Inhibition of SARS coronavirus infection in vitro with clinically approved antiviral drugs
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Tan, ELC, Ooi, EE, Lin, CY, Tan, HC, Ling, AE, Lim, B, Stanton, LW, Tan, ELC, Ooi, EE, Lin, CY, Tan, HC, Ling, AE, Lim, B, and Stanton, LW
- Published
- 2004
4. Laboratory-acquired severe acute respiratory syndrome.
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Lim PL, Kurup A, Gopalakrishna G, Chan KP, Wong CW, Ng LC, Se-Thoe SY, Oon L, Bai X, Stanton LW, Ruan Y, Miller LD, Vega VB, James L, Ooi PL, Kai CS, Olsen SJ, Ang B, and Leo Y
- Published
- 2004
5. Deletion in RMST lncRNA impairs hypothalamic neuronal development in a human stem cell-based model of Kallmann Syndrome.
- Author
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Ali G, Shin KC, Ahmed N, Habbab W, Alkhadairi G, Razzaq A, Bejaoui Y, El Hajj N, Mifsud B, Park Y, and Stanton LW
- Abstract
Rhabdomyosarcoma 2-associated transcript (RMST) long non-coding RNA has previously been shown to cause Kallmann syndrome (KS), a rare genetic disorder characterized by congenital hypogonadotropic hypogonadism (CHH) and olfactory dysfunction. In the present study, we generated large deletions of approximately 41.55 kb in the RMST gene in human pluripotent stem cells using CRISPR/Cas9 gene editing. To evaluate the impact of RMST deletion, these cells were differentiated into hypothalamic neurons that include 10-15% neurons that express gonadotrophin-releasing hormone (GnRH). We found that deletion in RMST did not impair the neurogenesis of GnRH neurons, however, the hypothalamic neurons were electro-physiologically hyperactive and had increased calcium influx activity compared to control. Transcriptomic and epigenetic analyses showed that RMST deletion caused altered expression of key genes involved in neuronal development, ion channels, synaptic signaling and cell adhesion. The in vitro generation of these RMST-deleted GnRH neurons provides an excellent cell-based model to dissect the molecular mechanism of RMST function in Kallmann syndrome and its role in hypothalamic neuronal development., (© 2024. The Author(s).)
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- 2024
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6. Characterization of a loss-of-function NSF attachment protein beta mutation in monozygotic triplets affected with epilepsy and autism using cortical neurons from proband-derived and CRISPR-corrected induced pluripotent stem cell lines.
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Ali G, Shin KC, Habbab W, Alkhadairi G, AbdelAleem A, AlShaban FA, Park Y, and Stanton LW
- Abstract
We investigated whether a homozygous recessive genetic variant of NSF attachment protein beta ( NAPB ) gene inherited by monozygotic triplets contributed to their phenotype of early-onset epilepsy and autism. Induced pluripotent stem cell (iPSC) lines were generated from all three probands and both parents. The NAPB genetic variation was corrected in iPSC lines from two probands by CRISPR/Cas9 gene editing. Cortical neurons were produced by directed, in vitro differentiation from all iPSC lines. These cell line-derived neurons enabled us to determine that the genetic variation in the probands causes exon skipping and complete absence of NAPB protein. Electrophysiological and transcriptomic comparisons of cortical neurons derived from parents and probands cell lines indicate that loss of NAPB function contributes to alterations in neuronal functions and likely contributed to the impaired neurodevelopment of the triplets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ali, Shin, Habbab, Alkhadairi, AbdelAleem, Alshaban, Park and Stanton.)
- Published
- 2024
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7. Deletion of TRPC6, an Autism Risk Gene, Induces Hyperexcitability in Cortical Neurons Derived from Human Pluripotent Stem Cells.
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Shin KC, Ali G, Ali Moussa HY, Gupta V, de la Fuente A, Kim HG, Stanton LW, and Park Y
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- Humans, TRPC6 Cation Channel genetics, Calcium metabolism, Neurons metabolism, Autistic Disorder genetics, Autism Spectrum Disorder genetics, Pluripotent Stem Cells metabolism
- Abstract
Autism spectrum disorder (ASD) is a complex and heterogeneous neurodevelopmental disorder linked to numerous rare, inherited, and arising de novo genetic variants. ASD often co-occurs with attention-deficit hyperactivity disorder and epilepsy, which are associated with hyperexcitability of neurons. However, the physiological and molecular mechanisms underlying hyperexcitability in ASD remain poorly understood. Transient receptor potential canonical-6 (TRPC6) is a Ca
2+ -permeable cation channel that regulates store-operated calcium entry (SOCE) and is a candidate risk gene for ASD. Using human pluripotent stem cell (hPSC)-derived cortical neurons, single-cell calcium imaging, and electrophysiological recording, we show that TRPC6 knockout (KO) reduces SOCE signaling and leads to hyperexcitability of neurons by increasing action potential frequency and network burst frequency. Our data provide evidence that reduction of SOCE by TRPC6 KO results in neuronal hyperexcitability, which we hypothesize is an important contributor to the cellular pathophysiology underlying hyperactivity in some ASD., (© 2023. The Author(s).)- Published
- 2023
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8. Identification of two novel autism genes, TRPC4 and SCFD2 , in Qatar simplex families through exome sequencing.
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Gupta V, Ben-Mahmoud A, Ku B, Velayutham D, Jan Z, Yousef Aden A, Kubbar A, Alshaban F, Stanton LW, Jithesh PV, Layman LC, and Kim HG
- Abstract
This study investigated the genetic underpinnings of autism spectrum disorder (ASD) in a Middle Eastern cohort in Qatar using exome sequencing. The study identified six candidate autism genes in independent simplex families, including both four known and two novel autosomal dominant and autosomal recessive genes associated with ASD. The variants consisted primarily of de novo and homozygous missense and splice variants. Multiple individuals displayed more than one candidate variant, suggesting the potential involvement of digenic or oligogenic models. These variants were absent in the Genome Aggregation Database (gnomAD) and exhibited extremely low frequencies in the local control population dataset. Two novel autism genes, TRPC4 and SCFD2 , were discovered in two Qatari autism individuals. Furthermore, the D651A substitution in CLCN3 and the splice acceptor variant in DHX30 were identified as likely deleterious mutations. Protein modeling was utilized to evaluate the potential impact of three missense variants in DEAF1 , CLCN3 , and SCFD2 on their respective structures and functions, which strongly supported the pathogenic natures of these variants. The presence of multiple de novo mutations across trios underscored the significant contribution of de novo mutations to the genetic etiology of ASD. Functional assays and further investigations are necessary to confirm the pathogenicity of the identified genes and determine their significance in ASD. Overall, this study sheds light on the genetic factors underlying ASD in Qatar and highlights the importance of considering diverse populations in ASD research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gupta, Ben-Mahmoud, Ku, Velayutham, Jan, Yousef Aden, Kubbar, Alshaban, Stanton, Jithesh, Layman and Kim.)
- Published
- 2023
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9. High-throughput autoantibody screening identifies differentially abundant autoantibodies in autism spectrum disorder.
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Mesleh A, Ehtewish H, Lennard K, Abdesselem HB, Al-Shaban F, Decock J, Alajez NM, Arredouani A, Emara MM, Albagha O, Stanton LW, Abdulla SA, Blackburnand JM, and El-Agnaf OMA
- Abstract
Introduction: Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by defects in two core domains, social/communication skills and restricted/repetitive behaviors or interests. There is no approved biomarker for ASD diagnosis, and the current diagnostic method is based on clinical manifestation, which tends to vary vastly between the affected individuals due to the heterogeneous nature of ASD. There is emerging evidence that supports the implication of the immune system in ASD, specifically autoimmunity; however, the role of autoantibodies in ASD children is not yet fully understood., Materials and Methods: In this study, we screened serum samples from 93 cases with ASD and 28 healthy controls utilizing high-throughput KoRectly Expressed (KREX) i-Ome protein-array technology. Our goal was to identify autoantibodies with differential expressions in ASD and to gain insights into the biological significance of these autoantibodies in the context of ASD pathogenesis., Result: Our autoantibody expression analysis identified 29 differential autoantibodies in ASD, 4 of which were upregulated and 25 downregulated. Subsequently, gene ontology (GO) and network analysis showed that the proteins of these autoantibodies are expressed in the brain and involved in axonal guidance, chromatin binding, and multiple metabolic pathways. Correlation analysis revealed that these autoantibodies negatively correlate with the age of ASD subjects., Conclusion: This study explored autoantibody reactivity against self-antigens in ASD individuals' serum using a high-throughput assay. The identified autoantibodies were reactive against proteins involved in axonal guidance, synaptic function, amino acid metabolism, fatty acid metabolism, and chromatin binding., Competing Interests: KL and JB are employees of Sengenics, who commercialize the KREX arrays used in this study. JB is also a board member of Sengenics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mesleh, Ehtewish, Lennard, Abdesselem, Al-Shaban, Decock, Alajez, Arredouani, Emara, Albagha, Stanton, Abdulla, Blackburnand and El-Agnaf.)
- Published
- 2023
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10. Blood Proteomics Analysis Reveals Potential Biomarkers and Convergent Dysregulated Pathways in Autism Spectrum Disorder: A Pilot Study.
