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1
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Hosseini K, Philippot G, Salomonsson SB, Cediel-Ulloa A, Gholizadeh E, Fredriksson R. Transcriptomic characterization of maturing neurons from human neural stem cells across developmental time points. IBRO Neurosci Rep 2025; 18:679-689. [PMID: 40336753 PMCID: PMC12056963 DOI: 10.1016/j.ibneur.2025.04.013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2024] [Revised: 04/16/2025] [Accepted: 04/17/2025] [Indexed: 05/09/2025] Open
Abstract
Neurodevelopmental studies employing animal models encounter challenges due to interspecies differences and ethical concerns. Maturing neurons of human origin, undergoing several developmental stages, present a powerful alternative. In this study, human embryonic stem cell (H9 cell line) was differentiated into neural stem cells and subsequently matured into neurons over 30 days. Ion AmpliSeq™ was used for transcriptomic characterization of human stem cell-derived neurons at multiple time points. Data analysis revealed a progressive increase of markers associated with neuronal development and astrocyte markers, indicating the establishment of a co-culture accommodating both glial and neurons. Transcriptomic and pathway enrichment analysis also revealed a more pronounced GABAergic phenotype in the neurons, signifying their specialization toward this cell type. The findings confirm the robustness of these cells across different passages and demonstrate detailed progression through stages of development. The model is intended for neurodevelopmental applications and can be adapted to investigate how genetic modifications or exposure to chemicals, pharmaceuticals, and other environmental factors influence neurons and glial maturation.
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Affiliation(s)
- Kimia Hosseini
- Department of Pharmaceutical Bioscience, Uppsala University, Sweden
| | - Gaëtan Philippot
- Department of Pharmaceutical Bioscience, Uppsala University, Sweden
| | | | | | - Elnaz Gholizadeh
- Department of Pharmaceutical Bioscience, Uppsala University, Sweden
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2
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Verma I, Seshagiri PB. Current Applications of Human Pluripotent Stem Cells in Neuroscience Research and Cell Transplantation Therapy for Neurological Disorders. Stem Cell Rev Rep 2025; 21:964-987. [PMID: 40186708 DOI: 10.1007/s12015-025-10851-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/05/2025] [Indexed: 04/07/2025]
Abstract
Many neurological diseases involving tissue damage cannot be treated with drug-based approaches, and the inaccessibility of human brain samples further hampers the study of these diseases. Human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an excellent model for studying neural development and function. PSCs can be differentiated into various neural cell types, providing a renewal source of functional human brain cells. Therefore, PSC-derived neural cells are increasingly used for multiple applications, including neurodevelopmental and neurotoxicological studies, neurological disease modeling, drug screening, and regenerative medicine. In addition, the neural cells generated from patient iPSCs can be used to study patient-specific disease signatures and progression. With the recent advances in genome editing technologies, it is possible to remove the disease-related mutations in the patient iPSCs to generate corrected iPSCs. The corrected iPSCs can differentiate into neural cells with normal physiological functions, which can be used for autologous transplantation. This review highlights the current progress in using PSCs to understand the fundamental principles of human neurodevelopment and dissect the molecular mechanisms of neurological diseases. This knowledge can be applied to develop better drugs and explore cell therapy options. We also discuss the basic requirements for developing cell transplantation therapies for neurological disorders and the current status of the ongoing clinical trials.
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Affiliation(s)
- Isha Verma
- Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India.
- Department of Neurology, University of Michigan, Ann Arbor, 48109, USA.
| | - Polani B Seshagiri
- Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India
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3
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Rasool D, Jahani-Asl A. Master regulators of neurogenesis: the dynamic roles of Ephrin receptors across diverse cellular niches. Transl Psychiatry 2024; 14:462. [PMID: 39505843 PMCID: PMC11541728 DOI: 10.1038/s41398-024-03168-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/11/2024] [Revised: 09/20/2024] [Accepted: 10/16/2024] [Indexed: 11/08/2024] Open
Abstract
The ephrin receptors (EphRs) are the largest family of receptor tyrosine kinases (RTKs) that are abundantly expressed in the developing brain and play important roles at different stages of neurogenesis ranging from neural stem cell (NSC) fate specification to neural migration, morphogenesis, and circuit assembly. Defects in EphR signalling have been associated with several pathologies including neurodevelopmental disorders (NDDs), intellectual disability (ID), and neurodegenerative diseases (NDs). Here, we review our current understanding of the complex and dynamic role of EphRs in the brain and discuss how deregulation of these receptors contributes to disease, highlighting their potential as valuable druggable targets.
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Affiliation(s)
- Dilan Rasool
- Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada
- University of Ottawa Brain and Mind Research Institute, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada
- Department of Medicine, Division of Experimental Medicine, McGill University, 1001 Decarie Boulevard, Montreal, QC, H4A 3J1, Canada
- Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Chemin de la Côte-Sainte-Catherine, Montréal, QC, H3T 1E2, Canada
| | - Arezu Jahani-Asl
- Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada.
- University of Ottawa Brain and Mind Research Institute, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada.
- Department of Medicine, Division of Experimental Medicine, McGill University, 1001 Decarie Boulevard, Montreal, QC, H4A 3J1, Canada.
- Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Chemin de la Côte-Sainte-Catherine, Montréal, QC, H3T 1E2, Canada.
- Gerald Bronfman Department of Oncology, McGill University, 5100 de Maisonneuve Blvd. West, Montréal, QC, H4A 3T2, Canada.
- Regenerative Medicine Program, and Cancer Therapeutics Program, Ottawa Hospital Research Institute, Ottawa, ON, K1H 8L6, Canada.
- Ottawa Institutes of System Biology, University of Ottawa, Health Sciences Campus, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada.
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4
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Bustos F, Findlay GM. Therapeutic validation and targeting of signalling networks that are dysregulated in intellectual disability. FEBS J 2023; 290:1454-1460. [PMID: 35212144 PMCID: PMC10952735 DOI: 10.1111/febs.16411] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2021] [Revised: 01/14/2022] [Accepted: 02/22/2022] [Indexed: 11/28/2022]
Abstract
Intellectual disability (ID) represents a major burden on healthcare systems in the developed world. However, there is a disconnect between our knowledge of genes that are mutated in ID and our understanding of the underpinning molecular mechanisms that cause these disorders. We argue that elucidating the signalling and transcriptional networks that are dysregulated in patients will afford new therapeutic opportunities.
