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Taiwo RO, Goldberg HS, Ilouz N, Singh PK, Shekh-Ahmad T, Levite M. Enigmatic intractable Epilepsy patients have antibodies that bind glutamate receptor peptides, kill neurons, damage the brain, and cause Generalized Tonic Clonic Seizures. J Neural Transm (Vienna) 2025; 132:663-688. [PMID: 39932550 PMCID: PMC12043744 DOI: 10.1007/s00702-024-02855-2] [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: 09/23/2024] [Accepted: 10/22/2024] [Indexed: 05/02/2025]
Abstract
Epilepsy affects 1-2% of the world population, is enigmatic in 30% of cases, and is often intractable, unresponsive to antiepileptic drugs, and accompanied by cognitive, psychiatric and behavioral problems. Tests for Autoimmune Epilepsy are not performed routinely, and limited to passive diagnosis of known autoimmune antibodies, without essential functional tests to reveal active pathogenic antibodies. We investigated two young Epilepsy patients with different Epilepsy characteristics, repeated intractable seizures, and enigmatic etiology. We suspected Autoimmune Epilepsy. We found that both patients have elevated IgG antibodies, and three types of glutamate receptor antibodies, to: AMPA-GluR3B, NMDA-NR1 and NMDA-NR2 peptides. In contrast, they lack autoantibodies to: LGI1, CASPR2, GABA-RB1, Amphiphysin, CV2, PNMA1, Ri, Yo, Hu, Recoverin, Soxi and Titin. IgG antibodies of both patients bound and killed human neural cells In vitro. Moreover, In vivo video EEG studies in naive rats revealed that patient's IgG antibodies, infused continually into rat brain, bound neural cells in the hippocampus and cortex, caused neural loss in these brain regions, and induced recurrent Generalized Tonic Clonic Seizures. We assume they can do so also in the patient's brain. This is the first model of human Autoimmune Epilepsy in rats. It can serve for discovery of patient's pathogenic antibodies, and drug development. Tests for autoimmune antibodies that bind glutamate receptor peptides, and functional diagnostic tests, are obligatory in all enigmatic intractable Epilepsy patients. Current diagnosis of Autoimmune Epilepsy is insufficient! If pathogenic antibodies are found, intractable patients must receive available, suitable and potentially life-changing immunotherapies for Autoimmune Epilepsy.
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Affiliation(s)
- Rhoda Olowe Taiwo
- Department of Pharmaceutics, Faculty of Medicine, The Institute for Drug Research, School of Pharmacy, The Hebrew University, Ein Karem, 91120, Jerusalem, Israel
| | - Hadassa Sterm Goldberg
- Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
- Institute of Pediatric Neurology, Epilepsy Center, Schneider Children's Medical Center, Petah Tiqva, Israel
| | - Nili Ilouz
- Faculty of Medicine, The Hebrew University, Ein Karem, 9112102, Jerusalem, Israel
| | - Prince Kumar Singh
- Department of Pharmaceutics, Faculty of Medicine, The Institute for Drug Research, School of Pharmacy, The Hebrew University, Ein Karem, 91120, Jerusalem, Israel
| | - Tawfeeq Shekh-Ahmad
- Department of Pharmaceutics, Faculty of Medicine, The Institute for Drug Research, School of Pharmacy, The Hebrew University, Ein Karem, 91120, Jerusalem, Israel.
| | - Mia Levite
- Faculty of Medicine, The Hebrew University, Ein Karem, 9112102, Jerusalem, Israel.
- Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, 9112001, Jerusalem, Israel.
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2
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Salmanzadeh H, Poojari A, Rabiee A, Zeitlin BD, Halliwell RF. Neuropharmacology of human TERA2.cl.SP12 stem cell-derived neurons in ultra-long-term culture for antiseizure drug discovery. Front Neurosci 2023; 17:1182720. [PMID: 37397467 PMCID: PMC10308080 DOI: 10.3389/fnins.2023.1182720] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2023] [Accepted: 05/25/2023] [Indexed: 07/04/2023] Open
Abstract
Modeling the complex and prolonged development of the mammalian central nervous system in vitro remains a profound challenge. Most studies of human stem cell derived neurons are conducted over days to weeks and may or may not include glia. Here we have utilized a single human pluripotent stem cell line, TERA2.cl.SP12 to derive both neurons and glial cells and determined their differentiation and functional maturation over 1 year in culture together with their ability to display epileptiform activity in response to pro-convulsant agents and to detect antiseizure drug actions. Our experiments show that these human stem cells differentiate in vitro into mature neurons and glia cells and form inhibitory and excitatory synapses and integrated neural circuits over 6-8 months, paralleling early human neurogenesis in vivo; these neuroglia cultures display complex electrochemical signaling including high frequency trains of action potentials from single neurons, neural network bursts and highly synchronized, rhythmical firing patterns. Neural activity in our 2D neuron-glia circuits is modulated by a variety of voltage-gated and ligand-gated ion channel acting drugs and these actions were consistent in both young and highly mature neuron cultures. We also show for the first time that spontaneous and epileptiform activity is modulated by first, second and third generation antiseizure agents consistent with animal and human studies. Together, our observations strongly support the value of long-term human stem cell-derived neuroglial cultures in disease modeling and neuropsychiatric drug discovery.
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Affiliation(s)
- Hamed Salmanzadeh
- Thomas J. Long School of Pharmacy, University of the Pacific, Stockton, CA, United States
| | - Ankita Poojari
- Thomas J. Long School of Pharmacy, University of the Pacific, Stockton, CA, United States
| | - Atefeh Rabiee
- Thomas J. Long School of Pharmacy, University of the Pacific, Stockton, CA, United States
| | - Benjamin D. Zeitlin
- Arthur A. Dugoni School of Dentistry, University of the Pacific, San Francisco, CA, United States
| | - Robert F. Halliwell
- Thomas J. Long School of Pharmacy, University of the Pacific, Stockton, CA, United States
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3
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Abstract
Although tumourigenesis occurs due to genetic mutations, the role of epigenetic dysregulations in cancer is also well established. Epigenetic dysregulations in cancer may occur as a result of mutations in genes encoding histone/DNA-modifying enzymes and chromatin remodellers or mutations in histone protein itself. It is also true that misregulated gene expression without genetic mutations in these factors could also support tumour initiation and progression. Interestingly, metabolic rewiring has emerged as a hallmark of cancer due to gene mutations in specific metabolic enzymes or dietary/environmental factors. Recent studies report an intricate cross-talk between epigenetic and metabolic reprogramming in cancer. This review discusses the role of epigenetic and metabolic dysregulations and their cross-talk in tumourigenesis with a special focus on gliomagenesis. We also discuss the role of recently developed human embryonic stem cells/induced pluripotent stem cells-derived organoid models of gliomas and how these models are proving instrumental in uncovering human-specific cellular and molecular complexities of gliomagenesis.
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Affiliation(s)
- Bismi Phasaludeen
- Department of Biochemistry and Molecular Biology, College of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, Abu Dhabi, United Arab Emirates
| | - Bright Starling Emerald
- Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, Abu Dhabi, United Arab Emirates,Zayed Center for Health Sciences, United Arab Emirates University, Al Ain, Abu Dhabi, United Arab Emirates
| | - Suraiya Anjum Ansari
- Department of Biochemistry and Molecular Biology, College of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, Abu Dhabi, United Arab Emirates,Zayed Center for Health Sciences, United Arab Emirates University, Al Ain, Abu Dhabi, United Arab Emirates
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4
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Little MH, Humphreys BD. Regrow or Repair: An Update on Potential Regenerative Therapies for the Kidney. J Am Soc Nephrol 2022; 33:15-32. [PMID: 34789545 PMCID: PMC8763179 DOI: 10.1681/asn.2021081073] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Fifteen years ago, this journal published a review outlining future options for regenerating the kidney. At that time, stem cell populations were being identified in multiple tissues, the concept of stem cell recruitment to a site of injury was of great interest, and the possibility of postnatal renal stem cells was growing in momentum. Since that time, we have seen the advent of human induced pluripotent stem cells, substantial advances in our capacity to both sequence and edit the genome, global and spatial transcriptional analysis down to the single-cell level, and a pandemic that has challenged our delivery of health care to all. This article will look back over this period of time to see how our view of kidney development, disease, repair, and regeneration has changed and envision a future for kidney regeneration and repair over the next 15 years.