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Mesleh A, Ehtewish H, de la Fuente A, Al-Shamari H, Ghazal I, Al-Faraj F, Al-Shaban F, Abdesselem HB, Emara M, Alajez NM, Arredouani A, Decock J, Albagha O, Stanton LW, Abdulla SA, and El-Agnaf OMA
- Subjects
- Humans, Pilot Projects, Proteomics, Biomarkers metabolism, Proteome metabolism, Autism Spectrum Disorder genetics
- Abstract
Autism spectrum disorder (ASD) is an umbrella term that encompasses several disabling neurodevelopmental conditions. These conditions are characterized by impaired manifestation in social and communication skills with repetitive and restrictive behaviors or interests. Thus far, there are no approved biomarkers for ASD screening and diagnosis; also, the current diagnosis depends heavily on a physician's assessment and family's awareness of ASD symptoms. Identifying blood proteomic biomarkers and performing deep blood proteome profiling could highlight common underlying dysfunctions between cases of ASD, given its heterogeneous nature, thus laying the foundation for large-scale blood-based biomarker discovery studies. This study measured the expression of 1196 serum proteins using proximity extension assay (PEA) technology. The screened serum samples included ASD cases (n = 91) and healthy controls (n = 30) between 6 and 15 years of age. Our findings revealed 251 differentially expressed proteins between ASD and healthy controls, of which 237 proteins were significantly upregulated and 14 proteins were significantly downregulated. Machine learning analysis identified 15 proteins that could be biomarkers for ASD with an area under the curve (AUC) = 0.876 using support vector machine (SVM). Gene Ontology (GO) analysis of the top differentially expressed proteins (TopDE) and weighted gene co-expression analysis (WGCNA) revealed dysregulation of SNARE vesicular transport and ErbB pathways in ASD cases. Furthermore, correlation analysis showed that proteins from those pathways correlate with ASD severity. Further validation and verification of the identified biomarkers and pathways are warranted.
- Published
- 2023
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11. Generation of induced pluripotent stem cell lines from nonaffected parents and monozygotic triplets affected with autism spectrum disorder and epilepsy.
- Author
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Ali G, Habbab W, Alkhadairi G, Al-Shaban FA, and Stanton LW
- Subjects
- Humans, Autism Spectrum Disorder, Induced Pluripotent Stem Cells, Epilepsy genetics
- Abstract
We have generated induced pluripotent stem cell (iPSC) lines from monozygotic triplets with a rare homozygous mutation in NAPB gene (c.354+2T>G). iPSC lines were also generated from their consanguineous parents who were both heterozygous for the inherited NAPB mutation. The iPSC lines were generated using non-integrating Sendai viral vectors. All iPSC lines showed prototypical stem cell morphology, expressed pluripotency markers and were able to differentiate to all three germ lineages. These iPSC lines will be useful to explore the molecular function of NAPB in neurophysiology and how its dysfunction potentially contributes to the progression of neurodevelopmental disorders associated with autism and epilepsy., 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 © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)
- Published
- 2022
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12. Upregulation of β-catenin due to loss of miR-139 contributes to motor neuron death in amyotrophic lateral sclerosis.
- Author
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Hawkins S, Namboori SC, Tariq A, Blaker C, Flaxman C, Dey NS, Henley P, Randall A, Rosa A, Stanton LW, and Bhinge A
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- DNA-Binding Proteins metabolism, Humans, Motor Neurons metabolism, Mutation, RNA-Binding Protein FUS genetics, RNA-Binding Protein FUS metabolism, Up-Regulation genetics, beta Catenin genetics, beta Catenin metabolism, Amyotrophic Lateral Sclerosis genetics, Amyotrophic Lateral Sclerosis metabolism, MicroRNAs genetics, MicroRNAs metabolism, Neurodegenerative Diseases metabolism
- Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons (MNs). There are no effective treatments and patients usually die within 2-5 years of diagnosis. Emerging commonalities between familial and sporadic cases of this complex multifactorial disorder include disruption to RNA processing and cytoplasmic inclusion bodies containing TDP-43 and/or FUS protein aggregates. Both TDP-43 and FUS have been implicated in RNA processing functions, including microRNA biogenesis, transcription, and splicing. In this study, we explore the misexpression of microRNAs in an iPSC-based disease model of FUS ALS. We identify the downregulation of miR-139, an MN-enriched microRNA, in FUS and sporadic ALS MN. We discover that miR-139 downregulation leads to the activation of canonical WNT signaling and demonstrate that the WNT transcriptional mediator β-catenin is a major driver of MN degeneration in ALS. Our results highlight the importance of homeostatic RNA networks in ALS., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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13. Single Extracellular Vesicle Analysis Using Flow Cytometry for Neurological Disorder Biomarkers.
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Ali Moussa HY, Manaph N, Ali G, Maacha S, Shin KC, Ltaief SM, Gupta V, Tong Y, Ponraj J, Salloum-Asfar S, Mansour S, Al-Shaban FA, Kim HG, Stanton LW, Grivel JC, Abdulla SA, Al-Shammari AR, and Park Y
- Abstract
Extracellular vesicles (EVs) are membrane vesicles released from cells to the extracellular space, involved in cell-to-cell communication by the horizontal transfer of biomolecules such as proteins and RNA. Because EVs can cross the blood-brain barrier (BBB), circulating through the bloodstream and reflecting the cell of origin in terms of disease prognosis and severity, the contents of plasma EVs provide non-invasive biomarkers for neurological disorders. However, neuronal EV markers in blood plasma remain unclear. EVs are very heterogeneous in size and contents, thus bulk analyses of heterogeneous plasma EVs using Western blot and ELISA have limited utility. In this study, using flow cytometry to analyze individual neuronal EVs, we show that our plasma EVs isolated by size exclusion chromatography are mainly CD63-positive exosomes of endosomal origin. As a neuronal EV marker, neural cell adhesion molecule (NCAM) is highly enriched in EVs released from induced pluripotent stem cells (iPSCs)-derived cortical neurons and brain organoids. We identified the subpopulations of plasma EVs that contain NCAM using flow cytometry-based individual EV analysis. Our results suggest that plasma NCAM-positive neuronal EVs can be used to discover biomarkers for neurological disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ali Moussa, Manaph, Ali, Maacha, Shin, Ltaief, Gupta, Tong, Ponraj, Salloum-Asfar, Mansour, Al-Shaban, Kim, Stanton, Grivel, Abdulla, Al-Shammari and Park.)
- Published
- 2022
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14. Single-cell transcriptomics identifies master regulators of neurodegeneration in SOD1 ALS iPSC-derived motor neurons.
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Namboori SC, Thomas P, Ames R, Hawkins S, Garrett LO, Willis CRG, Rosa A, Stanton LW, and Bhinge A
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- Gene Expression Regulation, Gene Regulatory Networks, Humans, Interneurons metabolism, Motor Neurons metabolism, Nerve Degeneration pathology, Signal Transduction, Transforming Growth Factor beta metabolism, Amyotrophic Lateral Sclerosis pathology, Gene Expression Profiling, Induced Pluripotent Stem Cells pathology, Motor Neurons pathology, Nerve Degeneration genetics, Single-Cell Analysis, Superoxide Dismutase-1 metabolism
- Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by the loss of motor neurons. We utilized single-cell transcriptomics to uncover dysfunctional pathways in degenerating motor neurons differentiated from SOD1 E100G ALS patient-derived induced pluripotent stem cells (iPSCs) and respective isogenic controls. Differential gene expression and network analysis identified activation of developmental pathways and core transcriptional factors driving the ALS motor neuron gene dysregulation. Specifically, we identified activation of SMAD2, a downstream mediator of the transforming growth factor β (TGF-β) signaling pathway as a key driver of SOD1 iPSC-derived motor neuron degeneration. Importantly, our analysis indicates that activation of TGFβ signaling may be a common mechanism shared between SOD1, FUS, C9ORF72, VCP, and sporadic ALS motor neurons. Our results demonstrate the utility of single-cell transcriptomics in mapping disease-relevant gene regulatory networks driving neurodegeneration in ALS motor neurons. We find that ALS-associated mutant SOD1 targets transcriptional networks that perturb motor neuron homeostasis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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15. In Reply.
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Cool SM, Sathiyanathan P, Samsonraj RM, Tan CLL, Ling L, Lezhava A, Nurcombe V, and Stanton LW
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- Biomarkers, Tumor, Bone Marrow, Genomics, Humans, Mesenchymal Stem Cells
- Published
- 2020
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16. A genomic biomarker that identifies human bone marrow-derived mesenchymal stem cells with high scalability.
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Sathiyanathan P, Samsonraj RM, Tan CLL, Ling L, Lezhava A, Nurcombe V, Stanton LW, and Cool SM
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- Bone Marrow Cells metabolism, Cell Differentiation genetics, Cell Lineage genetics, Cell Proliferation genetics, Clone Cells, Genotype, Glutathione Transferase genetics, Glutathione Transferase metabolism, Homozygote, Humans, Mesenchymal Stem Cells metabolism, Transcriptome genetics, Biomarkers metabolism, Bone Marrow Cells cytology, Genome, Human, Mesenchymal Stem Cells cytology
- Abstract
Although the application of human mesenchymal stem cells (hMSCs) to repair damaged or diseased tissues has proven relatively effective, both the donor-to-donor variability in ex vivo expansion rates and the maintenance of stemness remain a bottleneck to widespread translation. Previous work from this laboratory stratified donors into those yielding hMSCs with high- or low-growth capacity; global transcriptomic analysis revealed that high-growth-capacity hMSCs were characterized by a loss of the gene encoding glutathione S-transferase theta 1 (GSTT1). These GSTT1-null hMSCs demonstrated increased proliferative rates, clonogenic potential, and longer telomeres compared with low-growth capacity hMSCs that were GSTT1-positive. Thus, this study identifies GSTT1 as a novel genomic DNA biomarker for hMSC scalability., (© AlphaMed Press 2020.)
- Published
- 2020
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17. A Balanced Translocation in Kallmann Syndrome Implicates a Long Noncoding RNA, RMST, as a GnRH Neuronal Regulator.