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Affiliation(s)
- Francisco Bustos
- Pediatrics and Rare Diseases GroupSanford ResearchSioux FallsSDUSA
- Department of PediatricsSanford School of MedicineUniversity of South DakotaSioux FallsSDUSA
| | - Greg M. Findlay
- The MRC Protein Phosphorylation & Ubiquitylation UnitSchool of Life SciencesThe University of DundeeDundeeUK
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5
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Florentino RM, Li Q, Coard MC, Haep N, Motomura T, Diaz-Aragon R, Faccioli LAP, Amirneni S, Kocas-Kilicarslan ZN, Ostrowska A, Squires JE, Feranchak AP, Soto-Gutierrez A. Transmembrane channel activity in human hepatocytes and cholangiocytes derived from induced pluripotent stem cells. Hepatol Commun 2022; 6:1561-1573. [PMID: 35289126 PMCID: PMC9234678 DOI: 10.1002/hep4.1920] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/09/2021] [Revised: 01/06/2022] [Accepted: 01/22/2022] [Indexed: 11/10/2022] Open
Abstract
The initial creation of human-induced pluripotent stem cells (iPSCs) set the foundation for the future of regenerative medicine. Human iPSCs can be differentiated into a variety of cell types in order to study normal and pathological molecular mechanisms. Currently, there are well-defined protocols for the differentiation, characterization, and establishment of functionality in human iPSC-derived hepatocytes (iHep) and iPSC-derived cholangiocytes (iCho). Electrophysiological study on chloride ion efflux channel activity in iHep and iCho cells has not been previously reported. We generated iHep and iCho cells and characterized them based on hepatocyte-specific and cholangiocyte-specific markers. The relevant transmembrane channels were selected: cystic fibrosis transmembrane conductance regulator, leucine rich repeat-containing 8 subunit A, and transmembrane member 16 subunit A. To measure the activity in these channels, we used whole-cell patch-clamp techniques with a standard intracellular and extracellular solution. Our iHep and iCho cells demonstrated definitive activity in the selected transmembrane channels, and this approach may become an important tool for investigating human liver biology of cholestatic diseases.
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Affiliation(s)
- Rodrigo M Florentino
- Department of PathologyUniversity of Pittsburgh School of MedicinePittsburghPennsylvaniaUSA.,Pittsburgh Liver Research CenterUniversity of PittsburghPittsburghPennsylvaniaUSA
| | - Qin Li
- Department of PediatricsUniversity of Pittsburgh Medical CenterPittsburghPennsylvaniaUSA
| | - Michael C Coard
- Department of PathologyUniversity of Pittsburgh School of MedicinePittsburghPennsylvaniaUSA
| | - Nils Haep
- Department of PathologyUniversity of Pittsburgh School of MedicinePittsburghPennsylvaniaUSA
| | - Takashi Motomura
- Department of PathologyUniversity of Pittsburgh School of MedicinePittsburghPennsylvaniaUSA
| | - Ricardo Diaz-Aragon
- Department of PathologyUniversity of Pittsburgh School of MedicinePittsburghPennsylvaniaUSA
| | - Lanuza A P Faccioli
- Department of PathologyUniversity of Pittsburgh School of MedicinePittsburghPennsylvaniaUSA
| | - Sriram Amirneni
- Department of PathologyUniversity of Pittsburgh School of MedicinePittsburghPennsylvaniaUSA
| | | | - Alina Ostrowska
- Department of PathologyUniversity of Pittsburgh School of MedicinePittsburghPennsylvaniaUSA.,Pittsburgh Liver Research CenterUniversity of PittsburghPittsburghPennsylvaniaUSA
| | - James E Squires
- Pittsburgh Liver Research CenterUniversity of PittsburghPittsburghPennsylvaniaUSA.,Division of Gastroenterology, Hepatology, and NutritionUniversity of Pittsburgh Medical CenterPittsburghPennsylvaniaUSA
| | - Andrew P Feranchak
- Pittsburgh Liver Research CenterUniversity of PittsburghPittsburghPennsylvaniaUSA.,Department of PediatricsUniversity of Pittsburgh Medical CenterPittsburghPennsylvaniaUSA
| | - Alejandro Soto-Gutierrez
- Department of PathologyUniversity of Pittsburgh School of MedicinePittsburghPennsylvaniaUSA.,Pittsburgh Liver Research CenterUniversity of PittsburghPittsburghPennsylvaniaUSA.,McGowan Institute for Regenerative MedicinePittsburghPennsylvaniaUSA
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6
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Reed X, Cobb MM, Skinbinski G, Roosen D, Kaganovich A, Ding J, Finkbeiner S, Cookson MR. Transcriptional signatures in iPSC-derived neurons are reproducible across labs when differentiation protocols are closely matched. Stem Cell Res 2021; 56:102558. [PMID: 34626895 PMCID: PMC8655646 DOI: 10.1016/j.scr.2021.102558] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/12/2021] [Revised: 09/16/2021] [Accepted: 09/29/2021] [Indexed: 11/29/2022] Open
Abstract
Reproducibility of expression patterns in iPSC-derived cells from different labs is an important first step in ensuring replication of biochemical or functional assays that are performed in different labs. Here we show that reproducible gene expression patterns from iPSCs and iPSC-derived neurons matured and collected at two separate laboratory locations can be achieved by closely matching protocols and reagents. While there are significant differences in gene expression between iPSCs and differentiated neurons, as well as between different donor lines of the same cell type, transcriptional changes that vary with laboratory sites are relatively small. These results suggest that making great efforts to match protocols, reagents and technical methods between labs may improve the reproducibility of iPSC-derived cell models.
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Affiliation(s)
- Xylena Reed
- Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA
| | - Melanie M Cobb
- Center for Systems and Therapeutics & Taube/Koret Center for Neurodegenerative Disease, Gladstone Institutes, San Francisco, CA, 94158, USA
| | - Gaia Skinbinski
- Center for Systems and Therapeutics & Taube/Koret Center for Neurodegenerative Disease, Gladstone Institutes, San Francisco, CA, 94158, USA
| | - Dorien Roosen
- Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA
| | - Alice Kaganovich
- Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA
| | - Jinhui Ding
- Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA
| | - Steve Finkbeiner
- Center for Systems and Therapeutics & Taube/Koret Center for Neurodegenerative Disease, Gladstone Institutes, San Francisco, CA, 94158, USA; Departments of Neurology and Physiology, University of California San Francisco, San Francisco, CA 94158, USA
| | - Mark R Cookson
- Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA
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7
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Peteri UK, Pitkonen J, de Toma I, Nieminen O, Utami KH, Strandin TM, Corcoran P, Roybon L, Vaheri A, Ethell I, Casarotto P, Pouladi MA, Castrén ML. Urokinase plasminogen activator mediates changes in human astrocytes modeling fragile X syndrome. Glia 2021; 69:2947-2962. [PMID: 34427356 DOI: 10.1002/glia.24080] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2021] [Revised: 08/13/2021] [Accepted: 08/16/2021] [Indexed: 12/25/2022]
Abstract
The function of astrocytes intertwines with the extracellular matrix, whose neuron and glial cell-derived components shape neuronal plasticity. Astrocyte abnormalities have been reported in the brain of the mouse model for fragile X syndrome (FXS), the most common cause of inherited intellectual disability, and a monogenic cause of autism spectrum disorder. We compared human FXS and control astrocytes generated from human induced pluripotent stem cells and we found increased expression of urokinase plasminogen activator (uPA), which modulates degradation of extracellular matrix. Several pathways associated with uPA and its receptor function were activated in FXS astrocytes. Levels of uPA were also increased in conditioned medium collected from FXS hiPSC-derived astrocyte cultures and correlated inversely with intracellular Ca2+ responses to activation of L-type voltage-gated calcium channels in human astrocytes. Increased uPA augmented neuronal phosphorylation of TrkB within the docking site for the phospholipase-Cγ1 (PLCγ1), indicating effects of uPA on neuronal plasticity. Gene expression changes during neuronal differentiation preceding astrogenesis likely contributed to properties of astrocytes with FXS-specific alterations that showed specificity by not affecting differentiation of adenosine triphosphate (ATP)-responsive astrocyte population. To conclude, our studies identified uPA as an important regulator of astrocyte function and demonstrated that increased uPA in human FXS astrocytes modulated astrocytic responses and neuronal plasticity.