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Affiliation(s)
- Melissa H. Little
- Murdoch Children’s Research Institute, Parkville, Melbourne, Victoria, Australia,Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, Melbourne, Victoria, Australia,Department of Anatomy and Neuroscience, The University of Melbourne, Parkville, Melbourne, Victoria, Australia
| | - Benjamin D. Humphreys
- Division of Nephrology, Department of Medicine, Washington University in St. Louis School of Medicine, Missouri,Department of Developmental Biology, Washington University in St. Louis School of Medicine, Missouri
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5
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Daadi MM. Isolation and Purification of Self-Renewable Human Neural Stem Cells from iPSCs for Cell Therapy in Experimental Model of Ischemic Stroke. Methods Mol Biol 2022; 2389:165-175. [PMID: 34558010 DOI: 10.1007/978-1-0716-1783-0_14] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/24/2023]
Abstract
Neural stem cell therapy has been galvanized by the discovery of pluripotent stem cells. The possibility to generate specialized central nervous system-specific differentiated cells using human somatic cells engineered to become induced pluripotent stem cells (iPSCs) was a game changer. This technology has broad applications in the field of regenerative medicine, in vitro disease modeling, targeted drug discovery, and precision medicine. Currently, iPSCs are one of the most promising cell sources amenable for commercialization and off-the-shelf neural stem cell therapy products. iPSCs exhibit a strong self-renewable ability that supports the development of a virtually unlimited source of neural cells for structural repair in neurological disorders. However, along with this strong proliferative capacity of iPSCs comes the tumorigenic potential of these cells after transplantation. Thus, the isolation and purification of a homogeneous population of human neural stem cells (hNSCs) are of paramount importance to ensure consistency in the composition of the cellular product and to avoid tumor formation in the host brain. This chapter describes the isolation, neuralization, and long-term perpetuation of hNSCs derived from iPSCs through the use of specific growth medium and the preparation of hNSCs for transplantation in an experimental model of stroke. Additionally, we will describe methods to analyze the ischemic stroke and size of grafts using magnetic resonance imaging and OsiriX software and neuroanatomical tracing procedures to study axonal remodeling after ischemic stroke and cell transplantation.
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Affiliation(s)
- Marcel M Daadi
- Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA.
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6
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Talukdar S, Emdad L, Das SK, Fisher PB. GAP junctions: multifaceted regulators of neuronal differentiation. Tissue Barriers 2021; 10:1982349. [PMID: 34651545 DOI: 10.1080/21688370.2021.1982349] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022] Open
Abstract
Gap junctions are intercellular membrane channels consisting of connexin proteins, which contribute to direct cytoplasmic exchange of small molecules, substrates and metabolites between adjacent cells. These channels play important roles in neuronal differentiation, maintenance, survival and function. Gap junctions regulate differentiation of neurons from embryonic, neural and induced pluripotent stem cells. In addition, they control transdifferentiation of neurons from mesenchymal stem cells. The expression and levels of several connexins correlate with cell cycle changes and different stages of neurogenesis. Connexins such as Cx36, Cx45, and Cx26, play a crucial role in neuronal function. Several connexin knockout mice display lethal or severely impaired phenotypes. Aberrations in connexin expression is frequently associated with various neurodegenerative disorders. Gap junctions also act as promising therapeutic targets for neuronal regenerative medicine, because of their role in neural stem cell integration, injury and remyelination.
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Affiliation(s)
- Sarmistha Talukdar
- Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, VA, United States.,Vcu Institute of Molecular Medicine, Richmond, VA, United States
| | - Luni Emdad
- Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, VA, United States.,Vcu Institute of Molecular Medicine, Richmond, VA, United States.,Vcu Massey Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, VA, United States
| | - Swadesh K Das
- Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, VA, United States.,Vcu Institute of Molecular Medicine, Richmond, VA, United States.,Vcu Massey Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, VA, United States
| | - Paul B Fisher
- Department of Human and Molecular Genetics, Virginia Commonwealth University, School of Medicine, Richmond, VA, United States.,Vcu Institute of Molecular Medicine, Richmond, VA, United States.,Vcu Massey Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, VA, United States
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7
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Lestrell E, O'Brien CM, Elnathan R, Voelcker NH. Vertically Aligned Nanostructured Topographies for Human Neural Stem Cell Differentiation and Neuronal Cell Interrogation. ADVANCED THERAPEUTICS 2021. [DOI: 10.1002/adtp.202100061] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Affiliation(s)
- Esther Lestrell
- Faculty of Pharmacy and Pharmaceutical Sciences Monash University Parkville VIC 3052 Australia
- Melbourne Centre for Nanofabrication Victorian Node of the Australian National Fabrication Facility 151 Wellington Road Clayton Victoria 3168 Australia
- CSIRO Manufacturing Clayton Victoria 3168 Australia
| | - Carmel M. O'Brien
- CSIRO Manufacturing Clayton Victoria 3168 Australia
- Australian Regenerative Medicine Institute Monash University Clayton Victoria 3168 Australia
| | - Roey Elnathan
- Faculty of Pharmacy and Pharmaceutical Sciences Monash University Parkville VIC 3052 Australia
- Melbourne Centre for Nanofabrication Victorian Node of the Australian National Fabrication Facility 151 Wellington Road Clayton Victoria 3168 Australia
| | - Nicolas H. Voelcker
- Faculty of Pharmacy and Pharmaceutical Sciences Monash University Parkville VIC 3052 Australia
- Melbourne Centre for Nanofabrication Victorian Node of the Australian National Fabrication Facility 151 Wellington Road Clayton Victoria 3168 Australia
- CSIRO Manufacturing Clayton Victoria 3168 Australia
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8
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Galiakberova AA, Dashinimaev EB. Neural Stem Cells and Methods for Their Generation From Induced Pluripotent Stem Cells in vitro. Front Cell Dev Biol 2020; 8:815. [PMID: 33117792 PMCID: PMC7578226 DOI: 10.3389/fcell.2020.00815] [Citation(s) in RCA: 55] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2020] [Accepted: 07/31/2020] [Indexed: 12/11/2022] Open
Abstract
Neural stem cells (NSCs) provide promising approaches for investigating embryonic neurogenesis, modeling of the pathogenesis of diseases of the central nervous system, and for designing drug-screening systems. Such cells also have an application in regenerative medicine. The most convenient and acceptable source of NSCs is pluripotent stem cells (embryonic stem cells or induced pluripotent stem cells). However, there are many different protocols for the induction and differentiation of NSCs, and these result in a wide range of neural cell types. This review is intended to summarize the knowledge accumulated, to date, by workers in this field. It should be particularly useful for researchers who are beginning investigations in this area of cell biology.
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Affiliation(s)
- Adelya A Galiakberova
- Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia.,Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Moscow, Russia
| | - Erdem B Dashinimaev
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Moscow, Russia.,Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, Moscow, Russia
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9
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10
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Nakayama N, Pothiawala A, Lee JY, Matthias N, Umeda K, Ang BK, Huard J, Huang Y, Sun D. Human pluripotent stem cell-derived chondroprogenitors for cartilage tissue engineering. Cell Mol Life Sci 2020; 77:2543-2563. [PMID: 31915836 PMCID: PMC11104892 DOI: 10.1007/s00018-019-03445-2] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2019] [Revised: 12/24/2019] [Accepted: 12/27/2019] [Indexed: 02/06/2023]
Abstract
The cartilage of joints, such as meniscus and articular cartilage, is normally long lasting (i.e., permanent). However, once damaged, especially in large animals and humans, joint cartilage is not spontaneously repaired. Compensating the lack of repair activity by supplying cartilage-(re)forming cells, such as chondrocytes or mesenchymal stromal cells, or by transplanting a piece of normal cartilage, has been the basis of therapy for biological restoration of damaged joint cartilage. Unfortunately, current biological therapies face problems on a number of fronts. The joint cartilage is generated de novo from a specialized cell type, termed a 'joint progenitor' or 'interzone cell' during embryogenesis. Therefore, embryonic chondroprogenitors that mimic the property of joint progenitors might be the best type of cell for regenerating joint cartilage in the adult. Pluripotent stem cells (PSCs) are expected to differentiate in culture into any somatic cell type through processes that mimic embryogenesis, making human (h)PSCs a promising source of embryonic chondroprogenitors. The major research goals toward the clinical application of PSCs in joint cartilage regeneration are to (1) efficiently generate lineage-specific chondroprogenitors from hPSCs, (2) expand the chondroprogenitors to the number needed for therapy without loss of their chondrogenic activity, and (3) direct the in vivo or in vitro differentiation of the chondroprogenitors to articular or meniscal (i.e., permanent) chondrocytes rather than growth plate (i.e., transient) chondrocytes. This review is aimed at providing the current state of research toward meeting these goals. We also include our recent achievement of successful generation of "permanent-like" cartilage from long-term expandable, hPSC-derived ectomesenchymal chondroprogenitors.