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Stamou M, Ng SY, Brand H, Wang H, Plummer L, Best L, Havlicek S, Hibberd M, Khor CC, Gusella J, Balasubramanian R, Talkowski M, Stanton LW, and Crowley WF
- Subjects
- Adult, Chromosomes, Human, Pair 12 genetics, Chromosomes, Human, Pair 7 genetics, Genome-Wide Association Study, Gonadotropin-Releasing Hormone genetics, Humans, Induced Pluripotent Stem Cells metabolism, Induced Pluripotent Stem Cells pathology, Male, Neural Crest metabolism, Neural Crest pathology, Prognosis, Gonadotropin-Releasing Hormone metabolism, Kallmann Syndrome genetics, Kallmann Syndrome pathology, RNA, Long Noncoding genetics, Translocation, Genetic
- Abstract
Context: Kallmann syndrome (KS) is a rare, genetically heterogeneous Mendelian disorder. Structural defects in KS patients have helped define the genetic architecture of gonadotropin-releasing hormone (GnRH) neuronal development in this condition., Objective: Examine the functional role a novel structural defect affecting a long noncoding RNA (lncRNA), RMST, found in a KS patient., Design: Whole genome sequencing, induced pluripotent stem cells and derived neural crest cells (NCC) from the KS patient were contrasted with controls., Setting: The Harvard Reproductive Sciences Center, Massachusetts General Hospital Center for Genomic Medicine, and Singapore Genome Institute., Patient: A KS patient with a unique translocation, t(7;12)(q22;q24)., Interventions/main Outcome Measure/results: A novel translocation was detected affecting the lncRNA, RMST, on chromosome 12 in the absence of any other KS mutations. Compared with controls, the patient's induced pluripotent stem cells and NCC provided functional information regarding RMST. Whereas RMST expression increased during NCC differentiation in controls, it was substantially reduced in the KS patient's NCC coincident with abrogated NCC morphological development and abnormal expression of several "downstream" genes essential for GnRH ontogeny (SOX2, PAX3, CHD7, TUBB3, and MKRN3). Additionally, an intronic single nucleotide polymorphism in RMST was significantly implicated in a genome-wide association study associated with age of menarche., Conclusions: A novel deletion in RMST implicates the loss of function of a lncRNA as a unique cause of KS and suggests it plays a critical role in the ontogeny of GnRH neurons and puberty., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2020
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18. Rapid generation of purified human RPE from pluripotent stem cells using 2D cultures and lipoprotein uptake-based sorting.
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Michelet F, Balasankar A, Teo N, Stanton LW, and Singhal S
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- Cell Differentiation, Humans, Cell Culture Techniques methods, Lipoproteins metabolism, Pluripotent Stem Cells metabolism, Retinal Pigment Epithelium metabolism
- Abstract
Background: Despite increasing demand, current protocols for human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) remain time, labor, and cost intensive. Additionally, absence of robust methods for selective RPE purification and removal of non-RPE cell impurities prevents upscaling of clinical quality RPE production. We aimed to address these challenges by developing a simplified hPSC-derived RPE production and purification system that yields high-quality RPE monolayers within 90 days., Methods: Human pluripotent stem cells were differentiated into RPE using an innovative time and cost-effective protocol relying entirely on 2D cultures and minimal use of cytokines. Once RPE identity was obtained, cells were transferred onto permeable membranes to acquire mature RPE morphology. RPE differentiation was verified by electron microscopy, polarized VEGF expression, establishment of high transepithelial electrical resistance and photoreceptor phagocytosis assay. After 4 weeks on permeable membranes, RPE cell cultures were incubated with Dil-AcLDL (DiI-conjugated acetylated low-density lipoproteins) and subjected to fluorescence-activated cell sorting (FACS) for purification and subculture., Results: Using our 2D cytokine scarce protocol, hPSC-derived functional RPE cells can be obtained within 2 months. Nevertheless, at this stage, most samples contain a percentage of non-RPE/early RPE progenitor cells that make them unsuitable for clinical application. We demonstrate that functional RPE cells express high levels of lipoprotein receptors and that this correlates with their ability to uptake lipoproteins. Combining photoreceptor uptake assay with lipoprotein uptake assay further confirms that only functional RPE cells uptake AcLDL. Incubation of mixed RPE/non-RPE cell cultures with fluorophore conjugated AcLDL and subsequent FACS-based isolation of labeled cells allows selective purification of mature functional RPE. When subcultured, DiI-AcLDL-labeled cells rapidly form pure homogenous high-quality RPE monolayers., Conclusions: Pure functional RPE monolayers can be derived from hPSC within 90 days using simplified 2D cultures in conjunction with our RPE PLUS protocol (RPE Purification by Lipoprotein Uptake-based Sorting). The simplicity of this protocol makes it scalable, and the rapidity of production and purification allows for high-quality RPE to be produced in a short span of time making them ideally suited for downstream clinical and in vitro applications.
- Published
- 2020
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19. Phenotypic and molecular features underlying neurodegeneration of motor neurons derived from spinal and bulbar muscular atrophy patients.
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Sheila M, Narayanan G, Ma S, Tam WL, Chai J, and Stanton LW
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- Bulbo-Spinal Atrophy, X-Linked genetics, Cell Differentiation, Gene Expression Profiling, Humans, Induced Pluripotent Stem Cells metabolism, Induced Pluripotent Stem Cells pathology, Intracellular Signaling Peptides and Proteins genetics, Phenotype, Signal Transduction, Bulbo-Spinal Atrophy, X-Linked metabolism, Bulbo-Spinal Atrophy, X-Linked pathology, Intracellular Signaling Peptides and Proteins physiology, Motor Neurons metabolism, Motor Neurons pathology, Neurites metabolism, Neurites pathology
- Abstract
Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of polyglutamine region in the androgen receptor. To gain insights into mechanisms of SBMA, four wild-type and five SBMA iPSC lines were differentiated to spinal motor neurons (sMNs) with high efficiency. SBMA sMNs showed neurite defects, reduced sMN survival and decreased protein synthesis levels. Microarray analysis revealed a dysregulation in various neuronal-related signalling pathways in SBMA sMNs. Strikingly, FAM135B a novel gene of unknown function, was found drastically downregulated in SBMA sMNs. Knockdown of FAM135B in wild-type sMNs reduced their survival and contributed to neurite defects, similar to SBMA sMNs, suggesting a functional role of FAM135B in SBMA. The degenerative phenotypes and dysregulated genes revealed could be potential therapeutic targets for SBMA., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2019
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20. APP upregulation contributes to retinal ganglion cell degeneration via JNK3.
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Liu C, Zhang CW, Zhou Y, Wong WQ, Lee LC, Ong WY, Yoon SO, Hong W, Fu XY, Soong TW, Koo EH, Stanton LW, Lim KL, Xiao ZC, and Dawe GS
- Subjects
- Amyloid beta-Protein Precursor genetics, Animals, Axotomy, Mice, Mice, Mutant Strains, Mitogen-Activated Protein Kinase 10 genetics, Optic Nerve pathology, Optic Nerve Diseases genetics, Optic Nerve Diseases pathology, Retinal Ganglion Cells pathology, Amyloid beta-Protein Precursor biosynthesis, Gene Expression Regulation, Enzymologic, Mitogen-Activated Protein Kinase 10 biosynthesis, Optic Nerve metabolism, Optic Nerve Diseases metabolism, Retinal Ganglion Cells metabolism, Up-Regulation
- Abstract
Axonal injury is a common feature of central nervous system insults. Upregulation of amyloid precursor protein (APP) is observed following central nervous system neurotrauma and is regarded as a marker of central nervous system axonal injury. However, the underlying mechanism by which APP mediates neuronal death remains to be elucidated. Here, we used mouse optic nerve axotomy (ONA) to model central nervous system axonal injury replicating aspects of retinal ganglion cell (RGC) death in optic neuropathies. APP and APP intracellular domain (AICD) were upregulated in retina after ONA and APP knockout reduced Tuj1
+ RGC loss. Pathway analysis of microarray data combined with chromatin immunoprecipitation and a luciferase reporter assay demonstrated that AICD interacts with the JNK3 gene locus and regulates JNK3 expression. Moreover, JNK3 was found to be upregulated after ONA and to contribute to Tuj1+ RGC death. APP knockout reduced the ONA-induced enhanced expression of JNK3 and phosphorylated JNK (pJNK). Gamma-secretase inhibitors prevented production of AICD, reduced JNK3 and pJNK expression similarly, and protected Tuj1+ RGCs from ONA-induced cell death. Together these data indicate that ONA induces APP expression and that gamma-secretase cleavage of APP releases AICD, which upregulates JNK3 leading to RGC death. This pathway may be a novel target for neuronal protection in optic neuropathies and other forms of neurotrauma.- Published
- 2018
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21. Single-cell gene expression analysis reveals regulators of distinct cell subpopulations among developing human neurons.
- Author
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Wang J, Jenjaroenpun P, Bhinge A, Angarica VE, Del Sol A, Nookaew I, Kuznetsov VA, and Stanton LW
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- Cell Differentiation, Cell Lineage, Cells, Cultured, Gene Expression Regulation, Developmental, Humans, Neurogenesis, RNA, Long Noncoding genetics, Transcription Factors genetics, Gene Expression Profiling methods, Gene Regulatory Networks, Neural Stem Cells cytology, Sequence Analysis, RNA methods, Single-Cell Analysis methods
- Abstract
The stochastic dynamics and regulatory mechanisms that govern differentiation of individual human neural precursor cells (NPC) into mature neurons are currently not fully understood. Here, we used single-cell RNA-sequencing (scRNA-seq) of developing neurons to dissect/identify NPC subtypes and critical developmental stages of alternative lineage specifications. This study comprises an unsupervised, high-resolution strategy for identifying cell developmental bifurcations, tracking the stochastic transcript kinetics of the subpopulations, elucidating regulatory networks, and finding key regulators. Our data revealed the bifurcation and developmental tracks of the two NPC subpopulations, and we captured an early (24 h) transition phase that leads to alternative neuronal specifications. The consequent up-regulation and down-regulation of stage- and subpopulation-specific gene groups during the course of maturation revealed biological insights with regard to key regulatory transcription factors and lincRNAs that control cellular programs in the identified neuronal subpopulations., (© 2017 Wang et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2017
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22. Cyclin-Dependent Kinase-Dependent Phosphorylation of Sox2 at Serine 39 Regulates Neurogenesis.