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Affiliation(s)
- Ulla-Kaisa Peteri
- Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Juho Pitkonen
- Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Ilario de Toma
- Systems Neurobiology Laboratory, Barcelona Institute of Science and Technology, Barcelona, Spain
| | - Otso Nieminen
- Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Kagistia Hana Utami
- Department of Physiology, National University of Singapore (NUS), Singapore, Singapore
| | - Tomas M Strandin
- Department of Virology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Padraic Corcoran
- Array and Analysis Facility, Department of Medical Sciences, Uppsala University, Uppsala, Sweden
| | - Laurent Roybon
- iPSC Laboratory for CNS Disease Modeling, Department of Experimental Medical Science, BMC D10, and MultiPark and the Lund Stem Cell Center, Lund University, Lund, Sweden
| | - Antti Vaheri
- Department of Virology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Iryna Ethell
- Biomedical Sciences, University of California Riverside School of Medicine, Riverside, California, USA
| | | | - Mahmoud A Pouladi
- Department of Physiology, National University of Singapore (NUS), Singapore, Singapore.,British Columbia Children's Hospital Research Institute, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada
| | - Maija L Castrén
- Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
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8
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Yao H, Wu W, Cerf I, Zhao HW, Wang J, Negraes PD, Muotri AR, Haddad GG. Methadone interrupts neural growth and function in human cortical organoids. Stem Cell Res 2020; 49:102065. [PMID: 33137567 DOI: 10.1016/j.scr.2020.102065] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/29/2020] [Revised: 09/29/2020] [Accepted: 10/20/2020] [Indexed: 12/16/2022] Open
Abstract
Prenatal opioids exposure can lead to both neonatal abstinence syndrome in newborns and neurological deficits later in life. Although opioids have been well studied in general, the cellular and molecular mechanisms by which opioids affect human fetal brain development has not been well understood. In this work, we have taken advantage of a human 3D-brain cortical organoid (hCO) that facilitated enormously the investigation of early human brain development. Using imaging, immunofluorescence, multi-electrode array (MEA) and patch clamp recording techniques, we have investigated the effect of methadone, a frequently used opioid during pregnancy, on early neural development, including neuronal growth, neural network activity and synaptic transmission in hCOs. Our results demonstrated that methadone dose-dependently halted the growth of hCOs and induced organoid disintegration after a prolonged exposure. In addition, methadone dose-dependently suppressed the firing of spontaneous action potentials in hCOs and this suppression could be reversed upon methadone withdrawal in hCOs treated with lower dosages. Further investigation using patch clamp whole cell configuration revealed that, at clinically relevant concentrations, methadone decreased the frequency and amplitude of excitatory postsynaptic currents in neurons, indicating a critical role of methadone in weakening synaptic transmission in neural networks in hCOs. In addition, methadone significantly attenuated the voltage-dependent Na+ current in hCOs. We conclude that methadone interrupts neural growth and function in early brain development.
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Affiliation(s)
- Hang Yao
- Departments of Pediatrics, University of California San Diego, La Jolla, CA 92093, United States
| | - Wei Wu
- Departments of Pediatrics, University of California San Diego, La Jolla, CA 92093, United States
| | - Ines Cerf
- Departments of Pediatrics, University of California San Diego, La Jolla, CA 92093, United States
| | - Helen W Zhao
- Departments of Pediatrics, University of California San Diego, La Jolla, CA 92093, United States
| | - Juan Wang
- Departments of Pediatrics, University of California San Diego, La Jolla, CA 92093, United States
| | - Priscilla D Negraes
- Department of Cellular & Molecular Medicine, Stem Cell Program, La Jolla, CA 92093, United States
| | - Alysson R Muotri
- Departments of Pediatrics, University of California San Diego, La Jolla, CA 92093, United States; Department of Cellular & Molecular Medicine, Stem Cell Program, La Jolla, CA 92093, United States
| | - Gabriel G Haddad
- Departments of Pediatrics, University of California San Diego, La Jolla, CA 92093, United States; Department of Neurosciences, University of California San Diego, La Jolla, CA 92093, United States; Rady Children's Hospital San Diego, CA 92123, United States.
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9
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An efficient neuron-astrocyte differentiation protocol from human embryonic stem cell-derived neural progenitors to assess chemical-induced developmental neurotoxicity. Reprod Toxicol 2020; 98:107-116. [PMID: 32931842 DOI: 10.1016/j.reprotox.2020.09.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2020] [Revised: 08/13/2020] [Accepted: 09/07/2020] [Indexed: 01/10/2023]
Abstract
Human embryonic stem cell neuronal differentiation models provide promising in vitro tools for the prediction of developmental neurotoxicity of chemicals. Such models mimic essential elements of human relevant neuronal development, including the differentiation of a variety of brain cell types and their neuronal network formation as evidenced by specific gene and protein biomarkers. However, the reproducibility and lengthy culture duration of cell models present drawbacks and delay regulatory implementation. Here we present a relatively short and robust protocol to differentiate H9-derived neural progenitor cells (NPCs) into a neuron-astrocyte co-culture. When frozen-stored NPCs were re-cultured and induced into neuron-astrocyte differentiation, they showed gene- and protein expression typical for these cells, and most notably they exhibited spontaneous electrical activity within three days of culture as measured by a multi-well micro-electrode array. Modulating the ratio of astrocytes and neurons through different growth factors including glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNTF) did not compromise the ability to develop spontaneous electrical activity. This robust neuronal differentiation model may serve as a functional component of a testing strategy for unravelling mechanisms of developmental neurotoxicity.
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10
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Das Sharma S, Pal R, Reddy BK, Selvaraj BT, Raj N, Samaga KK, Srinivasan DJ, Ornelas L, Sareen D, Livesey MR, Bassell GJ, Svendsen CN, Kind PC, Chandran S, Chattarji S, Wyllie DJA. Cortical neurons derived from human pluripotent stem cells lacking FMRP display altered spontaneous firing patterns. Mol Autism 2020; 11:52. [PMID: 32560741 PMCID: PMC7304215 DOI: 10.1186/s13229-020-00351-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2019] [Accepted: 05/11/2020] [Indexed: 12/21/2022] Open
Abstract
BACKGROUND Fragile X syndrome (FXS), a neurodevelopmental disorder, is a leading monogenetic cause of intellectual disability and autism spectrum disorder. Notwithstanding the extensive studies using rodent and other pre-clinical models of FXS, which have provided detailed mechanistic insights into the pathophysiology of this disorder, it is only relatively recently that human stem cell-derived neurons have been employed as a model system to further our understanding of the pathophysiological events that may underlie FXS. Our study assesses the physiological properties of human pluripotent stem cell-derived cortical neurons lacking fragile X mental retardation protein (FMRP). METHODS Electrophysiological whole-cell voltage- and current-clamp recordings were performed on two control and three FXS patient lines of human cortical neurons derived from induced pluripotent stem cells. In addition, we also describe the properties of an isogenic pair of lines in one of which FMR1 gene expression has been silenced. RESULTS Neurons lacking FMRP displayed bursts of spontaneous action potential firing that were more frequent but shorter in duration compared to those recorded from neurons expressing FMRP. Inhibition of large conductance Ca2+-activated K+ currents and the persistent Na+ current in control neurons phenocopies action potential bursting observed in neurons lacking FMRP, while in neurons lacking FMRP pharmacological potentiation of voltage-dependent Na+ channels phenocopies action potential bursting observed in control neurons. Notwithstanding the changes in spontaneous action potential firing, we did not observe any differences in the intrinsic properties of neurons in any of the lines examined. Moreover, we did not detect any differences in the properties of miniature excitatory postsynaptic currents in any of the lines. CONCLUSIONS Pharmacological manipulations can alter the action potential burst profiles in both control and FMRP-null human cortical neurons, making them appear like their genetic counterpart. Our studies indicate that FMRP targets that have been found in rodent models of FXS are also potential targets in a human-based model system, and we suggest potential mechanisms by which activity is altered.