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Affiliation(s)
- Naoki Nakayama
- Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston Medical School, 1825 Pressler St., Houston, TX, 77030, USA.
- Department of Orthopaedic Surgery, The University of Texas Health Science Center at Houston Medical School, Houston, TX, USA.
| | - Azim Pothiawala
- Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston Medical School, 1825 Pressler St., Houston, TX, 77030, USA
| | - John Y Lee
- Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston Medical School, 1825 Pressler St., Houston, TX, 77030, USA
- Miller School of Medicine, University of Miami, Miami, FL, USA
| | - Nadine Matthias
- Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston Medical School, 1825 Pressler St., Houston, TX, 77030, USA
| | - Katsutsugu Umeda
- Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston Medical School, 1825 Pressler St., Houston, TX, 77030, USA
- Department of Pediatrics, Kyoto University School of Medicine, Kyoto, Japan
| | - Bryan K Ang
- Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston Medical School, 1825 Pressler St., Houston, TX, 77030, USA
- Weil Cornell Medicine, New York, NY, USA
| | - Johnny Huard
- Department of Orthopaedic Surgery, The University of Texas Health Science Center at Houston Medical School, Houston, TX, USA
- Steadman Philippon Research Institute, Vail, CO, USA
| | - Yun Huang
- Institute of Bioscience and Technology, Texas A&M University, Houston, TX, USA
| | - Deqiang Sun
- Institute of Bioscience and Technology, Texas A&M University, Houston, TX, USA
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11
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Sundaramoorthy V, Godde N, J. Farr R, Green D, M. Haynes J, Bingham J, O’Brien CM, Dearnley M. Modelling Lyssavirus Infections in Human Stem Cell-Derived Neural Cultures. Viruses 2020; 12:E359. [PMID: 32218146 PMCID: PMC7232326 DOI: 10.3390/v12040359] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2019] [Revised: 03/02/2020] [Accepted: 03/20/2020] [Indexed: 12/20/2022] Open
Abstract
Rabies is a zoonotic neurological infection caused by lyssavirus that continues to result in devastating loss of human life. Many aspects of rabies pathogenesis in human neurons are not well understood. Lack of appropriate ex-vivo models for studying rabies infection in human neurons has contributed to this knowledge gap. In this study, we utilize advances in stem cell technology to characterize rabies infection in human stem cell-derived neurons. We show key cellular features of rabies infection in our human neural cultures, including upregulation of inflammatory chemokines, lack of neuronal apoptosis, and axonal transmission of viruses in neuronal networks. In addition, we highlight specific differences in cellular pathogenesis between laboratory-adapted and field strain lyssavirus. This study therefore defines the first stem cell-derived ex-vivo model system to study rabies pathogenesis in human neurons. This new model system demonstrates the potential for enabling an increased understanding of molecular mechanisms in human rabies, which could lead to improved control methods.
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Affiliation(s)
- Vinod Sundaramoorthy
- Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australian Animal Health Laboratory (AAHL), East Geelong, VIC 3219, Australia; (V.S.); (N.G.); (R.J.F.); (D.G.); (J.B.)
| | - Nathan Godde
- Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australian Animal Health Laboratory (AAHL), East Geelong, VIC 3219, Australia; (V.S.); (N.G.); (R.J.F.); (D.G.); (J.B.)
| | - Ryan J. Farr
- Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australian Animal Health Laboratory (AAHL), East Geelong, VIC 3219, Australia; (V.S.); (N.G.); (R.J.F.); (D.G.); (J.B.)
| | - Diane Green
- Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australian Animal Health Laboratory (AAHL), East Geelong, VIC 3219, Australia; (V.S.); (N.G.); (R.J.F.); (D.G.); (J.B.)
| | - John M. Haynes
- Monash Institute of Pharmaceutical Sciences, 399 Royal Parade, Parkville, VIC 3052, Australia;
| | - John Bingham
- Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australian Animal Health Laboratory (AAHL), East Geelong, VIC 3219, Australia; (V.S.); (N.G.); (R.J.F.); (D.G.); (J.B.)
| | - Carmel M. O’Brien
- CSIRO Manufacturing, Research Way, Clayton, VIC 3168, Australia
- Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 3168, Australia
| | - Megan Dearnley
- Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australian Animal Health Laboratory (AAHL), East Geelong, VIC 3219, Australia; (V.S.); (N.G.); (R.J.F.); (D.G.); (J.B.)
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12
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Seranova E, Palhegyi AM, Verma S, Dimova S, Lasry R, Naama M, Sun C, Barrett T, Rosenstock TR, Kumar D, Cohen MA, Buganim Y, Sarkar S. Human Induced Pluripotent Stem Cell Models of Neurodegenerative Disorders for Studying the Biomedical Implications of Autophagy. J Mol Biol 2020; 432:2754-2798. [PMID: 32044344 DOI: 10.1016/j.jmb.2020.01.024] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2019] [Revised: 01/22/2020] [Accepted: 01/23/2020] [Indexed: 12/12/2022]
Abstract
Autophagy is an intracellular degradation process that is essential for cellular survival, tissue homeostasis, and human health. The housekeeping functions of autophagy in mediating the clearance of aggregation-prone proteins and damaged organelles are vital for post-mitotic neurons. Improper functioning of this process contributes to the pathology of myriad human diseases, including neurodegeneration. Impairment in autophagy has been reported in several neurodegenerative diseases where pharmacological induction of autophagy has therapeutic benefits in cellular and transgenic animal models. However, emerging studies suggest that the efficacy of autophagy inducers, as well as the nature of the autophagy defects, may be context-dependent, and therefore, studies in disease-relevant experimental systems may provide more insights for clinical translation to patients. With the advancements in human stem cell technology, it is now possible to establish disease-affected cellular platforms from patients for investigating disease mechanisms and identifying candidate drugs in the appropriate cell types, such as neurons that are otherwise not accessible. Towards this, patient-derived human induced pluripotent stem cells (hiPSCs) have demonstrated considerable promise in constituting a platform for effective disease modeling and drug discovery. Multiple studies have utilized hiPSC models of neurodegenerative diseases to study autophagy and evaluate the therapeutic efficacy of autophagy inducers in neuronal cells. This review provides an overview of the regulation of autophagy, generation of hiPSCs via cellular reprogramming, and neuronal differentiation. It outlines the findings in various neurodegenerative disorders where autophagy has been studied using hiPSC models.
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Affiliation(s)
- Elena Seranova
- Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
| | - Adina Maria Palhegyi
- Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
| | - Surbhi Verma
- Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom; Cellular Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India
| | - Simona Dimova
- Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
| | - Rachel Lasry
- Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, The Hebrew University Hadassah Medical School, Jerusalem, 91120, Israel
| | - Moriyah Naama
- Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, The Hebrew University Hadassah Medical School, Jerusalem, 91120, Israel
| | - Congxin Sun
- Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
| | - Timothy Barrett
- Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
| | - Tatiana Rosado Rosenstock
- Department of Physiological Science, Santa Casa de São Paulo School of Medical Sciences, São Paulo, SP, 01221-020, Brazil
| | - Dhiraj Kumar
- Cellular Immunology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India
| | - Malkiel A Cohen
- Whitehead Institute for Biomedical Research, Cambridge, MA, 02142, USA
| | - Yosef Buganim
- Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, The Hebrew University Hadassah Medical School, Jerusalem, 91120, Israel
| | - Sovan Sarkar
- Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
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13
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Zheng Y, Xue X, Resto-Irizarry AM, Li Z, Shao Y, Zheng Y, Zhao G, Fu J. Dorsal-ventral patterned neural cyst from human pluripotent stem cells in a neurogenic niche. SCIENCE ADVANCES 2019; 5:eaax5933. [PMID: 31844664 PMCID: PMC6905871 DOI: 10.1126/sciadv.aax5933] [Citation(s) in RCA: 60] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/05/2019] [Accepted: 10/09/2019] [Indexed: 05/22/2023]
Abstract
Despite its importance in central nervous system development, development of the human neural tube (NT) remains poorly understood, given the challenges of studying human embryos, and the developmental divergence between humans and animal models. We report a human NT development model, in which NT-like tissues, neuroepithelial (NE) cysts, are generated in a bioengineered neurogenic environment through self-organization of human pluripotent stem cells (hPSCs). NE cysts correspond to the neural plate in the dorsal ectoderm and have a default dorsal identity. Dorsal-ventral (DV) patterning of NE cysts is achieved using retinoic acid and/or sonic hedgehog and features sequential emergence of the ventral floor plate, P3, and pMN domains in discrete, adjacent regions and a dorsal territory progressively restricted to the opposite dorsal pole. This hPSC-based, DV patterned NE cyst system will be useful for understanding the self-organizing principles that guide NT patterning and for investigations of neural development and neural disease.