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Lim S, Bhinge A, Bragado Alonso S, Aksoy I, Aprea J, Cheok CF, Calegari F, Stanton LW, and Kaldis P
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- Amino Acid Sequence, Animals, Cell Differentiation, DNA metabolism, Gene Expression Regulation, Mice, Models, Biological, Mutant Proteins metabolism, NIH 3T3 Cells, Neural Stem Cells metabolism, Neurons cytology, Neurons metabolism, Phosphorylation, Protein Binding, Protein Stability, SOXB1 Transcription Factors chemistry, Serine Proteases metabolism, Cyclin-Dependent Kinases metabolism, Neurogenesis genetics, Phosphoserine metabolism, SOXB1 Transcription Factors metabolism
- Abstract
Sox2 is known to be important for neuron formation, but the precise mechanism through which it activates a neurogenic program and how this differs from its well-established function in self-renewal of stem cells remain elusive. In this study, we identified a highly conserved cyclin-dependent kinase (Cdk) phosphorylation site on serine 39 (S39) in Sox2. In neural stem cells (NSCs), phosphorylation of S39 enhances the ability of Sox2 to negatively regulate neuronal differentiation, while loss of phosphorylation is necessary for chromatin retention of a truncated form of Sox2 generated during neurogenesis. We further demonstrated that nonphosphorylated cleaved Sox2 specifically induces the expression of proneural genes and promotes neurogenic commitment in vivo Our present study sheds light on how the level of Cdk kinase activity directly regulates Sox2 to tip the balance between self-renewal and differentiation in NSCs., (Copyright © 2017 American Society for Microbiology.)
- Published
- 2017
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23. Phosphorylation of amyloid precursor protein by mutant LRRK2 promotes AICD activity and neurotoxicity in Parkinson's disease.
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Chen ZC, Zhang W, Chua LL, Chai C, Li R, Lin L, Cao Z, Angeles DC, Stanton LW, Peng JH, Zhou ZD, Lim KL, Zeng L, and Tan EK
- Subjects
- Animals, Cells, Cultured, Disease Models, Animal, Dopamine metabolism, Dopaminergic Neurons metabolism, Humans, Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 genetics, Male, Mice, Mice, Transgenic, Parkinson Disease genetics, Parkinson Disease metabolism, Phosphorylation, Amyloid beta-Protein Precursor metabolism, Dopaminergic Neurons pathology, Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 physiology, Mutation, Parkinson Disease pathology, Protein Interaction Domains and Motifs
- Abstract
Mutations in LRRK2 , which encodes leucine-rich repeat kinase 2, are the most common genetic cause of familial and sporadic Parkinson's disease (PD), a degenerative disease of the central nervous system that causes impaired motor function and, in advanced stages, dementia. Dementia is a common symptom of another neurodegenerative disease, Alzheimer's disease, and research suggests that there may be pathophysiological and genetic links between the two diseases. Aggregates of β amyloid [a protein produced through cleavage of amyloid precursor protein (APP)] are seen in both diseases and in PD patients carrying G2019S-mutant LRRK2. Using patient-derived cells, brain tissue, and PD model mice, we found that LRRK2 interacted with and phosphorylated APP at Thr
668 within its intracellular domain (AICD). Phosphorylation of APP at Thr668 promoted AICD transcriptional activity and correlated with increased nuclear abundance of AICD and decreased abundance of a dopaminergic neuron marker in cultures and brain tissue. The AICD regulates the transcription of genes involved in cytoskeletal dynamics and apoptosis. Overexpression of AICD, but not a phosphodeficient mutant (AICDT668A ), increased the loss of dopaminergic neurons in older mice expressing LRRK2G2019S Moreover, the amount of Thr668 -phosphorylated APP was substantially greater in postmortem brain tissue and dopaminergic neurons (generated by reprogramming skin cells) from LRRK2G2019S patients than in those from healthy individuals. LRRK2 inhibitors reduced the phosphorylation of APP at Thr668 in the patient-derived dopaminergic neurons and in the midbrains of LRRK2G2019S mice. Thus, APP is a substrate of LRRK2, and its phosphorylation promotes AICD function and neurotoxicity in PD., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)- Published
- 2017
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24. Long-Term Culture of Self-renewing Pancreatic Progenitors Derived from Human Pluripotent Stem Cells.
- Author
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Trott J, Tan EK, Ong S, Titmarsh DM, Denil SLIJ, Giam M, Wong CK, Wang J, Shboul M, Eio M, Cooper-White J, Cool SM, Rancati G, Stanton LW, Reversade B, and Dunn NR
- Subjects
- Animals, Cell Differentiation drug effects, Cell Line, Down-Regulation, Feeder Cells cytology, Feeder Cells metabolism, Homeodomain Proteins genetics, Homeodomain Proteins metabolism, Humans, Insulin pharmacology, Insulin-Secreting Cells cytology, Insulin-Secreting Cells metabolism, Kidney metabolism, Kidney pathology, Mice, Mice, Inbred NOD, Mice, SCID, Pancreas cytology, Pluripotent Stem Cells metabolism, SOX9 Transcription Factor metabolism, Stem Cells metabolism, Trans-Activators metabolism, Transplantation, Heterologous, Cell Self Renewal physiology, Pluripotent Stem Cells cytology, Stem Cells cytology
- Abstract
Pluripotent stem cells have been proposed as an unlimited source of pancreatic β cells for studying and treating diabetes. However, the long, multi-step differentiation protocols used to generate functional β cells inevitably exhibit considerable variability, particularly when applied to pluripotent cells from diverse genetic backgrounds. We have developed culture conditions that support long-term self-renewal of human multipotent pancreatic progenitors, which are developmentally more proximal to the specialized cells of the adult pancreas. These cultured pancreatic progenitor (cPP) cells express key pancreatic transcription factors, including PDX1 and SOX9, and exhibit transcriptomes closely related to their in vivo counterparts. Upon exposure to differentiation cues, cPP cells give rise to pancreatic endocrine, acinar, and ductal lineages, indicating multilineage potency. Furthermore, cPP cells generate insulin+ β-like cells in vitro and in vivo, suggesting that they offer a convenient alternative to pluripotent cells as a source of adult cell types for modeling pancreatic development and diabetes., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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25. Transcriptome analysis for the identification of cellular markers related to trabecular meshwork differentiation.
- Author
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Sathiyanathan P, Tay CY, and Stanton LW
- Subjects
- Aged, Biomarkers metabolism, Cornea metabolism, Humans, Middle Aged, Sclera metabolism, Stem Cells cytology, Cell Differentiation genetics, Gene Expression Profiling, Trabecular Meshwork cytology, Trabecular Meshwork metabolism
- Abstract
Background: Development of primary open-angle glaucoma (POAG) is associated with the malfunctioning trabecular meshwork (TM). Cell therapy offers great potential for the treatment of POAG, but requires the generation of functional TM cells in vitro to replace the lost/dysfunctional cells. TM differentiation in vitro from various stem cell types must be monitored by the expression of specific markers. However, no single definitive marker of the TM has been identified., Results: To identify robust markers of TM differentiation, we performed global transcriptome profiling using high-density oligonucleotide microarray on ex vivo TM tissue and cultured TM progenitors. Corneal and scleral tissues were also used in the analysis. After removal of genes expressed in the cornea and sclera, 18 genes were identified that were differentially expressed in the TM relative to the other samples. CDH23, F5, KCNAB1, FGF9, SPP1, and HEY1 were selected among the genes highly expressed in the TM, together with BDNF which was repressed, compared to progenitors for further investigation. Expression analysis by qPCR verified the differential expression and immunofluorescence of the anterior segment confirmed strong expression in the TM., Conclusions: Three independent cohort of expression studies have identified novel markers, fitting in identifying TM cells and in evaluating directed TM differentiation in vitro.
- Published
- 2017
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26. Genetic Correction of SOD1 Mutant iPSCs Reveals ERK and JNK Activated AP1 as a Driver of Neurodegeneration in Amyotrophic Lateral Sclerosis.
- Author
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Bhinge A, Namboori SC, Zhang X, VanDongen AMJ, and Stanton LW
- Subjects
- Amyotrophic Lateral Sclerosis metabolism, Cells, Cultured, Genetic Engineering, Humans, Induced Pluripotent Stem Cells cytology, Induced Pluripotent Stem Cells metabolism, Motor Neurons metabolism, Point Mutation, Proto-Oncogene Proteins c-jun metabolism, Superoxide Dismutase-1 metabolism, Amyotrophic Lateral Sclerosis genetics, Amyotrophic Lateral Sclerosis pathology, Induced Pluripotent Stem Cells pathology, MAP Kinase Signaling System, Motor Neurons pathology, Superoxide Dismutase-1 genetics, Transcription Factor AP-1 metabolism
- Abstract
Although mutations in several genes with diverse functions have been known to cause amyotrophic lateral sclerosis (ALS), it is unknown to what extent causal mutations impinge on common pathways that drive motor neuron (MN)-specific neurodegeneration. In this study, we combined induced pluripotent stem cells-based disease modeling with genome engineering and deep RNA sequencing to identify pathways dysregulated by mutant SOD1 in human MNs. Gene expression profiling and pathway analysis followed by pharmacological screening identified activated ERK and JNK signaling as key drivers of neurodegeneration in mutant SOD1 MNs. The AP1 complex member JUN, an ERK/JNK downstream target, was observed to be highly expressed in MNs compared with non-MNs, providing a mechanistic insight into the specific degeneration of MNs. Importantly, investigations of mutant FUS MNs identified activated p38 and ERK, indicating that network perturbations induced by ALS-causing mutations converge partly on a few specific pathways that are drug responsive and provide immense therapeutic potential., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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27. Generation of sibling-matched induced pluripotent stem cell lines from spinal and bulbar muscular atrophy patients.
- Author
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Narayanan G, Sheila M, Chai J, and Stanton LW
- Subjects
- Adult, Base Sequence, Cell Differentiation, Cell Line, DNA Fingerprinting, Humans, Induced Pluripotent Stem Cells metabolism, Karyotype, Leukocytes, Mononuclear cytology, Leukocytes, Mononuclear metabolism, Male, Microscopy, Fluorescence, Middle Aged, Muscular Atrophy, Spinal genetics, Muscular Atrophy, Spinal metabolism, Receptors, Androgen genetics, Transcription Factors genetics, Transcription Factors metabolism, Trinucleotide Repeats genetics, Cellular Reprogramming, Induced Pluripotent Stem Cells cytology, Muscular Atrophy, Spinal pathology
- Abstract
Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of CAG repeats in the Androgen Receptor gene (AR). We report the generation of induced pluripotent stem cell (iPSC) lines from two SBMA patients and their healthy siblings. The SBMA and healthy iPSC lines retain the number of AR CAG repeats, express pluripotency markers and are able to differentiate into the three germ layers. The iPSC lines are also free of Sendai virus transgenes and have normal karyotypes. The SBMA iPSC lines with their sibling-matched controls would serve as useful tools to study SBMA disease mechanism., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2017
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28. Re-engineered RNA-Guided FokI-Nucleases for Improved Genome Editing in Human Cells.