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Affiliation(s)
- Shreya Das Sharma
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India.,The University of Trans-Displinary Health Sciences and Technology, Bangalore, 560064, India
| | - Rakhi Pal
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India
| | - Bharath Kumar Reddy
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India
| | - Bhuvaneish T Selvaraj
- Centre for Clinical Brain Sciences, University of Edinburgh, Chancellor's Building, Edinburgh, EH16 4SB, UK.,UK Dementia Research Institute at the University of Edinburgh, Edinburgh Medical School, Chancellor's Building, Edinburgh, EH16 4SB, UK
| | - Nisha Raj
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, 30322, USA
| | - Krishna Kumar Samaga
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India
| | - Durga J Srinivasan
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India.,The University of Trans-Displinary Health Sciences and Technology, Bangalore, 560064, India
| | - Loren Ornelas
- The Board of Governors Regenerative Medicine Institute and Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.,iPSC Core, The David Janet Polak Foundation Stem Cell Core Laboratory, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.,Cedars-Sinai Biomanufacturing Center, West Hollywood, CA, 90069, USA
| | - Dhruv Sareen
- The Board of Governors Regenerative Medicine Institute and Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.,iPSC Core, The David Janet Polak Foundation Stem Cell Core Laboratory, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.,Cedars-Sinai Biomanufacturing Center, West Hollywood, CA, 90069, USA.,Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA
| | - Matthew R Livesey
- Centre for Discovery Brain Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK
| | - Gary J Bassell
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, 30322, USA
| | - Clive N Svendsen
- The Board of Governors Regenerative Medicine Institute and Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.,Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA
| | - Peter C Kind
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India.,Centre for Discovery Brain Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK.,Patrick Wild Centre, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK.,Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK
| | - Siddharthan Chandran
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India.,Centre for Clinical Brain Sciences, University of Edinburgh, Chancellor's Building, Edinburgh, EH16 4SB, UK.,UK Dementia Research Institute at the University of Edinburgh, Edinburgh Medical School, Chancellor's Building, Edinburgh, EH16 4SB, UK.,Patrick Wild Centre, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK.,Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK
| | - Sumantra Chattarji
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India. .,Centre for Discovery Brain Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK. .,Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK. .,National Centre for Biological Sciences, Tata Institute for Fundamental Research, Bangalore, 560065, India.
| | - David J A Wyllie
- Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, 560065, India. .,Centre for Discovery Brain Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK. .,Patrick Wild Centre, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK. .,Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, Edinburgh, EH8 9XD, UK.
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11
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Bieder A, Yoshihara M, Katayama S, Krjutškov K, Falk A, Kere J, Tapia-Páez I. Dyslexia Candidate Gene and Ciliary Gene Expression Dynamics During Human Neuronal Differentiation. Mol Neurobiol 2020; 57:2944-2958. [PMID: 32445086 PMCID: PMC7320047 DOI: 10.1007/s12035-020-01905-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2019] [Accepted: 03/19/2020] [Indexed: 11/30/2022]
Abstract
Developmental dyslexia (DD) is a neurodevelopmental condition with complex genetic mechanisms. A number of candidate genes have been identified, some of which are linked to neuronal development and migration and to ciliary functions. However, expression and regulation of these genes in human brain development and neuronal differentiation remain uncharted. Here, we used human long-term self-renewing neuroepithelial stem (lt-NES, here termed NES) cells derived from human induced pluripotent stem cells to study neuronal differentiation in vitro. We characterized gene expression changes during differentiation by using RNA sequencing and validated dynamics for selected genes by qRT-PCR. Interestingly, we found that genes related to cilia were significantly enriched among upregulated genes during differentiation, including genes linked to ciliopathies with neurodevelopmental phenotypes. We confirmed the presence of primary cilia throughout neuronal differentiation. Focusing on dyslexia candidate genes, 33 out of 50 DD candidate genes were detected in NES cells by RNA sequencing, and seven candidate genes were upregulated during differentiation to neurons, including DYX1C1 (DNAAF4), a highly replicated DD candidate gene. Our results suggest a role of ciliary genes in differentiating neuronal cells and show that NES cells provide a relevant human neuronal model to study ciliary and DD candidate genes.
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Affiliation(s)
- Andrea Bieder
- Department of Biosciences and Nutrition, Karolinska Institutet, Hälsovägen 9, 141 57, Huddinge, Sweden.
| | - Masahito Yoshihara
- Department of Biosciences and Nutrition, Karolinska Institutet, Hälsovägen 9, 141 57, Huddinge, Sweden
| | - Shintaro Katayama
- Department of Biosciences and Nutrition, Karolinska Institutet, Hälsovägen 9, 141 57, Huddinge, Sweden
| | - Kaarel Krjutškov
- Department of Biosciences and Nutrition, Karolinska Institutet, Hälsovägen 9, 141 57, Huddinge, Sweden.,Competence Centre on Health Technologies, Tartu, Estonia.,Research Program of Molecular Neurology, Research Programs Unit, University of Helsinki, Helsinki, Finland.,Folkhälsan Institute of Genetics, Helsinki, Finland
| | - Anna Falk
- Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
| | - Juha Kere
- Department of Biosciences and Nutrition, Karolinska Institutet, Hälsovägen 9, 141 57, Huddinge, Sweden. .,Research Program of Molecular Neurology, Research Programs Unit, University of Helsinki, Helsinki, Finland. .,Folkhälsan Institute of Genetics, Helsinki, Finland. .,School of Basic and Medical Biosciences, King's College London, London, UK.
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12
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Liu G, David BT, Trawczynski M, Fessler RG. Advances in Pluripotent Stem Cells: History, Mechanisms, Technologies, and Applications. Stem Cell Rev Rep 2020; 16:3-32. [PMID: 31760627 PMCID: PMC6987053 DOI: 10.1007/s12015-019-09935-x] [Citation(s) in RCA: 303] [Impact Index Per Article: 60.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Over the past 20 years, and particularly in the last decade, significant developmental milestones have driven basic, translational, and clinical advances in the field of stem cell and regenerative medicine. In this article, we provide a systemic overview of the major recent discoveries in this exciting and rapidly developing field. We begin by discussing experimental advances in the generation and differentiation of pluripotent stem cells (PSCs), next moving to the maintenance of stem cells in different culture types, and finishing with a discussion of three-dimensional (3D) cell technology and future stem cell applications. Specifically, we highlight the following crucial domains: 1) sources of pluripotent cells; 2) next-generation in vivo direct reprogramming technology; 3) cell types derived from PSCs and the influence of genetic memory; 4) induction of pluripotency with genomic modifications; 5) construction of vectors with reprogramming factor combinations; 6) enhancing pluripotency with small molecules and genetic signaling pathways; 7) induction of cell reprogramming by RNA signaling; 8) induction and enhancement of pluripotency with chemicals; 9) maintenance of pluripotency and genomic stability in induced pluripotent stem cells (iPSCs); 10) feeder-free and xenon-free culture environments; 11) biomaterial applications in stem cell biology; 12) three-dimensional (3D) cell technology; 13) 3D bioprinting; 14) downstream stem cell applications; and 15) current ethical issues in stem cell and regenerative medicine. This review, encompassing the fundamental concepts of regenerative medicine, is intended to provide a comprehensive portrait of important progress in stem cell research and development. Innovative technologies and real-world applications are emphasized for readers interested in the exciting, promising, and challenging field of stem cells and those seeking guidance in planning future research direction.