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Affiliation(s)
- Y. Zheng
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
- Center for Biomedical Engineering, Department of Electronic Science and Technology, University of Science and Technology of China, Hefei 230027, Anhui, China
| | - X. Xue
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - A. M. Resto-Irizarry
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - Z. Li
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - Y. Shao
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - Y. Zheng
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
| | - G. Zhao
- Center for Biomedical Engineering, Department of Electronic Science and Technology, University of Science and Technology of China, Hefei 230027, Anhui, China
- Anhui Provincial Engineering Technology Research Center for Biopreservation and Artificial Organs, Hefei 230022, Anhui, China
| | - J. Fu
- Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
- Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
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14
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Continuous Immune-Modulatory Effects of Human Olig2+ Precursor Cells Attenuating a Chronic-Active Model of Multiple Sclerosis. Mol Neurobiol 2019; 57:1021-1034. [DOI: 10.1007/s12035-019-01802-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2018] [Accepted: 07/10/2019] [Indexed: 01/17/2023]
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15
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Jung-Klawitter S, Opladen T. Induced pluripotent stem cells (iPSCs) as model to study inherited defects of neurotransmission in inborn errors of metabolism. J Inherit Metab Dis 2018; 41:1103-1116. [PMID: 29980968 DOI: 10.1007/s10545-018-0225-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/28/2018] [Revised: 05/08/2018] [Accepted: 06/25/2018] [Indexed: 11/29/2022]
Abstract
The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs) has revolutionized the way of modeling human disease. Especially for the modeling of rare human monogenetic diseases with limited numbers of patients available worldwide and limited access to the mostly affected tissues, iPSCs have become an invaluable tool. To study rare diseases affecting neurotransmitter biosynthesis and neurotransmission, stem cell models carrying patient-specific mutations have become highly important as most of the cell types present in the human brain and the central nervous system (CNS), including motoneurons, neurons, oligodendrocytes, astrocytes, and microglia, can be differentiated from iPSCs following distinct developmental programs. Differentiation can be performed using classical 2D differentiation protocols, thereby generating specific subtypes of neurons or glial cells in a dish. On the other side, 3D differentiation into "organoids" opened new ways to study misregulated developmental processes associated with rare neurological and neurometabolic diseases. For the analysis of defects in neurotransmission associated with rare neurometabolic diseases, different types of brain organoids have been made available during the last years including forebrain, midbrain and cerebral organoids. In this review, we illustrate reprogramming of somatic cells to iPSCs, differentiation in 2D and 3D, as well as already available disease-specific iPSC models, and discuss current and future applications of these techniques.
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Affiliation(s)
- Sabine Jung-Klawitter
- Department of General Pediatrics, Division of Neuropediatrics and Metabolic Medicine, University Hospital Heidelberg, Im Neuenheimer Feld 669, 69120, Heidelberg, Germany.
| | - Thomas Opladen
- Department of General Pediatrics, Division of Neuropediatrics and Metabolic Medicine, University Hospital Heidelberg, Im Neuenheimer Feld 669, 69120, Heidelberg, Germany
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16
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Liu Y, Antonic A, Yang X, Korte N, Lim K, Michalska AE, Dottori M, Howells DW. Derivation of phenotypically diverse neural culture from hESC by combining adherent and dissociation methods. J Neurosci Methods 2018; 308:286-293. [PMID: 30003885 DOI: 10.1016/j.jneumeth.2018.07.005] [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] [Received: 01/13/2018] [Revised: 07/05/2018] [Accepted: 07/05/2018] [Indexed: 11/18/2022]
Abstract
BACKGROUND Differentiation of human embryonic stem cells (hESCs) into distinct neural lineages has been widely studied. However, preparation of mixed yet neurochemically mature populations, for the study of neurological diseases involving mixed cell types has received less attention. NEW METHOD We combined two commonly used differentiation methods to provide robust and reproducible cultures in which a mixture of primarily GABAergic and Glutamatergic neurons was obtained. Detailed characterisation by immunocytochemistry (ICC) and quantitative real-time PCR (qPCR) assessed the neurochemical phenotype, and the maturation state of these neurons. RESULTS We found that once neurospheres (NSs) had attached to the culture plates, proliferation of neural stem cell was suppressed. Neuronal differentiation and synaptic development then occurred after 21 days in vitro (DIV). By 49DIV, there were large numbers of neurochemically and structurally mature neurons. The qPCR studies indicated that expression of GABAergic genes increased the most (93.3-fold increase), followed by glutamatergic (51-fold increase), along with smaller changes in expression of cholinergic (3-fold increase) and dopaminergic genes (6-fold increase), as well as a small change in glial cell marker expression (5-fold increase). COMPARISON WITH EXISTING METHOD (S) Existing methods isolate hESC-derived neural progenitors for onward differentiation to mature neurons using either migration or dissociative paradigms. These give poor survival or yield. By combining these approaches, we obtain high yields of morphologically and neurochemically mature neurons. These can be maintained in culture for extended periods. CONCLUSION Our method provides a novel, effective and robust neural culture system with structurally and neurochemically mature cell populations and neural networks, suitable for studying a range of neurological diseases from a human perspective.
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Affiliation(s)
- Ye Liu
- Florey Institute of Neuroscience and Mental Health, 30 Royal Parade, The University of Melbourne, Victoria, 3010, Australia; Department of Neurology, Fudan University, Huashan Hospital, Shanghai, 200040, China
| | - Ana Antonic
- Department of Neuroscience, Central Clinical School, Monash University, The Alfred Centre, VIC, 3004, Australia
| | - Xuan Yang
- Institute for Geriatrics and Rehabilitation, Beijing Geriatric Hospital, Beijing, 100095, China
| | - Nils Korte
- Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London, WC1E6BT, UK
| | - Katherine Lim
- Stem Cell Core Facility, Stem Cells Australia, The University of Melbourne, Victoria, 3010, Australia
| | - Anna E Michalska
- Stem Cell Core Facility, Stem Cells Australia, The University of Melbourne, Victoria, 3010, Australia
| | - Mirella Dottori
- Illawarra Health and Medical Research Institute Centre for Molecular and Medical Bioscience Building 32, University of Wollongong, NSW, 2522 Australia
| | - David W Howells
- School of Medicine, University of Tasmania, Hobart, Tasmania, 7001, Australia.
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17
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Towards Multi-Organoid Systems for Drug Screening Applications. Bioengineering (Basel) 2018; 5:bioengineering5030049. [PMID: 29933623 PMCID: PMC6163436 DOI: 10.3390/bioengineering5030049] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2018] [Revised: 06/15/2018] [Accepted: 06/19/2018] [Indexed: 12/13/2022] Open
Abstract
A low percentage of novel drug candidates succeed and reach the end of the drug discovery pipeline, mainly due to poor initial screening and assessment of the effects of the drug and its metabolites over various tissues in the human body. For that, emerging technologies involving the production of organoids from human pluripotent stem cells (hPSCs) and the use of organ-on-a-chip devices are showing great promise for developing a more reliable, rapid and cost-effective drug discovery process when compared with the current use of animal models. In particular, the possibility of virtually obtaining any type of cell within the human body, in combination with the ability to create patient-specific tissues using human induced pluripotent stem cells (hiPSCs), broadens the horizons in the fields of drug discovery and personalized medicine. In this review, we address the current progress and challenges related to the process of obtaining organoids from different cell lineages emerging from hPSCs, as well as how to create devices that will allow a precise examination of the in vitro effects generated by potential drugs in different organ systems.