- Author
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Havlicek S, Shen Y, Alpagu Y, Bruntraeger MB, Zufir NB, Phuah ZY, Fu Z, Dunn NR, and Stanton LW
- Subjects
- Bacterial Proteins metabolism, Base Sequence, Binding Sites, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Deoxyribonucleases, Type II Site-Specific chemistry, Deoxyribonucleases, Type II Site-Specific genetics, Endonucleases metabolism, Genetic Variation, Humans, Models, Biological, Mutation, Pluripotent Stem Cells metabolism, Protein Binding, Protein Multimerization, Regulatory Factor X Transcription Factors chemistry, Regulatory Factor X Transcription Factors genetics, Deoxyribonucleases, Type II Site-Specific metabolism, Gene Editing, Protein Engineering, RNA, Guide, CRISPR-Cas Systems
- Abstract
Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 enables us to generate targeted sequence changes in the genomes of cells and organisms. However, off-target effects have been a persistent problem hampering the development of therapeutics based on CRISPR/Cas9 and potentially confounding research results. Efforts to improve Cas9 specificity, like the development of RNA-guided FokI-nucleases (RFNs), usually come at the cost of editing efficiency and/or genome targetability. To overcome these limitations, we engineered improved chimeras of RFNs that enable higher cleavage efficiency and provide broader genome targetability, while retaining high fidelity for genome editing in human cells. Furthermore, we developed a new RFN ortholog derived from Staphylococcus aureus Cas9 and characterize its utility for efficient genome engineering. Finally, we demonstrate the feasibility of RFN orthologs to functionally hetero-dimerize to modify endogenous genes, unveiling a new dimension of RFN target design opportunities., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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29. Personalized genome sequencing coupled with iPSC technology identifies GTDC1 as a gene involved in neurodevelopmental disorders.
- Author
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Aksoy I, Utami KH, Winata CL, Hillmer AM, Rouam SL, Briault S, Davila S, Stanton LW, and Cacheux V
- Subjects
- Animals, Autism Spectrum Disorder metabolism, Autism Spectrum Disorder pathology, Cell Differentiation genetics, Central Nervous System growth & development, Central Nervous System pathology, Disease Models, Animal, Gene Expression Regulation, Developmental, Genome, Human, Glycosyltransferases biosynthesis, High-Throughput Nucleotide Sequencing, Humans, Induced Pluripotent Stem Cells pathology, Neural Stem Cells pathology, Neurons metabolism, Neurons pathology, Precision Medicine, Zebrafish genetics, Zebrafish growth & development, Autism Spectrum Disorder genetics, Glycosyltransferases genetics, Induced Pluripotent Stem Cells metabolism, Neural Stem Cells metabolism
- Abstract
The cellular and molecular mechanisms underlying neurodevelopmental conditions such as autism spectrum disorders have been studied intensively for decades. The ability to generate patient-specific induced pluripotent stem cells (iPSCs) now offers a novel strategy for modelling human diseases. Recent studies have reported the derivation of iPSCs from patients with neurological disorders. The key challenge remains the demonstration of disease-related phenotypes and the ability to model the disease. Here we report a case study with signs of neurodevelopmental disorders (NDDs) harbouring chromosomal rearrangements that were sequenced using long-insert DNA paired-end tag (DNA-PET) sequencing approach. We identified the disruption of a specific gene, GTDC1. By deriving iPSCs from this patient and differentiating them into neural progenitor cells (NPCs) and neurons we dissected the disease process at the cellular level and observed defects in both NPCs and neuronal cells. We also showed that disruption of GTDC1 expression in wild type human NPCs and neurons showed a similar phenotype as patient's iPSCs. Finally, we utilized a zebrafish model to demonstrate a role for GTDC1 in the development of the central nervous system. Our findings highlight the importance of combining sequencing technologies with the iPSC technology for NDDs modelling that could be applied for personalized medicine., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2017
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30. A pharmacogenomic profile of human neural progenitors undergoing differentiation in the presence of the traditional Chinese medicine NeuroAiD.
- Author
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Chan HY and Stanton LW
- Subjects
- Cell Line, Gene Expression Profiling methods, Gene Expression Regulation drug effects, Gene Regulatory Networks drug effects, Genome-Wide Association Study, Humans, Neural Stem Cells metabolism, Neurogenesis genetics, Neuroglia drug effects, Neuroglia metabolism, Neurons drug effects, Neurons metabolism, Oligonucleotide Array Sequence Analysis, Pharmacogenetics, Time Factors, Drugs, Chinese Herbal pharmacology, Neural Stem Cells drug effects, Neurogenesis drug effects, Neuroprotective Agents pharmacology
- Abstract
NeuroAiD, a traditional Chinese medicine widely used to treat stroke patients in China, was recently demonstrated in rodent models and in clinical trials to possess neuroregenerative and neuroprotective properties. In order to understand the mechanisms employed by NeuroAiD to bring about its neuroproliferative and neuroprotective effects, we investigated the impact of MLC901, a reformulated version of MLC601, on human neural progenitors undergoing neural differentiation at the molecular level by performing three independent microarray experiments. Functional annotations of the genes regulated by MLC901 that were associated with neurogenesis were found to be enriched. We also identified potential targets (FGF19, GALR2, MMP10, FGF3 and TDO2) of MLC901 that could promote neurogenesis and neuroprotection in the human brain. This work highlighted some interesting targets and offered some insights into the possible mechanism of action of MLC901. The discovery could also provide a platform to the development of future therapeutic targets.
- Published
- 2016
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31. The Neurogenic Potential of Astrocytes Is Regulated by Inflammatory Signals.
- Author
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Michelucci A, Bithell A, Burney MJ, Johnston CE, Wong KY, Teng SW, Desai J, Gumbleton N, Anderson G, Stanton LW, Williams BP, and Buckley NJ
- Subjects
- Animals, Animals, Newborn, Astrocytes drug effects, Astrocytes metabolism, Bone Morphogenetic Protein 4 metabolism, Cell Dedifferentiation drug effects, Cell Dedifferentiation genetics, Cell Line, Cell Proliferation drug effects, Central Nervous System pathology, Epigenesis, Genetic drug effects, Gene Expression Profiling, Gene Expression Regulation drug effects, Histones metabolism, Inflammation metabolism, Mice, Models, Biological, NF-kappa B metabolism, Neural Stem Cells drug effects, Neural Stem Cells metabolism, Phenotype, Promoter Regions, Genetic genetics, Protein Processing, Post-Translational drug effects, Signal Transduction drug effects, Signal Transduction genetics, Time Factors, Transcriptome drug effects, Transcriptome genetics, Tumor Necrosis Factor-alpha pharmacology, Astrocytes pathology, Inflammation pathology, Neurogenesis drug effects
- Abstract
Although the adult brain contains neural stem cells (NSCs) that generate new neurons throughout life, these astrocyte-like populations are restricted to two discrete niches. Despite their terminally differentiated phenotype, adult parenchymal astrocytes can re-acquire NSC-like characteristics following injury, and as such, these 'reactive' astrocytes offer an alternative source of cells for central nervous system (CNS) repair following injury or disease. At present, the mechanisms that regulate the potential of different types of astrocytes are poorly understood. We used in vitro and ex vivo astrocytes to identify candidate pathways important for regulation of astrocyte potential. Using in vitro neural progenitor cell (NPC)-derived astrocytes, we found that exposure of more lineage-restricted astrocytes to either tumor necrosis factor alpha (TNF-α) (via nuclear factor-κB (NFκB)) or the bone morphogenetic protein (BMP) inhibitor, noggin, led to re-acquisition of NPC properties accompanied by transcriptomic and epigenetic changes consistent with a more neurogenic, NPC-like state. Comparative analyses of microarray data from in vitro-derived and ex vivo postnatal parenchymal astrocytes identified several common pathways and upstream regulators associated with inflammation (including transforming growth factor (TGF)-β1 and peroxisome proliferator-activated receptor gamma (PPARγ)) and cell cycle control (including TP53) as candidate regulators of astrocyte phenotype and potential. We propose that inflammatory signalling may control the normal, progressive restriction in potential of differentiating astrocytes as well as under reactive conditions and represent future targets for therapies to harness the latent neurogenic capacity of parenchymal astrocytes.
- Published
- 2016
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32. Molecular Features Underlying Neurodegeneration Identified through In Vitro Modeling of Genetically Diverse Parkinson's Disease Patients.
- Author
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Lin L, Göke J, Cukuroglu E, Dranias MR, VanDongen AM, and Stanton LW
- Subjects
- Alternative Splicing genetics, Cell Differentiation drug effects, Cell Line, Dopaminergic Neurons drug effects, Dopaminergic Neurons metabolism, Dopaminergic Neurons pathology, Genes, Mitochondrial, Humans, Induced Pluripotent Stem Cells drug effects, Induced Pluripotent Stem Cells metabolism, Mesencephalon pathology, Models, Biological, Nerve Degeneration pathology, Neural Stem Cells drug effects, Neural Stem Cells metabolism, Neurites drug effects, Neurites metabolism, Neurotoxins toxicity, Oxidative Stress drug effects, Parkinson Disease pathology, Phenotype, RNA Splicing Factors genetics, RNA Splicing Factors metabolism, Sequence Analysis, RNA, Transcriptome genetics, alpha-Synuclein metabolism, Genetic Heterogeneity, Nerve Degeneration genetics, Parkinson Disease genetics
- Abstract
The fact that Parkinson's disease (PD) can arise from numerous genetic mutations suggests a unifying molecular pathology underlying the various genetic backgrounds. To address this hypothesis, we took an integrated approach utilizing in vitro disease modeling and comprehensive transcriptome profiling to advance our understanding of PD progression and the concordant downstream signaling pathways across divergent genetic predispositions. To model PD in vitro, we generated neurons harboring disease-causing mutations from patient-specific, induced pluripotent stem cells (iPSCs). We observed signs of degeneration in midbrain dopaminergic neurons, reflecting the cardinal feature of PD. Gene expression signatures of PD neurons provided molecular insights into disease phenotypes observed in vitro, including oxidative stress vulnerability and altered neuronal activity. Notably, PD neurons show that elevated RBFOX1, a gene previously linked to neurodevelopmental diseases, underlies a pattern of alternative RNA-processing associated with PD-specific phenotypes., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
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33. A Role for RE-1-Silencing Transcription Factor in Embryonic Stem Cells Cardiac Lineage Specification.