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Affiliation(s)
- Gele Liu
- Department of Neurosurgery, Rush University Medical College, 1725 W. Harrison St., Suite 855, Chicago, IL, 60612, USA.
| | - Brian T David
- Department of Neurosurgery, Rush University Medical College, 1725 W. Harrison St., Suite 855, Chicago, IL, 60612, USA
| | - Matthew Trawczynski
- Department of Neurosurgery, Rush University Medical College, 1725 W. Harrison St., Suite 855, Chicago, IL, 60612, USA
| | - Richard G Fessler
- Department of Neurosurgery, Rush University Medical College, 1725 W. Harrison St., Suite 855, Chicago, IL, 60612, USA
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13
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Patch-Clamp Recordings from Human Embryonic Stem Cells-Derived Fragile X Neurons. Methods Mol Biol 2019. [PMID: 30900181 DOI: 10.1007/978-1-4939-9080-1_11] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
Abstract
Performing electrophysiological recordings from human neurons that have been differentiated in vitro, as compared to primary cultures, raises many challenges. However, patch-clamp recording from neurons derived from stem cells provides an abundance of valuable information, reliably and fast. Here, we describe a protocol that is used successfully in our lab for recording from both control and Fragile X neurons, derived in vitro from human embryonic stem cells.
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14
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Telias M. Molecular Mechanisms of Synaptic Dysregulation in Fragile X Syndrome and Autism Spectrum Disorders. Front Mol Neurosci 2019; 12:51. [PMID: 30899214 PMCID: PMC6417395 DOI: 10.3389/fnmol.2019.00051] [Citation(s) in RCA: 58] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2018] [Accepted: 02/12/2019] [Indexed: 12/21/2022] Open
Abstract
Fragile X syndrome (FXS) is the most common form of monogenic hereditary cognitive impairment. FXS patient exhibit a high comorbidity rate with autism spectrum disorders (ASDs). This makes FXS a model disease for understanding how synaptic dysregulation alters neuronal excitability, learning and memory, social behavior, and more. Since 1991, with the discovery of fragile X mental retardation 1 (FMR1) as the sole gene that is mutated in FXS, thousands of studies into the function of the gene and its encoded protein FMR1 protein (FMRP), have been conducted, yielding important information regarding the pathophysiology of the disease, as well as insight into basic synaptic mechanisms that control neuronal networking and circuitry. Among the most important, are molecular mechanisms directly involved in plasticity, including glutamate and γ-aminobutyric acid (GABA) receptors, which can control synaptic transmission and signal transduction, including short- and long-term plasticity. More recently, several novel mechanisms involving growth factors, enzymatic cascades and transcription factors (TFs), have been proposed to have the potential of explaining some of the synaptic dysregulation in FXS. In this review article, I summarize the main mechanisms proposed to underlie synaptic disruption in FXS and ASDs. I focus on studies conducted on the Fmr1 knock-out (KO) mouse model and on FXS-human pluripotent stem cells (hPSCs), emphasizing the differences and even contradictions between mouse and human, whenever possible. As FXS and ASDs are both neurodevelopmental disorders that follow a specific time-course of disease progression, I highlight those studies focusing on the differential developmental regulation of synaptic abnormalities in these diseases.
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Affiliation(s)
- Michael Telias
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, United States
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15
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Yellapragada V, Liu X, Lund C, Känsäkoski J, Pulli K, Vuoristo S, Lundin K, Tuuri T, Varjosalo M, Raivio T. MKRN3 Interacts With Several Proteins Implicated in Puberty Timing but Does Not Influence GNRH1 Expression. Front Endocrinol (Lausanne) 2019; 10:48. [PMID: 30800097 PMCID: PMC6375840 DOI: 10.3389/fendo.2019.00048] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/12/2018] [Accepted: 01/21/2019] [Indexed: 12/12/2022] Open
Abstract
Paternally-inherited loss-of-function mutations in makorin ring finger protein 3 gene (MKRN3) underlie central precocious puberty. To investigate the puberty-related mechanism(s) of MKRN3 in humans, we generated two distinct bi-allelic MKRN3 knock-out human pluripotent stem cell lines, Del 1 and Del 2, and differentiated them into GNRH1-expressing neurons. Both Del 1 and Del 2 clones could be differentiated into neuronal progenitors and GNRH1-expressing neurons, however, the relative expression of GNRH1 did not differ from wild type cells (P = NS). Subsequently, we investigated stable and dynamic protein-protein interaction (PPI) partners of MKRN3 by stably expressing it in HEK cells followed by mass spectrometry analyses. We found 81 high-confidence novel protein interaction partners, which are implicated in cellular processes such as insulin signaling, RNA metabolism and cell-cell adhesion. Of the identified interactors, 20 have been previously implicated in puberty timing. In conclusion, our stem cell model for generation of GNRH1-expressing neurons did not offer mechanistic insight for the role of MKRN3 in puberty initiation. The PPI data, however, indicate that MKRN3 may regulate puberty by interacting with other puberty-related proteins. Further studies are required to elucidate the possible mechanisms and outcomes of these interactions.
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Affiliation(s)
- Venkatram Yellapragada
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
- Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Xiaonan Liu
- Molecular Systems Biology Research Group, Institute of Biotechnology & HiLIFE, University of Helsinki, Helsinki, Finland
- Proteomics Unit, Institute of Biotechnology, University of Helsinki, Helsinki, Finland
| | - Carina Lund
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
- Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Johanna Känsäkoski
- Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Kristiina Pulli
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
- Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Sanna Vuoristo
- Department of Obstetrics and Gynecology, Helsinki University Hospital, HUH, Helsinki, Finland
| | - Karolina Lundin
- Department of Obstetrics and Gynecology, Helsinki University Hospital, HUH, Helsinki, Finland
| | - Timo Tuuri
- Department of Obstetrics and Gynecology, Helsinki University Hospital, HUH, Helsinki, Finland
| | - Markku Varjosalo
- Molecular Systems Biology Research Group, Institute of Biotechnology & HiLIFE, University of Helsinki, Helsinki, Finland
- Proteomics Unit, Institute of Biotechnology, University of Helsinki, Helsinki, Finland
| | - Taneli Raivio
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
- Department of Physiology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
- New Children's Hospital, Pediatric Research Center, Helsinki University Hospital, HUH, Helsinki, Finland
- *Correspondence: Taneli Raivio
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16
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Telias M. Fragile X Syndrome Pre-Clinical Research: Comparing Mouse- and Human-Based Models. Methods Mol Biol 2019; 1942:155-162. [PMID: 30900183 DOI: 10.1007/978-1-4939-9080-1_13] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Despite almost 30 years of biomedical research, a treatment or cure for fragile X syndrome (FXS) is not yet available. The reasons behind this are varied, and among them are discrepancies in both research methodologies and research models. For many years, the fmr1 knockout mouse model dominated the field, and was used to draw important conclusions. The establishment of FXS-human cellular models called these conclusions into question, showing conflicting evidence. Discrepancies in FXS research, between mouse and human, might arise from differences inherent to each species, and from the use of different methodologies. This chapter summarizes these discrepancies and evaluates their impact on the current status of clinical trials.