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18
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Effect of prolonged differentiation on functional maturation of human pluripotent stem cell-derived neuronal cultures. Stem Cell Res 2018; 27:151-161. [PMID: 29414606 DOI: 10.1016/j.scr.2018.01.018] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/10/2017] [Revised: 01/09/2018] [Accepted: 01/17/2018] [Indexed: 01/15/2023] Open
Abstract
Long-term neural differentiation of human pluripotent stem cells (hPSCs) is associated with enhanced neuronal maturation, which is a necessity for creation of representative in vitro models. It also induces neurogenic-to-gliogenic fate switch, increasing proportion of endogenous astrocytes formed from the common neural progenitors. However, the significance of prolonged differentiation on the neural cell type composition and functional development of hPSC-derived neuronal cells has not been well characterized. Here, we studied two hPSC lines, both of which initially showed good neuronal differentiation capacity. However, the propensity for endogenous astrogenesis and maturation state after extended differentiation varied. Live cell calcium imaging revealed that prolonged differentiation facilitated maturation of GABAergic signaling. According to extracellular recordings with microelectrode array (MEA), neuronal activity was limited to fewer areas of the culture, which expressed more frequent burst activity. Efficient maturation after prolonged differentiation also promoted organization of spontaneous activity by burst compaction. These results suggest that although prolonged neural differentiation can be challenging, it has beneficial effect on functional maturation, which can also improve transition to different neural in vitro models and applications.
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19
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Zhu JD, Wang JJ, Ge G, Kang CS. Effects of Noggin-Transfected Neural Stem Cells on Neural Functional Recovery and Underlying Mechanism in Rats with Cerebral Ischemia Reperfusion Injury. J Stroke Cerebrovasc Dis 2017; 26:1547-1559. [PMID: 28478981 DOI: 10.1016/j.jstrokecerebrovasdis.2017.02.034] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2016] [Revised: 01/23/2017] [Accepted: 02/02/2017] [Indexed: 01/06/2023] Open
Abstract
OBJECTIVE To investigate neuroprotection of noggin-transfected neural stem cells (NSCs) against focal cerebral ischemia reperfusion injury (IRI) in rats. METHODS Eighty Wistar rats were randomly divided into the sham, IRI, NSCs, and noggin + NSCs groups. Noggin containing adenoviral vectors was transfected into rat NSCs. Rats were subjected to 2.0 hours middle cerebral artery occlusion and reperfusion 1.0 hour, followed by infusion into the lateral ventricles of NSCs alone, noggin-transfected NSCs, and saline at 3 days in the NSCs, noggin + NSCs, and sham groups, respectively. All rats were sacrificed on 1, 3, 7, and 28 days after transplantation; the colorimetric method was used to detect the levels of superoxide dismutase (SOD) and the malondialdehyde (MDA) content after the behavior capability determined. Western blot was performed for detecting the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) proteins. The TUNEL-positive and BrdU/nestin double-positive cells were observed under a light microscope and quantitative analysis was performed by morphometric technique. RESULTS Noggin-transfected NSCs significantly decreased the infarct volume and improved the neurological scores. Noggin-transfected NSCs also reduced the percentage of apoptotic neurons and relieved neuronal morphological damage. Noggin-transfected NSC transplantation markedly decreased the MDA levels and increased the SOD activity, and simultaneously downregulated the BMP4 (bone morphogenesis protein), VEGF, and bFGF proteins. CONCLUSIONS The present study demonstrates that grafting NSCs modified by noggin gene provides better neuroprotection for cerebrovascular disease.
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Affiliation(s)
- Jun-de Zhu
- Department of Anatomy, School of Basic Medicine, Guizhou Medical University, Guian New Area, China.
| | - Jun-Jie Wang
- Department of Anatomy, School of Basic Medicine, Guizhou Medical University, Guian New Area, China
| | - Guo Ge
- Department of Anatomy, School of Basic Medicine, Guizhou Medical University, Guian New Area, China
| | - Chao-Sheng Kang
- Department of Anatomy, School of Basic Medicine, Guizhou Medical University, Guian New Area, China
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20
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Lukovic D, Diez Lloret A, Stojkovic P, Rodríguez-Martínez D, Perez Arago MA, Rodriguez-Jimenez FJ, González-Rodríguez P, López-Barneo J, Sykova E, Jendelova P, Kostic J, Moreno-Manzano V, Stojkovic M, Bhattacharya SS, Erceg S. Highly Efficient Neural Conversion of Human Pluripotent Stem Cells in Adherent and Animal-Free Conditions. Stem Cells Transl Med 2017; 6:1217-1226. [PMID: 28213969 PMCID: PMC5442830 DOI: 10.1002/sctm.16-0371] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2016] [Revised: 10/20/2016] [Accepted: 11/16/2016] [Indexed: 12/23/2022] Open
Abstract
Neural differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can produce a valuable and robust source of human neural cell subtypes, holding great promise for the study of neurogenesis and development, and for treating neurological diseases. However, current hESCs and hiPSCs neural differentiation protocols require either animal factors or embryoid body formation, which decreases efficiency and yield, and strongly limits medical applications. Here we develop a simple, animal-free protocol for neural conversion of both hESCs and hiPSCs in adherent culture conditions. A simple medium formula including insulin induces the direct conversion of >98% of hESCs and hiPSCs into expandable, transplantable, and functional neural progenitors with neural rosette characteristics. Further differentiation of neural progenitors into dopaminergic and spinal motoneurons as well as astrocytes and oligodendrocytes indicates that these neural progenitors retain responsiveness to instructive cues revealing the robust applicability of the protocol in the treatment of different neurodegenerative diseases. The fact that this protocol includes animal-free medium and human extracellular matrix components avoiding embryoid bodies makes this protocol suitable for the use in clinic. Stem Cells Translational Medicine 2017;6:1217-1226.
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Affiliation(s)
- Dunja Lukovic
- Stem Cells Therapies in Neurodegenerative Diseases Lab.,National Stem Cell Bank-Valencia Node, Biomolecular and Bioinformatics Resources Platform PRB2,ISCIII
| | - Andrea Diez Lloret
- CABIMER (Centro Andaluz de Biología Molecular y Medicina Regenerativa), Avda. Americo Vespucio s/n, Parque Científico y Tecnológico Cartuja, Sevilla, Spain
| | | | - Daniel Rodríguez-Martínez
- CABIMER (Centro Andaluz de Biología Molecular y Medicina Regenerativa), Avda. Americo Vespucio s/n, Parque Científico y Tecnológico Cartuja, Sevilla, Spain
| | | | | | - Patricia González-Rodríguez
- Instituto de Biomedicina de Sevilla (IBiS) and Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain
| | - José López-Barneo
- Instituto de Biomedicina de Sevilla (IBiS) and Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain
| | - Eva Sykova
- Department of Neuroscience, Institute of Experimental Medicine, Academy of Science of the Czech Republic, Prague, Czech Republic
| | - Pavla Jendelova
- Department of Neuroscience, Institute of Experimental Medicine, Academy of Science of the Czech Republic, Prague, Czech Republic
| | - Jelena Kostic
- Stem Cells Therapies in Neurodegenerative Diseases Lab
| | | | - Miodrag Stojkovic
- Spebo Medical, Leskovac, Serbia.,Faculty of Medical Sciences, Human Genetics Department, University of Kragujevac, Serbia
| | - Shomi S Bhattacharya
- CABIMER (Centro Andaluz de Biología Molecular y Medicina Regenerativa), Avda. Americo Vespucio s/n, Parque Científico y Tecnológico Cartuja, Sevilla, Spain
| | - Slaven Erceg
- Stem Cells Therapies in Neurodegenerative Diseases Lab.,National Stem Cell Bank-Valencia Node, Biomolecular and Bioinformatics Resources Platform PRB2,ISCIII.,Department of Neuroscience, Institute of Experimental Medicine, Academy of Science of the Czech Republic, Prague, Czech Republic
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21
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Murphy AR, Ghobrial I, Jamshidi P, Laslett A, O'Brien CM, Cameron NR. Tailored emulsion-templated porous polymer scaffolds for iPSC-derived human neural precursor cell culture. Polym Chem 2017. [DOI: 10.1039/c7py01375b] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
The work here describes the synthesis of tailor-made, porous, polymeric materials with elastic moduli in the range associated with mammalian brain tissue (0.1–24 kPa).