- Author
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Aksoy I, Marcy G, Chen J, Divakar U, Kumar V, John-Sanchez D, Rahmani M, Buckley NJ, and Stanton LW
- Subjects
- Animals, Cell Lineage genetics, GATA4 Transcription Factor genetics, Gene Expression Regulation, Developmental, Mice, Mice, Knockout, Myocytes, Cardiac metabolism, Wnt Signaling Pathway, Cell Differentiation genetics, GATA4 Transcription Factor biosynthesis, Mouse Embryonic Stem Cells metabolism, Repressor Proteins genetics
- Abstract
During development, lineage specification is controlled by several signaling pathways involving various transcription factors (TFs). Here, we studied the RE-1-silencing transcription factor (REST) and identified an important role of this TF in cardiac differentiation. Using mouse embryonic stem cells (ESC) to model development, we found that REST knockout cells lost the ability to differentiate into the cardiac lineage. Detailed analysis of specific lineage markers expression showed selective downregulation of endoderm markers in REST-null cells, thus contributing to a loss of cardiogenic signals. REST regulates cardiac differentiation of ESCs by negatively regulating the Wnt/β-catenin signaling pathway and positively regulating the cardiogenic TF Gata4. We propose here a new role for REST in cell fate specification besides its well-known repressive role of neuronal differentiation., (© 2016 AlphaMed Press.)
- Published
- 2016
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34. MiR-375 is Essential for Human Spinal Motor Neuron Development and May Be Involved in Motor Neuron Degeneration.
- Author
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Bhinge A, Namboori SC, Bithell A, Soldati C, Buckley NJ, and Stanton LW
- Subjects
- Animals, Apoptosis genetics, Base Sequence, Humans, Mice, MicroRNAs metabolism, Molecular Sequence Data, Muscular Atrophy, Spinal genetics, Nerve Degeneration genetics, Neurogenesis genetics, Signal Transduction genetics, Tumor Suppressor Protein p53 metabolism, MicroRNAs genetics, Motor Neurons metabolism, Motor Neurons pathology, Nerve Degeneration pathology
- Abstract
The transcription factor REST is a key suppressor of neuronal genes in non-neuronal tissues. REST has been shown to suppress proneuronal microRNAs in neural progenitors indicating that REST-mediated neurogenic suppression may act in part via microRNAs. We used neural differentiation of Rest-null mouse ESC to identify dozens of microRNAs regulated by REST during neural development. One of the identified microRNAs, miR-375, was upregulated during human spinal motor neuron development. We found that miR-375 facilitates spinal motor neurogenesis by targeting the cyclin kinase CCND2 and the transcription factor PAX6. Additionally, miR-375 inhibits the tumor suppressor p53 and protects neurons from apoptosis in response to DNA damage. Interestingly, motor neurons derived from a spinal muscular atrophy patient displayed depressed miR-375 expression and elevated p53 protein levels. Importantly, SMA motor neurons were significantly more susceptible to DNA damage induced apoptosis suggesting that miR-375 may play a protective role in motor neurons., (© 2015 AlphaMed Press.)
- Published
- 2016
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35. Chromatin and RNA Maps Reveal Regulatory Long Noncoding RNAs in Mouse.
- Author
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Bogu GK, Vizán P, Stanton LW, Beato M, Di Croce L, and Marti-Renom MA
- Subjects
- Animals, Chromosome Mapping, Gene Expression Profiling, Promoter Regions, Genetic, Sequence Analysis, RNA, Transcriptome, Chromatin genetics, Mice genetics, RNA, Long Noncoding genetics
- Abstract
Discovering and classifying long noncoding RNAs (lncRNAs) across all mammalian tissues and cell lines remains a major challenge. Previously, mouse lncRNAs were identified using transcriptome sequencing (RNA-seq) data from a limited number of tissues or cell lines. Additionally, associating a few hundred lncRNA promoters with chromatin states in a single mouse cell line has identified two classes of chromatin-associated lncRNA. However, the discovery and classification of lncRNAs is still pending in many other tissues in mouse. To address this, we built a comprehensive catalog of lncRNAs by combining known lncRNAs with high-confidence novel lncRNAs identified by mapping and de novo assembling billions of RNA-seq reads from eight tissues and a primary cell line in mouse. Next, we integrated this catalog of lncRNAs with multiple genome-wide chromatin state maps and found two different classes of chromatin state-associated lncRNAs, including promoter-associated (plncRNAs) and enhancer-associated (elncRNAs) lncRNAs, across various tissues. Experimental knockdown of an elncRNA resulted in the downregulation of the neighboring protein-coding Kdm8 gene, encoding a histone demethylase. Our findings provide 2,803 novel lncRNAs and a comprehensive catalog of chromatin-associated lncRNAs across different tissues in mouse., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
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36. Global mapping of the regulatory interactions of histone residues.
- Author
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Jung I, Seo J, Lee HS, Stanton LW, Kim D, and Choi JK
- Subjects
- Acetylation, Acetyltransferases chemistry, Acetyltransferases genetics, Acetyltransferases metabolism, Databases, Nucleic Acid, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Histone-Lysine N-Methyltransferase chemistry, Histone-Lysine N-Methyltransferase genetics, Histone-Lysine N-Methyltransferase metabolism, Histones chemistry, Histones genetics, Jumonji Domain-Containing Histone Demethylases chemistry, Jumonji Domain-Containing Histone Demethylases genetics, Jumonji Domain-Containing Histone Demethylases metabolism, Lysine metabolism, Methylation, Methyltransferases chemistry, Methyltransferases genetics, Methyltransferases metabolism, Oligonucleotide Array Sequence Analysis, Point Mutation, Protein Interaction Mapping, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae growth & development, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins genetics, Chromatin Assembly and Disassembly, Histones metabolism, Nucleosomes metabolism, Protein Processing, Post-Translational, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Transcription, Genetic
- Abstract
Histone residues can serve as platforms for specific regulatory function. Here we constructed a map of regulatory associations between histone residues and a wide spectrum of chromatin regulation factors based on gene expression changes by histone point mutations in Saccharomyces cerevisiae. Detailed analyses of this map revealed novel associations. Regarding the modulation of H3K4 and K36 methylation by Set1, Set2, or Jhd2, we proposed a role for H4K91 acetylation in early Pol II elongation, and for H4K16 deacetylation in late elongation and crosstalk with H3K4 demethylation for gene silencing. The association of H3K56 with nucleosome positioning suggested that this lysine residue and its acetylation might contribute to nucleosome mobility for transcription activation. Further insights into chromatin regulation are expected from this approach., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2015
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37. RE1 silencing transcription factor/neuron-restrictive silencing factor regulates expansion of adult mouse subventricular zone-derived neural stem/progenitor cells in vitro.
- Author
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Soldati C, Caramanica P, Burney MJ, Toselli C, Bithell A, Augusti-Tocco G, Stanton LW, Biagioni S, Buckley NJ, and Cacci E
- Subjects
- Animals, Bone Morphogenetic Protein 6 physiology, Cell Differentiation physiology, Cell Proliferation physiology, Cells, Cultured, Humans, Male, Mice, Transcription Factors physiology, Adult Stem Cells physiology, Gene Silencing physiology, Lateral Ventricles cytology, Lateral Ventricles physiology, Neural Stem Cells physiology, Repressor Proteins physiology
- Abstract
Adult neural stem cell (aNSC) activity is tuned by external stimuli through the recruitment of transcription factors. This study examines the RE1 silencing transcription factor (REST) in neural stem/progenitor cells isolated from the subventricular zone of adult mouse brain and provides the first extensive characterization of REST-mediated control of the cellular and molecular properties. This study shows that REST knockdown affects the capacity of progenitor cells to generate neurospheres, reduces cell proliferation, and triggers cell differentiation despite the presence of growth factors. Genome- and transcriptome-wide analyses show that REST binding sites are significantly enriched in genes associated with synaptic transmission and nervous system development and function. Seeking candidate regulators of aNSC function, this study identifies a member of the bone morphogenetic protein (BMP) family, BMP6, the mRNA and protein of which increased after REST knockdown. The results of this study extend previous findings, demonstrating a reciprocal control of REST expression by BMPs. Administration of exogenous BMP6 inhibits aNSC proliferation and induces the expression of the astrocytic marker glial fibrillary acidic protein, highlighting its antimitogenic and prodifferentiative effects. This study suggests that BMP6 produced in a REST-regulated manner together with other signals can contribute to regulation of NSC maintenance and fate., (© 2015 Wiley Periodicals, Inc.)
- Published
- 2015
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38. Pluripotency Activity of Nanog Requires Biochemical Stabilization by Variant Histone Protein H2A.Z.
- Author
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Wang J, Qiao M, He Q, Shi R, Loh SJ, Stanton LW, and Wu M
- Subjects
- Animals, Cell Differentiation, Humans, Mice, Nanog Homeobox Protein, Pluripotent Stem Cells metabolism, Histones metabolism, Homeodomain Proteins metabolism
- Abstract
The variant histone protein H2A.Z plays a critical role in early development. Likewise, Nanog, a master regulator of embryonic stem cells (ESCs), is essential for proper development in early embryogenesis. In this study, we establish that these two factors work together to maintain pluripotency. It is shown that H2A.Z influences the protein level of Nanog through the ubiquitin-proteasome pathway. Knockdown of H2A.Z causes differentiation of mouse ESCs and disrupts the reprogramming of somatic cells, which can be partially rescued by overexpression of Nanog. We conclude that the H2A.Z-Nanog partnership is involved in ESC pluripotency and reprogramming of somatic cells. Stem Cells 2015;33:2126-2134., (© 2015 AlphaMed Press.)