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Affiliation(s)
- Michael Telias
- Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
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17
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Verma I, Seshagiri PB. Directed differentiation of mouse P19 embryonal carcinoma cells to neural cells in a serum- and retinoic acid-free culture medium. In Vitro Cell Dev Biol Anim 2018; 54:567-579. [PMID: 30030768 DOI: 10.1007/s11626-018-0275-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2018] [Accepted: 06/15/2018] [Indexed: 11/25/2022]
Abstract
P19 embryonal carcinoma cells (EC-cells) provide a simple and robust culture system for studying neural development. Most protocols developed so far for directing neural differentiation of P19 cells depend on the use of culture medium supplemented with retinoic acid (RA) and serum, which has an undefined composition. Hence, such protocols are not suitable for many molecular studies. In this study, we achieved neural differentiation of P19 cells in a serum- and RA-free culture medium by employing the knockout serum replacement (KSR) supplement. In the KSR-containing medium, P19 cells underwent predominant differentiation into neural lineage and by day 12 of culture, neural cells were present in 100% of P19-derived embryoid bodies (EBs). This was consistently accompanied by the increased expression of various neural lineage-associated markers during the course of differentiation. P19-derived neural cells comprised of NES+ neural progenitors (~ 46%), TUBB3+ immature neurons (~ 6%), MAP2+ mature neurons (~ 2%), and GFAP+ astrocytes (~ 50%). A heterogeneous neuronal population consisting of glutamatergic, GABAergic, serotonergic, and dopaminergic neurons was generated. Taken together, our study shows that the KSR medium is suitable for the differentiation of P19 cells to neural lineage without requiring additional (serum and RA) supplements. This stem cell differentiation system could be utilized for gaining mechanistic insights into neural differentiation and for identifying potential neuroactive compounds.
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Affiliation(s)
- Isha Verma
- Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Sir CV Raman Road, Bangalore, 560012, India
| | - Polani B Seshagiri
- Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Sir CV Raman Road, Bangalore, 560012, India.
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18
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Bustos F, Segarra-Fas A, Chaugule VK, Brandenburg L, Branigan E, Toth R, Macartney T, Knebel A, Hay RT, Walden H, Findlay GM. RNF12 X-Linked Intellectual Disability Mutations Disrupt E3 Ligase Activity and Neural Differentiation. Cell Rep 2018; 23:1599-1611. [PMID: 29742418 PMCID: PMC5976579 DOI: 10.1016/j.celrep.2018.04.022] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2017] [Revised: 03/13/2018] [Accepted: 04/03/2018] [Indexed: 11/29/2022] Open
Abstract
X-linked intellectual disability (XLID) is a heterogeneous syndrome affecting mainly males. Human genetics has identified >100 XLID genes, although the molecular and developmental mechanisms underpinning this disorder remain unclear. Here, we employ an embryonic stem cell model to explore developmental functions of a recently identified XLID gene, the RNF12/RLIM E3 ubiquitin ligase. We show that RNF12 catalytic activity is required for proper stem cell maintenance and neural differentiation, and this is disrupted by patient-associated XLID mutation. We further demonstrate that RNF12 XLID mutations specifically impair ubiquitylation of developmentally relevant substrates. XLID mutants disrupt distinct RNF12 functional modules by either inactivating the catalytic RING domain or interfering with a distal regulatory region required for efficient ubiquitin transfer. Our data thereby uncover a key function for RNF12 E3 ubiquitin ligase activity in stem cell and neural development and identify mechanisms by which this is disrupted in intellectual disability.
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Affiliation(s)
- Francisco Bustos
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Anna Segarra-Fas
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Viduth K Chaugule
- Institute of Molecular Cell and Systems Biology, The University of Glasgow, Glasgow G12 8QQ, UK
| | - Lennart Brandenburg
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Emma Branigan
- Centre for Gene Regulation and Expression, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Rachel Toth
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Thomas Macartney
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Axel Knebel
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Ronald T Hay
- Centre for Gene Regulation and Expression, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK
| | - Helen Walden
- Institute of Molecular Cell and Systems Biology, The University of Glasgow, Glasgow G12 8QQ, UK
| | - Greg M Findlay
- The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, UK.
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19
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Hessel EVS, Staal YCM, Piersma AH. Design and validation of an ontology-driven animal-free testing strategy for developmental neurotoxicity testing. Toxicol Appl Pharmacol 2018; 354:136-152. [PMID: 29544899 DOI: 10.1016/j.taap.2018.03.013] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2017] [Revised: 02/26/2018] [Accepted: 03/11/2018] [Indexed: 12/26/2022]
Abstract
Developmental neurotoxicity entails one of the most complex areas in toxicology. Animal studies provide only limited information as to human relevance. A multitude of alternative models have been developed over the years, providing insights into mechanisms of action. We give an overview of fundamental processes in neural tube formation, brain development and neural specification, aiming at illustrating complexity rather than comprehensiveness. We also give a flavor of the wealth of alternative methods in this area. Given the impressive progress in mechanistic knowledge of human biology and toxicology, the time is right for a conceptual approach for designing testing strategies that cover the integral mechanistic landscape of developmental neurotoxicity. The ontology approach provides a framework for defining this landscape, upon which an integral in silico model for predicting toxicity can be built. It subsequently directs the selection of in vitro assays for rate-limiting events in the biological network, to feed parameter tuning in the model, leading to prediction of the toxicological outcome. Validation of such models requires primary attention to coverage of the biological domain, rather than classical predictive value of individual tests. Proofs of concept for such an approach are already available. The challenge is in mining modern biology, toxicology and chemical information to feed intelligent designs, which will define testing strategies for neurodevelopmental toxicity testing.
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Affiliation(s)
- Ellen V S Hessel
- Center for Health Protection, National Institute for Public Health and The Environment (RIVM), P.O. Box 1, 3720BA Bilthoven, The Netherlands.
| | - Yvonne C M Staal
- Center for Health Protection, National Institute for Public Health and The Environment (RIVM), P.O. Box 1, 3720BA Bilthoven, The Netherlands
| | - Aldert H Piersma
- Center for Health Protection, National Institute for Public Health and The Environment (RIVM), P.O. Box 1, 3720BA Bilthoven, The Netherlands; Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, The Netherlands
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20
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Teixeira FG, Vasconcelos NL, Gomes ED, Marques F, Sousa JC, Sousa N, Silva NA, Assunção-Silva R, Lima R, Salgado AJ. Bioengineered cell culture systems of central nervous system injury and disease. Drug Discov Today 2016; 21:1456-1463. [DOI: 10.1016/j.drudis.2016.04.020] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2016] [Revised: 04/02/2016] [Accepted: 04/21/2016] [Indexed: 01/10/2023]
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21
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Telias M, Mayshar Y, Amit A, Ben-Yosef D. Molecular mechanisms regulating impaired neurogenesis of fragile X syndrome human embryonic stem cells. Stem Cells Dev 2016; 24:2353-65. [PMID: 26393806 DOI: 10.1089/scd.2015.0220] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Fragile X syndrome (FXS) is the most common form of inherited cognitive impairment. It is caused by developmental inactivation of the FMR1 gene and the absence of its encoded protein FMRP, which plays pivotal roles in brain development and function. In FXS embryos with full FMR1 mutation, FMRP is expressed during early embryogenesis and is gradually downregulated at the third trimester of pregnancy. FX-human embryonic stem cells (FX-hESCs), derived from FX human blastocysts, demonstrate the same pattern of developmentally regulated FMR1 inactivation when subjected to in vitro neural differentiation (IVND). In this study, we used this in vitro human platform to explore the molecular mechanisms downstream to FMRP in the context of early human embryonic neurogenesis. Our results show a novel role for the SOX superfamily of transcription factors, specifically for SOX2 and SOX9, which could explain the reduced and delayed neurogenesis observed in FX cells. In addition, we assess in this study the "GSK3β theory of FXS" for the first time in a human-based model. We found no evidence for a pathological increase in GSK3β protein levels upon cellular loss of FMRP, in contrast to what was found in the brain of Fmr1 knockout mice. Our study adds novel data on potential downstream targets of FMRP and highlights the importance of the FX-hESC IVND system.