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Affiliation(s)
- Ashley R. Murphy
- Department of Materials Science and Engineering
- Monash University
- Clayton
- Australia
| | - Irene Ghobrial
- Australian Regenerative Medicine Institute
- Science
- Technology
- Research and Innovation Precinct (STRIP)
- Monash University
| | - Pegah Jamshidi
- Australian Regenerative Medicine Institute
- Science
- Technology
- Research and Innovation Precinct (STRIP)
- Monash University
| | - Andrew Laslett
- Australian Regenerative Medicine Institute
- Science
- Technology
- Research and Innovation Precinct (STRIP)
- Monash University
| | - Carmel M. O'Brien
- Australian Regenerative Medicine Institute
- Science
- Technology
- Research and Innovation Precinct (STRIP)
- Monash University
| | - Neil R. Cameron
- Department of Materials Science and Engineering
- Monash University
- Clayton
- Australia
- School of Engineering
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22
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Chandrasekaran A, Avci HX, Leist M, Kobolák J, Dinnyés A. Astrocyte Differentiation of Human Pluripotent Stem Cells: New Tools for Neurological Disorder Research. Front Cell Neurosci 2016; 10:215. [PMID: 27725795 PMCID: PMC5035736 DOI: 10.3389/fncel.2016.00215] [Citation(s) in RCA: 114] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2016] [Accepted: 08/30/2016] [Indexed: 12/22/2022] Open
Abstract
Astrocytes have a central role in brain development and function, and so have gained increasing attention over the past two decades. Consequently, our knowledge about their origin, differentiation and function has increased significantly, with new research showing that astrocytes cultured alone or co-cultured with neurons have the potential to improve our understanding of various central nervous system diseases, such as amyotrophic lateral sclerosis, Alzheimer’s disease, or Alexander disease. The generation of astrocytes derived from pluripotent stem cells (PSCs) opens up a new area for studying neurologic diseases in vitro; these models could be exploited to identify and validate potential drugs by detecting adverse effects in the early stages of drug development. However, as it is now known that a range of astrocyte populations exist in the brain, it will be important in vitro to develop standardized protocols for the in vitro generation of astrocyte subsets with defined maturity status and phenotypic properties. This will then open new possibilities for co-cultures with neurons and the generation of neural organoids for research purposes. The aim of this review article is to compare and summarize the currently available protocols and their strategies to generate human astrocytes from PSCs. Furthermore, we discuss the potential role of human-induced PSCs derived astrocytes in disease modeling.
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Affiliation(s)
| | - Hasan X Avci
- BioTalentum LtdGödöllő, Hungary; Department of Medical Chemistry, University of SzegedSzeged, Hungary
| | - Marcel Leist
- Dorenkamp-Zbinden Chair, Faculty of Mathematics and Sciences, University of Konstanz Konstanz, Germany
| | | | - Andras Dinnyés
- BioTalentum LtdGödöllő, Hungary; Molecular Animal Biotechnology Laboratory, Szent Istvan UniversityGödöllő, Hungary
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23
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Fazeli Z, Omrani MD, Ghaderian SMH. CD29/CD184 expression analysis provides a signature for identification of neuronal like cells differentiated from PBMSCs. Neurosci Lett 2016; 630:189-193. [DOI: 10.1016/j.neulet.2016.07.056] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2016] [Revised: 07/23/2016] [Accepted: 07/28/2016] [Indexed: 12/31/2022]
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24
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Human Embryonic Stem Cells: A Model for the Study of Neural Development and Neurological Diseases. Stem Cells Int 2016; 2016:2958210. [PMID: 27239201 PMCID: PMC4864561 DOI: 10.1155/2016/2958210] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2015] [Accepted: 03/14/2016] [Indexed: 01/05/2023] Open
Abstract
Although the mechanism of neurogenesis has been well documented in other organisms, there might be fundamental differences between human and those species referring to species-specific context. Based on principles learned from other systems, it is found that the signaling pathways required for neural induction and specification of human embryonic stem cells (hESCs) recapitulated those in the early embryo development in vivo at certain degree. This underscores the usefulness of hESCs in understanding early human neural development and reinforces the need to integrate the principles of developmental biology and hESC biology for an efficient neural differentiation.
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25
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Yuen SM, Kwok HF. Temporal establishment of neural cell identity in vivo and in vitro. J Tissue Eng Regen Med 2016; 11:2582-2589. [PMID: 27061786 DOI: 10.1002/term.2158] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2015] [Accepted: 01/29/2016] [Indexed: 01/09/2023]
Abstract
Understanding cell fate specification is particularly useful because it enables biologists to generate specific neural cell types for treating currently untreatable neurological diseases. Traditionally, lineage-specific progenitors are generated in vitro from pluripotent cells, after which they may be channeled into more mature cell types in a stage-specific manner, which is similar to the way cells behave during development. However, the emergence of induced pluripotent stem cells means that specific cell types can be generated directly from fibroblasts or other somatic cell types, thus bypassing all of the necessary steps that happen in vivo. Based on this information, the present review first explores the regulatory circuitry that drives cell fate specification over time in vivo. In particular, it describes how the appearance of specific neuronal and glial cell types is governed by an intrinsic biological clock, followed by a discussion of how this can be achieved through the temporal expression of intracellular regulators in relation to cell-specific Dnase I hypersensitivity sites, promoters and enhancers. Cell fate acquisition in vitro was then examined in an attempt to evaluate whether the temporal regulation neural cell fate in vivo is still relevant to the generation of reprogrammed neural stem cells and neurons. Copyright © 2016 John Wiley & Sons, Ltd.
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Affiliation(s)
- Shun Ming Yuen
- Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau, SAR, China
| | - Hang Fai Kwok
- Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau, SAR, China
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26
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Lu H, Liu X, Zhang N, Zhu X, Liang H, Sun L, Cheng Y. Neuroprotective Effects of Brain-Derived Neurotrophic Factor and Noggin-Modified Bone Mesenchymal Stem Cells in Focal Cerebral Ischemia in Rats. J Stroke Cerebrovasc Dis 2016; 25:410-8. [DOI: 10.1016/j.jstrokecerebrovasdis.2015.10.013] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2015] [Revised: 09/09/2015] [Accepted: 10/17/2015] [Indexed: 11/24/2022] Open
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27
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Generating Diverse Spinal Motor Neuron Subtypes from Human Pluripotent Stem Cells. Stem Cells Int 2015; 2016:1036974. [PMID: 26823667 PMCID: PMC4707335 DOI: 10.1155/2016/1036974] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2015] [Accepted: 09/14/2015] [Indexed: 12/18/2022] Open
Abstract
Resolving the mechanisms underlying human neuronal diversification remains a major challenge in developmental and applied neurobiology. Motor neurons (MNs) represent a diverse pool of neuronal subtypes exhibiting differential vulnerability in different human neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). The ability to predictably manipulate MN subtype lineage restriction from human pluripotent stem cells (PSCs) will form the essential basis to establishing accurate, clinically relevant in vitro disease models. I first overview motor neuron developmental biology to provide some context for reviewing recent studies interrogating pathways that influence the generation of MN diversity. I conclude that motor neurogenesis from PSCs provides a powerful reductionist model system to gain insight into the developmental logic of MN subtype diversification and serves more broadly as a leading exemplar of potential strategies to resolve the molecular basis of neuronal subclass differentiation within the nervous system. These studies will in turn permit greater mechanistic understanding of differential MN subtype vulnerability using in vitro human disease models.
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28
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Yang H, Liu Y, Hai Y, Guo Y, Yang S, Li Z, Gao WQ, He Z. Efficient Conversion of Spermatogonial Stem Cells to Phenotypic and Functional Dopaminergic Neurons via the PI3K/Akt and P21/Smurf2/Nolz1 Pathway. Mol Neurobiol 2015; 52:1654-1669. [PMID: 25373443 DOI: 10.1007/s12035-014-8966-4] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2014] [Accepted: 10/24/2014] [Indexed: 01/10/2023]
Abstract
Parkinson's disease (PD) is a common neurodegenerative syndrome characterized by loss of midbrain dopaminergic (DA) neurons. Generation of functional dopaminergic (DA) neurons is of unusual significance for treating Parkinson's disease (PD). However, direct conversion of spermatogonial stem cells (SSCs) to functional DA neurons without being reprogrammed to a pluripotent status has not been achieved. Here, we report an efficient approach to obtain morphological, phenotypic, and functional DA neurons from SSCs using a specific combination of olfactory ensheathing cell-conditioned medium (OECCM) and several defined growth factors (DGF). By following the current protocol, direct conversion of SSCs (both SSC line and primary SSCs) to neural cells and DA neurons was demonstrated by expression of numerous phenotypic genes and proteins for neural cells, as well as cell morphological features. More significantly, SSCs-derived DA neurons acquired neuronal functional properties such as synapse formation, electrophysiology activity, and dopamine secretion. Furthermore, PI3K/Akt pathway and p21/Nolz1 cascades were activated whereas Smurf2 was inactivated, leading to cell cycle exit during the conversion of SSCs into DA neurons. Collectively, this study could provide sufficient neural cells from SSCs for applications in the treatment of PD and offers novel insights into mechanisms underlying neural system development from the line of germ cells.