- Published
- 2015
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39. Establishing criteria for human mesenchymal stem cell potency.
- Author
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Samsonraj RM, Rai B, Sathiyanathan P, Puan KJ, Rötzschke O, Hui JH, Raghunath M, Stanton LW, Nurcombe V, and Cool SM
- Subjects
- Animals, Cells, Cultured, Cytokines metabolism, Humans, Mice, Wound Healing physiology, Bone Marrow Cells cytology, Cell Differentiation physiology, Cell Proliferation physiology, Mesenchymal Stem Cells cytology
- Abstract
This study sought to identify critical determinants of mesenchymal stem cell (MSC) potency using in vitro and in vivo attributes of cells isolated from the bone marrow of age- and sex-matched donors. Adherence to plastic was not indicative of potency, yet capacity for long-term expansion in vitro varied considerably between donors, allowing the grouping of MSCs from the donors into either those with high-growth capacity or low-growth capacity. Using this grouping strategy, high-growth capacity MSCs were smaller in size, had greater colony-forming efficiency, and had longer telomeres. Cell-surface biomarker analysis revealed that the International Society for Cellular Therapy (ISCT) criteria did not distinguish between high-growth capacity and low-growth capacity MSCs, whereas STRO-1 and platelet-derived growth factor receptor alpha were preferentially expressed on high-growth capacity MSCs. These cells also had the highest mean expression of the mRNA transcripts TWIST-1 and DERMO-1. Irrespective of these differences, both groups of donor MSCs produced similar levels of key growth factors and cytokines involved in tissue regeneration and were capable of multilineage differentiation. However, high-growth capacity MSCs produced approximately double the volume of mineralized tissue compared to low-growth capacity MSCs when assessed for ectopic bone-forming ability. The additional phenotypic criteria presented in this study when combined with the existing ISCT minimum criteria and working proposal will permit an improved assessment of MSC potency and provide a basis for establishing the quality of MSCs prior to their therapeutic application., (© 2015 AlphaMed Press.)
- Published
- 2015
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40. Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency.
- Author
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Chen H, Aksoy I, Gonnot F, Osteil P, Aubry M, Hamela C, Rognard C, Hochard A, Voisin S, Fontaine E, Mure M, Afanassieff M, Cleroux E, Guibert S, Chen J, Vallot C, Acloque H, Genthon C, Donnadieu C, De Vos J, Sanlaville D, Guérin JF, Weber M, Stanton LW, Rougeulle C, Pain B, Bourillot PY, and Savatier P
- Subjects
- Animals, Embryonic Stem Cells cytology, Embryonic Stem Cells drug effects, Feeder Cells, Fibroblast Growth Factor 2 genetics, Fibroblast Growth Factor 2 metabolism, Fibroblasts cytology, Fibroblasts physiology, Humans, Leukemia Inhibitory Factor genetics, Leukemia Inhibitory Factor metabolism, Mice, Protein Array Analysis, STAT3 Transcription Factor genetics, Signal Transduction, Tamoxifen pharmacology, Embryonic Stem Cells physiology, Gene Expression Regulation physiology, Pluripotent Stem Cells physiology, STAT3 Transcription Factor metabolism
- Abstract
Leukemia inhibitory factor (LIF)/STAT3 signalling is a hallmark of naive pluripotency in rodent pluripotent stem cells (PSCs), whereas fibroblast growth factor (FGF)-2 and activin/nodal signalling is required to sustain self-renewal of human PSCs in a condition referred to as the primed state. It is unknown why LIF/STAT3 signalling alone fails to sustain pluripotency in human PSCs. Here we show that the forced expression of the hormone-dependent STAT3-ER (ER, ligand-binding domain of the human oestrogen receptor) in combination with 2i/LIF and tamoxifen allows human PSCs to escape from the primed state and enter a state characterized by the activation of STAT3 target genes and long-term self-renewal in FGF2- and feeder-free conditions. These cells acquire growth properties, a gene expression profile and an epigenetic landscape closer to those described in mouse naive PSCs. Together, these results show that temporarily increasing STAT3 activity is sufficient to reprogramme human PSCs to naive-like pluripotent cells.
- Published
- 2015
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41. PDX1 binds and represses hepatic genes to ensure robust pancreatic commitment in differentiating human embryonic stem cells.
- Author
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Teo AK, Tsuneyoshi N, Hoon S, Tan EK, Stanton LW, Wright CV, and Dunn NR
- Subjects
- Binding Sites, Biomarkers, Cell Line, Cluster Analysis, Computational Biology, Gene Expression Profiling, Humans, Liver metabolism, Nucleotide Motifs, Organ Specificity genetics, Organogenesis genetics, Position-Specific Scoring Matrices, Protein Binding, Response Elements, Transcription, Genetic, Cell Differentiation genetics, Gene Expression Regulation, Homeodomain Proteins metabolism, Human Embryonic Stem Cells cytology, Human Embryonic Stem Cells metabolism, Pancreas cytology, Pancreas metabolism, Trans-Activators metabolism
- Abstract
Inactivation of the Pancreatic and Duodenal Homeobox 1 (PDX1) gene causes pancreatic agenesis, which places PDX1 high atop the regulatory network controlling development of this indispensable organ. However, little is known about the identity of PDX1 transcriptional targets. We simulated pancreatic development by differentiating human embryonic stem cells (hESCs) into early pancreatic progenitors and subjected this cell population to PDX1 chromatin immunoprecipitation sequencing (ChIP-seq). We identified more than 350 genes bound by PDX1, whose expression was upregulated on day 17 of differentiation. This group included known PDX1 targets and many genes not previously linked to pancreatic development. ChIP-seq also revealed PDX1 occupancy at hepatic genes. We hypothesized that simultaneous PDX1-driven activation of pancreatic and repression of hepatic programs underlie early divergence between pancreas and liver. In HepG2 cells and differentiating hESCs, we found that PDX1 binds and suppresses expression of endogenous liver genes. These findings rebrand PDX1 as a context-dependent transcriptional repressor and activator within the same cell type., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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42. APP intracellular domain acts as a transcriptional regulator of miR-663 suppressing neuronal differentiation.
- Author
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Shu R, Wong W, Ma QH, Yang ZZ, Zhu H, Liu FJ, Wang P, Ma J, Yan S, Polo JM, Bernard CC, Stanton LW, Dawe GS, and Xiao ZC
- Subjects
- Amyloid beta-Protein Precursor genetics, Cell Differentiation physiology, Cell Line, Chromatin Immunoprecipitation, DNA-Binding Proteins metabolism, Gene Expression Regulation, Humans, MicroRNAs genetics, Protein Binding, Amyloid beta-Protein Precursor metabolism, Cell Differentiation genetics, MicroRNAs metabolism, Neurons cytology, Neurons metabolism
- Abstract
Amyloid precursor protein (APP) is best known for its involvement in the pathogenesis of Alzheimer's disease. We have previously demonstrated that APP intracellular domain (AICD) regulates neurogenesis; however, the mechanisms underlying AICD-mediated regulation of neuronal differentiation are not yet fully characterized. Using genome-wide chromatin immunoprecipitation approaches, we found that AICD is specifically recruited to the regulatory regions of several microRNA genes, and acts as a transcriptional regulator for miR-663, miR-3648 and miR-3687 in human neural stem cells. Functional assays show that AICD negatively modulates neuronal differentiation through miR-663, a primate-specific microRNA. Microarray data further demonstrate that miR-663 suppresses the expression of multiple genes implicated in neurogenesis, including FBXL18 and CDK6. Our results indicate that AICD has a novel role in suppression of neuronal differentiation via transcriptional regulation of miR-663 in human neural stem cells.
- Published
- 2015
- Full Text
- View/download PDF
43. NeuO: a fluorescent chemical probe for live neuron labeling.
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Er JC, Leong C, Teoh CL, Yuan Q, Merchant P, Dunn M, Sulzer D, Sames D, Bhinge A, Kim D, Kim SM, Yoon MH, Stanton LW, Je SH, Yun SW, and Chang YT
- Subjects
- Animals, Boron Compounds chemistry, Boron Compounds metabolism, Cells, Cultured, Fluorescent Dyes chemistry, Microscopy, Confocal, Microscopy, Video, Neurons cytology, Rats, Staining and Labeling, Fluorescent Dyes metabolism, Neurons metabolism
- Abstract
To address existing limitations in live neuron imaging, we have developed NeuO, a novel cell-permeable fluorescent probe with an unprecedented ability to label and image live neurons selectively over other cells in the brain. NeuO enables robust live neuron imaging and isolation in vivo and in vitro across species; its versatility and ease of use sets the basis for its development in a myriad of neuronal targeting applications., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2015
- Full Text
- View/download PDF
44. Pleiotropic functions for transcription factor zscan10.
- Author
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Kraus P, V S, Yu HB, Xing X, Lim SL, Adler T, Pimentel JA, Becker L, Bohla A, Garrett L, Hans W, Hölter SM, Janas E, Moreth K, Prehn C, Puk O, Rathkolb B, Rozman J, Adamski J, Bekeredjian R, Busch DH, Graw J, Klingenspor M, Klopstock T, Neff F, Ollert M, Stoeger T, Yildrim AÖ, Eickelberg O, Wolf E, Wurst W, Fuchs H, Gailus-Durner V, de Angelis MH, Lufkin T, and Stanton LW
- Subjects
- Animals, Behavior, Animal, Body Weight genetics, Bone Density genetics, Bone and Bones physiology, Codon, Initiator genetics, Eye growth & development, Female, Gene Expression Regulation genetics, Homozygote, Male, Mice, Mutation, Organ Size genetics, Pluripotent Stem Cells cytology, Pluripotent Stem Cells metabolism, Pregnancy, Weaning, Genetic Pleiotropy, Transcription Factors genetics
- Abstract
The transcription factor Zscan10 had been attributed a role as a pluripotency factor in embryonic stem cells based on its interaction with Oct4 and Sox2 in in vitro assays. Here we suggest a potential role of Zscan10 in controlling progenitor cell populations in vivo. Mice homozygous for a Zscan10 mutation exhibit reduced weight, mild hypoplasia in the spleen, heart and long bones and phenocopy an eye malformation previously described for Sox2 hypomorphs. Phenotypic abnormalities are supported by the nature of Zscan10 expression in midgestation embryos and adults suggesting a role for Zscan10 in either maintaining progenitor cell subpopulation or impacting on fate choice decisions thereof.