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Affiliation(s)
- Michael Telias
- 1 The Wolfe PGD-SC Lab, Racine IVF Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center , Tel Aviv, Israel .,2 Department of Cell and Developmental Biology Sackler Medical School, Tel Aviv University , Tel Aviv, Israel
| | - Yoav Mayshar
- 1 The Wolfe PGD-SC Lab, Racine IVF Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center , Tel Aviv, Israel
| | - Ami Amit
- 1 The Wolfe PGD-SC Lab, Racine IVF Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center , Tel Aviv, Israel
| | - Dalit Ben-Yosef
- 1 The Wolfe PGD-SC Lab, Racine IVF Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center , Tel Aviv, Israel .,2 Department of Cell and Developmental Biology Sackler Medical School, Tel Aviv University , Tel Aviv, Israel
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22
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Mungenast AE, Siegert S, Tsai LH. Modeling Alzheimer's disease with human induced pluripotent stem (iPS) cells. Mol Cell Neurosci 2016; 73:13-31. [PMID: 26657644 PMCID: PMC5930170 DOI: 10.1016/j.mcn.2015.11.010] [Citation(s) in RCA: 92] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2015] [Revised: 11/05/2015] [Accepted: 11/25/2015] [Indexed: 02/08/2023] Open
Abstract
In the last decade, induced pluripotent stem (iPS) cells have revolutionized the utility of human in vitro models of neurological disease. The iPS-derived and differentiated cells allow researchers to study the impact of a distinct cell type in health and disease as well as performing therapeutic drug screens on a human genetic background. In particular, clinical trials for Alzheimer's disease (AD) have been failing. Two of the potential reasons are first, the species gap involved in proceeding from initial discoveries in rodent models to human studies, and second, an unsatisfying patient stratification, meaning subgrouping patients based on the disease severity due to the lack of phenotypic and genetic markers. iPS cells overcome this obstacles and will improve our understanding of disease subtypes in AD. They allow researchers conducting in depth characterization of neural cells from both familial and sporadic AD patients as well as preclinical screens on human cells. In this review, we briefly outline the status quo of iPS cell research in neurological diseases along with the general advantages and pitfalls of these models. We summarize how genome-editing techniques such as CRISPR/Cas9 will allow researchers to reduce the problem of genomic variability inherent to human studies, followed by recent iPS cell studies relevant to AD. We then focus on current techniques for the differentiation of iPS cells into neural cell types that are relevant to AD research. Finally, we discuss how the generation of three-dimensional cell culture systems will be important for understanding AD phenotypes in a complex cellular milieu, and how both two- and three-dimensional iPS cell models can provide platforms for drug discovery and translational studies into the treatment of AD.
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Affiliation(s)
- Alison E Mungenast
- Picower Institute for Learning and Memory, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA; Department of Brain and Cognitive Sciences, MIT, Cambridge, MA, USA
| | - Sandra Siegert
- Picower Institute for Learning and Memory, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA; Department of Brain and Cognitive Sciences, MIT, Cambridge, MA, USA.
| | - Li-Huei Tsai
- Picower Institute for Learning and Memory, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA; Department of Brain and Cognitive Sciences, MIT, Cambridge, MA, USA
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23
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Abstract
UNLABELLED Fragile X syndrome (FXS), the most common form of inherited mental retardation, is a neurodevelopmental disorder caused by silencing of the FMR1 gene, which in FXS becomes inactivated during human embryonic development. We have shown recently that this process is recapitulated by in vitro neural differentiation of FX human embryonic stem cells (FX-hESCs), derived from FXS blastocysts. In the present study, we analyzed morphological and functional properties of neurons generated from FX-hESCs. Human FX neurons can fire single action potentials (APs) to depolarizing current commands, but are unable to discharge trains of APs. Their APs are of a reduced amplitudes and longer durations than controls. These are reflected in reduced inward Na(+) and outward K(+) currents. In addition, human FX neurons contain fewer synaptic vesicles and lack spontaneous synaptic activity. Notably, synaptic activity in these neurons can be restored by coculturing them with normal rat hippocampal neurons, demonstrating a critical role for synaptic mechanisms in FXS pathology. This is the first extensive functional analysis of human FX neurons derived in vitro from hESCs that provides a convenient tool for studying molecular mechanisms underlying the impaired neuronal functions in FXS. SIGNIFICANCE STATEMENT Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by silencing of the FMR1 gene. In this study, we describe for the first time the properties of neurons developed from human embryonic stem cells (hESCs) that carry the FMR1 mutation and are grown in culture for extended periods. These neurons are retarded compared with controls in several morphological and functional properties. In vitro neural differentiation of FX hESCs can thus serve as a most relevant system for the analysis of molecular mechanisms underlying the impaired neuronal functions in FXS.
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24
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Lin M, Lachman HM, Zheng D. Transcriptomics analysis of iPSC-derived neurons and modeling of neuropsychiatric disorders. Mol Cell Neurosci 2015; 73:32-42. [PMID: 26631648 DOI: 10.1016/j.mcn.2015.11.009] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2015] [Revised: 10/31/2015] [Accepted: 11/25/2015] [Indexed: 12/19/2022] Open
Abstract
Induced pluripotent stem cell (iPSC)-derived neurons and neural progenitors are great resources for studying neural development and differentiation and their disruptions in disease conditions, and hold the promise of future cell therapy. In general, iPSC lines can be established either specifically from patients with neuropsychiatric disorders or from healthy subjects. The iPSCs can then be induced to differentiate into neural lineages and the iPSC-derived neurons are valuable for various types of cell-based assays that seek to understand disease mechanisms and identify and test novel therapies. In addition, it is an ideal system for gene expression profiling (i.e., transcriptomic analysis), an efficient and cost-effective way to explore the genetic programs regulating neurodevelopment. Moreover, transcriptomic comparison, which can be performed between patient-derived samples and controls, or in control lines in which the expression of specific genes has been disrupted, can uncover convergent gene targets and pathways that are downstream of the hundreds of candidate genes that have been associated with neuropsychiatric disorders. The results, especially after integration with spatiotemporal transcriptomic profiles of normal human brain development, have indeed helped to uncover gene networks, molecular pathways, and cellular signaling that likely play critical roles in disease development and progression. On the other hand, despite the great promise, many challenges remain in the usage of iPSC-derived neurons for modeling neuropsychiatric disorders, for example, how to generate relatively homogenous populations of specific neuronal subtypes that are affected in a particular disorder and how to better address the genetic heterogeneity that exists in the patient population.