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Affiliation(s)
- Hao Yang
- State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China.
| | - Yang Liu
- State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China
| | - Yanan Hai
- State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China
| | - Ying Guo
- State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China
| | - Shi Yang
- Department of Urology, Shanghai Human Sperm Bank, Shanghai Institute of Andrology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 145 Shangdong Road, Shanghai, 200001, China
| | - Zheng Li
- Department of Urology, Shanghai Human Sperm Bank, Shanghai Institute of Andrology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 145 Shangdong Road, Shanghai, 200001, China
| | - Wei-Qiang Gao
- State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China
| | - Zuping He
- State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China.
- Department of Urology, Shanghai Human Sperm Bank, Shanghai Institute of Andrology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 145 Shangdong Road, Shanghai, 200001, China.
- Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai, 200127, China.
- Shanghai Key Laboratory of Reproductive Medicine, Shanghai, 200025, China.
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Current Neurogenic and Neuroprotective Strategies to Prevent and Treat Neurodegenerative and Neuropsychiatric Disorders. Neuromolecular Med 2015; 17:404-22. [PMID: 26374113 DOI: 10.1007/s12017-015-8369-3] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2015] [Accepted: 08/22/2015] [Indexed: 12/31/2022]
Abstract
The adult central nervous system is commonly known to have a very limited regenerative capacity. The presence of functional stem cells in the brain can therefore be seen as a paradox, since in other organs these are known to counterbalance cell loss derived from pathological conditions. This fact has therefore raised the possibility to stimulate neural stem cell differentiation and proliferation or survival by either stem cell replacement therapy or direct administration of neurotrophic factors or other proneurogenic molecules, which in turn has also originated regenerative medicine for the treatment of otherwise incurable neurodegenerative and neuropsychiatric disorders that take a huge toll on society. This may be facilitated by the fact that many of these disorders converge on similar pathophysiological pathways: excitotoxicity, oxidative stress, neuroinflammation, mitochondrial failure, excessive intracellular calcium and apoptosis. This review will therefore focus on the most promising achievements in promoting neuroprotection and neuroregeneration reported to date.
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Gallegos-Cárdenas A, Webb R, Jordan E, West R, West FD, Yang JY, Wang K, Stice SL. Pig Induced Pluripotent Stem Cell-Derived Neural Rosettes Developmentally Mimic Human Pluripotent Stem Cell Neural Differentiation. Stem Cells Dev 2015; 24:1901-11. [DOI: 10.1089/scd.2015.0025] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Affiliation(s)
- Amalia Gallegos-Cárdenas
- Regenerative Bioscience Center, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
- Department of Animal and Dairy Science, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
- Departamento de Producción Animal, Facultad de Zootecnia, Universidad Nacional Agraria La Molina, Girona, Perú
| | - Robin Webb
- Regenerative Bioscience Center, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
- Department of Animal and Dairy Science, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
| | - Erin Jordan
- Regenerative Bioscience Center, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
- Department of Animal and Dairy Science, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
| | - Rachel West
- Regenerative Bioscience Center, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
- Department of Animal and Dairy Science, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
| | - Franklin D. West
- Regenerative Bioscience Center, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
- Department of Animal and Dairy Science, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
| | - Jeong-Yeh Yang
- Regenerative Bioscience Center, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
- Department of Animal and Dairy Science, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
| | - Kai Wang
- Regenerative Bioscience Center, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
- Department of Animal and Dairy Science, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
| | - Steven L. Stice
- Regenerative Bioscience Center, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
- Department of Animal and Dairy Science, University of Georgia, Rhodes Center for Animal and Dairy Science, Athens, Georgia
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31
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Mulloy B, Rider CC. The Bone Morphogenetic Proteins and Their Antagonists. VITAMINS AND HORMONES 2015; 99:63-90. [PMID: 26279373 DOI: 10.1016/bs.vh.2015.06.004] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The bone morphogenetic proteins (BMPs) and the growth and differentiation factors comprise a single family of some 20 homologous, dimeric cytokines which share the cystine-knot domain typical of the TGF-β superfamily. They control the differentiation and activity of a range of cell types, including many outside bone and cartilage. They serve as developmental morphogens, but are also important in chronic pathologies, including tissue fibrosis and cancer. One mechanism for enabling tight spatiotemporal control of their activities is through a number of antagonist proteins, including Noggin, Follistatin, Chordin, Twisted gastrulation (TSG), and the seven members of the Cerberus and Dan family. These antagonists are secreted proteins that bind selectively to particular BMPs with high affinity, thereby blocking receptor engagement and signaling. Most of these antagonists also possess a TGF-β cystine-knot domain. Here, we discuss current knowledge and understanding of the structures and activities of the BMPs and their antagonists, with a particular focus on the latter proteins. Recent advances in structural biology of BMP antagonists have begun the process of elucidating the molecular basis of their activity, displaying a surprising variety between the modes of action of these closely related proteins. We also discuss the interactions of the antagonists with the glycosaminoglycan heparan sulfate, which is found ubiquitously on cell surfaces and in the extracellular matrix.
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Affiliation(s)
- Barbara Mulloy
- Centre for Biomedical Sciences, School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, United Kingdom
| | - Chris C Rider
- Centre for Biomedical Sciences, School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, United Kingdom.
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Neural Progenitor Cells Derived from Human Embryonic Stem Cells as an Origin of Dopaminergic Neurons. Stem Cells Int 2015; 2015:647437. [PMID: 26064138 PMCID: PMC4430666 DOI: 10.1155/2015/647437] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2015] [Revised: 04/11/2015] [Accepted: 04/14/2015] [Indexed: 12/14/2022] Open
Abstract
Human embryonic stem cells (hESCs) are able to proliferate in vitro indefinitely without losing their ability to differentiate into multiple cell types upon exposure to appropriate signals. Particularly, the ability of hESCs to differentiate into neuronal subtypes is fundamental to develop cell-based therapies for several neurodegenerative disorders, such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. In this study, we differentiated hESCs to dopaminergic neurons via an intermediate stage, neural progenitor cells (NPCs). hESCs were induced to neural progenitor cells by Dorsomorphin, a small molecule that inhibits BMP signalling. The resulting neural progenitor cells exhibited neural bipolarity with high expression of neural progenitor genes and possessed multipotential differentiation ability. CBF1 and bFGF responsiveness of these hES-NP cells suggested their similarity to embryonic neural progenitor cells. A substantial number of dopaminergic neurons were derived from hES-NP cells upon supplementation of FGF8 and SHH, key dopaminergic neuron inducers. Importantly, multiple markers of midbrain neurons were detected, including NURR1, PITX3, and EN1, suggesting that hESC-derived dopaminergic neurons attained the midbrain identity. Altogether, this work underscored the generation of neural progenitor cells that retain the properties of embryonic neural progenitor cells. These cells will serve as an unlimited source for the derivation of dopaminergic neurons, which might be applicable for treating patients with Parkinson's disease.
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Miranda CC, Fernandes TG, Pascoal JF, Haupt S, Brüstle O, Cabral JMS, Diogo MM. Spatial and temporal control of cell aggregation efficiently directs human pluripotent stem cells towards neural commitment. Biotechnol J 2015; 10:1612-24. [PMID: 25866360 DOI: 10.1002/biot.201400846] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2014] [Revised: 03/12/2015] [Accepted: 04/04/2015] [Indexed: 02/03/2023]
Abstract
3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work, we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precursors using 3D suspension conditions and chemically-defined culture media. We evaluated different inoculation methodologies for hPSC expansion as 3D aggregates and characterized the resulting cultures in terms of aggregate size distribution. It was demonstrated that upon single-cell inoculation, after four days of culture, 3D aggregates were composed of homogenous populations of hPSC and were characterized by an average diameter of 139 ± 26 μm, which was determined to be the optimal size to initiate neural commitment. Temporal analysis revealed that upon neural specification it is possible to maximize the percentage of neural precursor cells expressing the neural markers Sox1 and Pax6 after nine days of culture. These results highlight our ability to define a robust method for production of hPSC-derived neural precursors that minimizes processing steps and that constitutes a promising alternative to the traditional planar adherent culture system due to a high potential for scaling-up.