- Published
- 2014
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45. MiR-135b is a direct PAX6 target and specifies human neuroectoderm by inhibiting TGF-β/BMP signaling.
- Author
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Bhinge A, Poschmann J, Namboori SC, Tian X, Jia Hui Loh S, Traczyk A, Prabhakar S, and Stanton LW
- Subjects
- Binding Sites, Bone Morphogenetic Proteins genetics, Cell Differentiation, Cell Line, Embryonic Stem Cells cytology, Embryonic Stem Cells metabolism, Eye Proteins genetics, Gene Expression Profiling, Homeodomain Proteins genetics, Humans, MicroRNAs metabolism, Models, Molecular, Mutation, Neural Plate, PAX6 Transcription Factor, Paired Box Transcription Factors genetics, Repressor Proteins genetics, Sequence Analysis, DNA, Transcription Factors genetics, Transcription Factors metabolism, Transforming Growth Factor beta genetics, Bone Morphogenetic Proteins metabolism, Eye Proteins metabolism, Gene Expression Regulation, Developmental, Homeodomain Proteins metabolism, MicroRNAs genetics, Paired Box Transcription Factors metabolism, Repressor Proteins metabolism, Signal Transduction, Transforming Growth Factor beta metabolism
- Abstract
Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development, and recently, the TF PAX6 was shown to be critical for human NE specification. However, microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach, we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs, including hsa-miR-135b. MiR-135b was activated during NE development, and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-β and BMP signaling pathways, thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage., (© 2014 The Authors.)
- Published
- 2014
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- View/download PDF
46. Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in embryonic stem cells.
- Author
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Aksoy I, Giudice V, Delahaye E, Wianny F, Aubry M, Mure M, Chen J, Jauch R, Bogu GK, Nolden T, Himmelbauer H, Xavier Doss M, Sachinidis A, Schulz H, Hummel O, Martinelli P, Hübner N, Stanton LW, Real FX, Bourillot PY, and Savatier P
- Subjects
- Animals, Blotting, Western, Chromatin Immunoprecipitation, Flow Cytometry, Gene Knockdown Techniques, High-Throughput Nucleotide Sequencing, Kruppel-Like Factor 4, Kruppel-Like Transcription Factors genetics, Mice, Microarray Analysis, Real-Time Polymerase Chain Reaction, Cell Differentiation physiology, Embryonic Stem Cells physiology, Endoderm embryology, Gene Expression Regulation, Developmental physiology, Kruppel-Like Transcription Factors metabolism, Mesoderm embryology
- Abstract
Krüppel-like factors (Klf) 4 and 5 are two closely related members of the Klf family, known to play key roles in cell cycle regulation, somatic cell reprogramming and pluripotency. Here we focus on the functional divergence between Klf4 and Klf5 in the inhibition of mouse embryonic stem (ES) cell differentiation. Using microarrays and chromatin immunoprecipitation coupled to ultra-high-throughput DNA sequencing, we show that Klf4 negatively regulates the expression of endodermal markers in the undifferentiated ES cells, including transcription factors involved in the commitment of pluripotent stem cells to endoderm differentiation. Knockdown of Klf4 enhances differentiation towards visceral and definitive endoderm. In contrast, Klf5 negatively regulates the expression of mesodermal markers, some of which control commitment to the mesoderm lineage, and knockdown of Klf5 specifically enhances differentiation towards mesoderm. We conclude that Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in murine ES cells.
- Published
- 2014
- Full Text
- View/download PDF
47. Transcription factor-induced lineage programming of noradrenaline and motor neurons from embryonic stem cells.
- Author
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Mong J, Panman L, Alekseenko Z, Kee N, Stanton LW, Ericson J, and Perlmann T
- Subjects
- Adrenergic Neurons metabolism, Animals, Cell Line, Embryonic Stem Cells metabolism, Gene Expression Regulation, Genetic Engineering, Genome genetics, Homeodomain Proteins metabolism, Humans, Mice, Motor Neurons metabolism, Signal Transduction, Adrenergic Neurons cytology, Cell Lineage, Embryonic Stem Cells cytology, Motor Neurons cytology, Transcription Factors metabolism
- Abstract
An important goal in stem cell biology is to develop methods for efficient generation of clinically interesting cell types from relevant stem cell populations. This is particularly challenging for different types of neurons of the central nervous system where hundreds of distinct neuronal cell types are generated during embryonic development. We previously used a strategy based on forced transcription factor expression in embryonic stem cell-derived neural progenitors to generate specific types of neurons, including dopamine and serotonin neurons. Here, we extend these studies and show that noradrenergic neurons can also be generated from pluripotent embryonic stem cells by forced expression of the homeobox transcription factor Phox2b under the signaling influence of fibroblast growth factor 8 (FGF8) and bone morphogenetic proteins. In neural progenitors exposed to FGF8 and sonic hedgehog both Phox2b and the related Phox2a instead promoted the generation of neurons with the characteristics of mid- and hindbrain motor neurons. The efficient generation of these neuron types enabled a comprehensive genome-wide gene expression analysis that provided further validation of the identity of generated cells. Moreover, we also demonstrate that the generated cell types are amenable to drug testing in vitro and we show that variants of the differentiation protocols can be applied to cultures of human pluripotent stem cells for the generation of human noradrenergic and visceral motor neurons. Thus, these studies provide a basis for characterization of yet an additional highly clinically relevant neuronal cell type., (© 2013 AlphaMed Press.)
- Published
- 2014
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- View/download PDF
48. Sox transcription factors require selective interactions with Oct4 and specific transactivation functions to mediate reprogramming.
- Author
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Aksoy I, Jauch R, Eras V, Chng WB, Chen J, Divakar U, Ng CK, Kolatkar PR, and Stanton LW
- Subjects
- Alkaline Phosphatase metabolism, Animals, Cell Culture Techniques, Cellular Reprogramming genetics, Cellular Reprogramming physiology, HMGB Proteins genetics, HMGB Proteins metabolism, Humans, Induced Pluripotent Stem Cells metabolism, Induced Pluripotent Stem Cells transplantation, Mice, Mice, SCID, Octamer Transcription Factor-3 genetics, Point Mutation, SOXF Transcription Factors genetics, Transcriptional Activation, beta Catenin metabolism, Induced Pluripotent Stem Cells physiology, Octamer Transcription Factor-3 metabolism, SOXF Transcription Factors metabolism
- Abstract
The unique ability of Sox2 to cooperate with Oct4 at selective binding sites in the genome is critical for reprogramming somatic cells into induced pluripotent stem cells (iPSCs). We have recently demonstrated that Sox17 can be converted into a reprogramming factor by alteration of a single amino acid (Sox17EK) within its DNA binding HMG domain. Here we expanded this study by introducing analogous mutations to 10 other Sox proteins and interrogated the role of N-and C-termini on the reprogramming efficiency. We found that point-mutated Sox7 and Sox17 can convert human and mouse fibroblasts into iPSCs, but Sox4, Sox5, Sox6, Sox8, Sox9, Sox11, Sox12, Sox13, and Sox18 cannot. Next we studied regions outside the HMG domain and found that the C-terminal transactivation domain of Sox17 and Sox7 enhances the potency of Sox2 in iPSC assays and confers weak reprogramming potential to the otherwise inactive Sox4EK and Sox18EK proteins. These results suggest that the glutamate (E) to lysine (K) mutation in the HMG domain is necessary but insufficient to swap the function of Sox factors. Moreover, the HMG domain alone fused to the VP16 transactivation domain is able to induce reprogramming, albeit at low efficiency. By molecular dissection of the C-terminus of Sox17, we found that the β-catenin interaction region contributes to the enhanced reprogramming efficiency of Sox17EK. To mechanistically understand the enhanced reprogramming potential of Sox17EK, we analyzed ChIP-sequencing and expression data and identified a subset of candidate genes specifically regulated by Sox17EK and not by Sox2., (© AlphaMed Press.)
- Published
- 2013
- Full Text
- View/download PDF
49. Genome wide analysis reveals Zic3 interaction with distal regulatory elements of stage specific developmental genes in zebrafish.
- Author
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Winata CL, Kondrychyn I, Kumar V, Srinivasan KG, Orlov Y, Ravishankar A, Prabhakar S, Stanton LW, Korzh V, and Mathavan S
- Subjects
- Animals, Binding Sites, Gene Expression Regulation, Developmental, Genomics, Homeodomain Proteins metabolism, Transcription Factors metabolism, Wnt Signaling Pathway genetics, Zebrafish genetics, Zebrafish growth & development, Zebrafish Proteins metabolism, Body Patterning genetics, Homeodomain Proteins genetics, Regulatory Elements, Transcriptional genetics, Regulatory Sequences, Nucleic Acid genetics, Transcription Factors genetics, Zebrafish Proteins genetics
- Abstract
Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2013
- Full Text
- View/download PDF
50. An epigenetic signature of developmental potential in neural stem cells and early neurons.
- Author
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Burney MJ, Johnston C, Wong KY, Teng SW, Beglopoulos V, Stanton LW, Williams BP, Bithell A, and Buckley NJ
- Subjects
- Animals, Base Sequence, Cell Differentiation genetics, Cell Lineage genetics, Flow Cytometry, Gene Expression Regulation, Developmental, Histones metabolism, Mice, Molecular Sequence Data, Neural Stem Cells cytology, Neurogenesis genetics, Neurons cytology, Promoter Regions, Genetic, Protein Processing, Post-Translational genetics, Transcription Factors metabolism, Transcriptome genetics, Epigenesis, Genetic, Neural Stem Cells metabolism, Neurons metabolism
- Abstract
A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur before any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons., (© AlphaMed Press.)
- Published
- 2013
- Full Text
- View/download PDF
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