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Affiliation(s)
- Mingyan Lin
- Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY, USA
| | - Herbert M Lachman
- Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY, USA; Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY, USA; Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY, USA; Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY, USA
| | - Deyou Zheng
- Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY, USA; Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY, USA; Department of Neurology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY, USA.
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Bradford AB, McNutt PM. Importance of being Nernst: Synaptic activity and functional relevance in stem cell-derived neurons. World J Stem Cells 2015; 7:899-921. [PMID: 26240679 PMCID: PMC4515435 DOI: 10.4252/wjsc.v7.i6.899] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/19/2014] [Revised: 02/28/2015] [Accepted: 05/11/2015] [Indexed: 02/06/2023] Open
Abstract
Functional synaptogenesis and network emergence are signature endpoints of neurogenesis. These behaviors provide higher-order confirmation that biochemical and cellular processes necessary for neurotransmitter release, post-synaptic detection and network propagation of neuronal activity have been properly expressed and coordinated among cells. The development of synaptic neurotransmission can therefore be considered a defining property of neurons. Although dissociated primary neuron cultures readily form functioning synapses and network behaviors in vitro, continuously cultured neurogenic cell lines have historically failed to meet these criteria. Therefore, in vitro-derived neuron models that develop synaptic transmission are critically needed for a wide array of studies, including molecular neuroscience, developmental neurogenesis, disease research and neurotoxicology. Over the last decade, neurons derived from various stem cell lines have shown varying ability to develop into functionally mature neurons. In this review, we will discuss the neurogenic potential of various stem cells populations, addressing strengths and weaknesses of each, with particular attention to the emergence of functional behaviors. We will propose methods to functionally characterize new stem cell-derived neuron (SCN) platforms to improve their reliability as physiological relevant models. Finally, we will review how synaptically active SCNs can be applied to accelerate research in a variety of areas. Ultimately, emphasizing the critical importance of synaptic activity and network responses as a marker of neuronal maturation is anticipated to result in in vitro findings that better translate to efficacious clinical treatments.
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In vitro modeling of hyperpigmentation associated to neurofibromatosis type 1 using melanocytes derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2015; 112:9034-9. [PMID: 26150484 DOI: 10.1073/pnas.1501032112] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
"Café-au-lait" macules (CALMs) and overall skin hyperpigmentation are early hallmarks of neurofibromatosis type 1 (NF1). One of the most frequent monogenic diseases, NF1 has subsequently been characterized with numerous benign Schwann cell-derived tumors. It is well established that neurofibromin, the NF1 gene product, is an antioncogene that down-regulates the RAS oncogene. In contrast, the molecular mechanisms associated with alteration of skin pigmentation have remained elusive. We have reassessed this issue by differentiating human embryonic stem cells into melanocytes. In the present study, we demonstrate that NF1 melanocytes reproduce the hyperpigmentation phenotype in vitro, and further characterize the link between loss of heterozygosity and the typical CALMs that appear over the general hyperpigmentation. Molecular mechanisms associated with these pathological phenotypes correlate with an increased activity of cAMP-mediated PKA and ERK1/2 signaling pathways, leading to overexpression of the transcription factor MITF and of the melanogenic enzymes tyrosinase and dopachrome tautomerase, all major players in melanogenesis. Finally, the hyperpigmentation phenotype can be rescued using specific inhibitors of these signaling pathways. These results open avenues for deciphering the pathological mechanisms involved in pigmentation diseases, and provide a robust assay for the development of new strategies for treating these diseases.
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Telias M, Ben-Yosef D. Neural stem cell replacement: a possible therapy for neurodevelopmental disorders? Neural Regen Res 2015; 10:180-2. [PMID: 25883606 PMCID: PMC4392655 DOI: 10.4103/1673-5374.152361] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/21/2014] [Indexed: 11/04/2022] Open
Affiliation(s)
- Michael Telias
- Wolfe PGD-Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center; Department of Cell and Developmental Biology, Sackler Medical School, Tel Aviv University, Tel Aviv, Israel
| | - Dalit Ben-Yosef
- Wolfe PGD-Stem Cell Lab, Racine IVF Unit, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center; Department of Cell and Developmental Biology, Sackler Medical School, Tel Aviv University, Tel Aviv, Israel
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28
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Scheenen WJJM, Celikel T. Nanophysiology: Bridging synapse ultrastructure, biology, and physiology using scanning ion conductance microscopy. Synapse 2015; 69:233-41. [PMID: 25655013 DOI: 10.1002/syn.21807] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2014] [Accepted: 01/22/2015] [Indexed: 01/01/2023]
Abstract
Synaptic communication is at the core of neural circuit function, and its plasticity allows the nervous system to adapt to the changes in its environment. Understanding the mechanisms of this synaptic (re)organization will benefit from novel methodologies that enable simultaneous study of synaptic ultrastructure, biology, and physiology in identified circuits. Here, we describe one of these methodologies, i.e., scanning ion conductance microscopy (SICM), for electrical mapping of the membrane anatomy in tens of nanometers resolution in living neurons. When combined with traditional patch-clamp and fluorescence microscopy techniques, and the newly emerging nanointerference methodologies, SICM has the potential to mechanistically bridge the synaptic structure and function longitudinally throughout the life of a synapse.
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Affiliation(s)
- Wim J J M Scheenen
- Department of Neurophysiology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, The Netherlands
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Telias M, Segal M, Ben-Yosef D. Electrical maturation of neurons derived from human embryonic stem cells. F1000Res 2014; 3:196. [PMID: 25309736 PMCID: PMC4184377 DOI: 10.12688/f1000research.4943.2] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 10/01/2014] [Indexed: 11/20/2022] Open
Abstract
In-vitro neuronal differentiation of human pluripotent stem cells has become a widely used tool in disease modeling and prospective regenerative medicine. Most studies evaluate neurons molecularly and only a handful of them use electrophysiological tools to directly indicate that these are genuine neurons. Therefore, the specific timing of development of intrinsic electrophysiological properties and synaptic capabilities remains poorly understood. Here we describe a systematic analysis of developing neurons derived in-vitro from human embryonic stem cells (hESCs). We show that hESCs differentiated in-vitro into early embryonic neurons, displaying basically mature morphological and electrical features as early as day 37. This early onset of action potential discharges suggests that first stages of neurogenesis in humans are already associated with electrical maturation. Spike frequency, amplitude, duration, threshold and after hyperpolarization were found to be the most predictive parameters for electrical maturity. Furthermore, we were able to detect spontaneous synaptic activity already at these early time-points, demonstrating that neuronal connectivity can develop concomitantly with the gradual process of electrical maturation. These results highlight the functional properties of hESCs in the process of their development into neurons. Moreover, our results provide practical tools for the direct measurement of functional maturity, which can be reproduced and implemented for stem cell research of neurogenesis in general, and neurodevelopmental disorders in particular.
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Affiliation(s)
- Michael Telias
- Wolfe PGD-SC Lab, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Tel-Aviv, 64239, Israel ; Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, 64239, Israel
| | - Menahem Segal
- Department of Neurobiology, Weizmann Institute of Science, Rehovot, 76100, Israel
| | - Dalit Ben-Yosef
- Wolfe PGD-SC Lab, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, Tel-Aviv, 64239, Israel ; Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, 64239, Israel
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