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Affiliation(s)
- Cláudia C Miranda
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
| | - Tiago G Fernandes
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
| | - Jorge F Pascoal
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
| | - Simone Haupt
- Institute of Reconstructive Neurobiology, University of Bonn and Hertie Foundation, Bonn, Germany.,LIFE & BRAIN GmbH, Bonn, Germany
| | - Oliver Brüstle
- Institute of Reconstructive Neurobiology, University of Bonn and Hertie Foundation, Bonn, Germany.,LIFE & BRAIN GmbH, Bonn, Germany
| | - Joaquim M S Cabral
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
| | - Maria Margarida Diogo
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
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Nicaise C, Mitrecic D, Falnikar A, Lepore AC. Transplantation of stem cell-derived astrocytes for the treatment of amyotrophic lateral sclerosis and spinal cord injury. World J Stem Cells 2015; 7:380-398. [PMID: 25815122 PMCID: PMC4369494 DOI: 10.4252/wjsc.v7.i2.380] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/29/2014] [Revised: 10/07/2014] [Accepted: 11/19/2014] [Indexed: 02/06/2023] Open
Abstract
Neglected for years, astrocytes are now recognized to fulfill and support many, if not all, homeostatic functions of the healthy central nervous system (CNS). During neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and spinal cord injury (SCI), astrocytes in the vicinity of degenerating areas undergo both morphological and functional changes that might compromise their intrinsic properties. Evidence from human and animal studies show that deficient astrocyte functions or loss-of-astrocytes largely contribute to increased susceptibility to cell death for neurons, oligodendrocytes and axons during ALS and SCI disease progression. Despite exciting advances in experimental CNS repair, most of current approaches that are translated into clinical trials focus on the replacement or support of spinal neurons through stem cell transplantation, while none focus on the specific replacement of astroglial populations. Knowing the important functions carried out by astrocytes in the CNS, astrocyte replacement-based therapies might be a promising approach to alleviate overall astrocyte dysfunction, deliver neurotrophic support to degenerating spinal tissue and stimulate endogenous CNS repair abilities. Enclosed in this review, we gathered experimental evidence that argue in favor of astrocyte transplantation during ALS and SCI. Based on their intrinsic properties and according to the cell type transplanted, astrocyte precursors or stem cell-derived astrocytes promote axonal growth, support mechanisms and cells involved in myelination, are able to modulate the host immune response, deliver neurotrophic factors and provide protective molecules against oxidative or excitotoxic insults, amongst many possible benefits. Embryonic or adult stem cells can even be genetically engineered in order to deliver missing gene products and therefore maximize the chance of neuroprotection and functional recovery. However, before broad clinical translation, further preclinical data on safety, reliability and therapeutic efficiency should be collected. Although several technical challenges need to be overcome, we discuss the major hurdles that have already been met or solved by targeting the astrocyte population in experimental ALS and SCI models and we discuss avenues for future directions based on latest molecular findings regarding astrocyte biology.
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35
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Xiao Y, Fu H, Han X, Hu X, Gu H, Chen Y, Wei Q, Hu Q. Role of synaptic structural plasticity in impairments of spatial learning and memory induced by developmental lead exposure in Wistar rats. PLoS One 2014; 9:e115556. [PMID: 25536363 PMCID: PMC4275220 DOI: 10.1371/journal.pone.0115556] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2014] [Accepted: 11/24/2014] [Indexed: 11/19/2022] Open
Abstract
Lead (Pb) is found to impair cognitive function. Synaptic structural plasticity is considered to be the physiological basis of synaptic functional plasticity and has been recently found to play important roles in learning and memory. To study the effect of Pb on spatial learning and memory at different developmental stages, and its relationship with alterations of synaptic structural plasticity, postnatal rats were randomly divided into three groups: Control; Pre-weaning Pb (Parents were exposed to 2 mM PbCl2 3 weeks before mating until weaning of pups); Post-weaning Pb (Weaned pups were exposed to 2 mM PbCl2 for 9 weeks). The spatial learning and memory of rats was measured by Morris water maze (MWM) on PND 85–90. Rat pups in Pre-weaning Pb and Post-weaning Pb groups performed significantly worse than those in Control group (p<0.05). However, there was no significant difference in the performance of MWM between the two Pb-exposure groups. Before MWM (PND 84), the number of neurons and synapses significantly decreased in Pre-weaning Pb group, but not in Post-weaning Pb group. After MWM (PND 91), the number of synapses in Pre-weaning Pb group increased significantly, but it was still less than that of Control group (p<0.05); the number of synapses in Post-weaning Pb group was also less than that of Control group (p<0.05), although the number of synapses has no differences between Post-weaning Pb and Control groups before MWM. In both Pre-weaning Pb and Post-weaning Pb groups, synaptic structural parameters such as thickness of postsynaptic density (PSD), length of synaptic active zone and synaptic curvature increased significantly while width of synaptic cleft decreased significantly compared to Control group (p<0.05). Our data demonstrated that both early and late developmental Pb exposure impaired spatial learning and memory as well as synaptic structural plasticity in Wistar rats.
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Affiliation(s)
- Yongmei Xiao
- Department of Preventive Medicine, School of Public Health, Sun Yat-Sen University, Guangzhou 510080, China
| | - Hongjun Fu
- The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609, United States of America
| | - Xiaojie Han
- Department of Preventive Medicine, School of Public Health, Sun Yat-Sen University, Guangzhou 510080, China
| | - Xiaoxia Hu
- Department of Preventive Medicine, School of Public Health, Sun Yat-Sen University, Guangzhou 510080, China
| | - Huaiyu Gu
- School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China
| | - Yilin Chen
- Department of Preventive Medicine, School of Public Health, Sun Yat-Sen University, Guangzhou 510080, China
| | - Qing Wei
- Department of Preventive Medicine, School of Public Health, Sun Yat-Sen University, Guangzhou 510080, China
| | - Qiansheng Hu
- Department of Preventive Medicine, School of Public Health, Sun Yat-Sen University, Guangzhou 510080, China
- * E-mail:
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36
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Katsirntaki K, Mauritz C, Olmer R, Schmeckebier S, Sgodda M, Puppe V, Eggenschwiler R, Duerr J, Schubert SC, Schmiedl A, Ochs M, Cantz T, Salwig I, Szibor M, Braun T, Rathert C, Martens A, Mall MA, Martin U. Bronchoalveolar sublineage specification of pluripotent stem cells: effect of dexamethasone plus cAMP-elevating agents and keratinocyte growth factor. Tissue Eng Part A 2014; 21:669-82. [PMID: 25316003 DOI: 10.1089/ten.tea.2014.0097] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Respiratory progenitors can be efficiently generated from pluripotent stem cells (PSCs). However, further targeted differentiation into bronchoalveolar sublineages is still in its infancy, and distinct specifying effects of key differentiation factors are not well explored. Focusing on airway epithelial Clara cell generation, we analyzed the effect of the glucocorticoid dexamethasone plus cAMP-elevating agents (DCI) on the differentiation of murine embryonic and induced pluripotent stem cells (iPSCs) into bronchoalveolar epithelial lineages, and whether keratinocyte growth factor (KGF) might further influence lineage decisions. We demonstrate that DCI strongly induce expression of the Clara cell marker Clara cell secretory protein (CCSP). While KGF synergistically supports the inducing effect of DCI on alveolar markers with increased expression of surfactant protein (SP)-C and SP-B, an inhibitory effect on CCSP expression was shown. In contrast, neither KGF nor DCI seem to have an inducing effect on ciliated cell markers. Furthermore, the use of iPSCs from transgenic mice with CCSP promoter-dependent lacZ expression or a knockin of a YFP reporter cassette in the CCSP locus enabled detection of derivatives with Clara cell typical features. Collectively, DCI was shown to support bronchoalveolar specification of mouse PSCs, in particular Clara-like cells, and KGF to inhibit bronchial epithelial differentiation. The targeted in vitro generation of Clara cells with their important function in airway protection and regeneration will enable the evaluation of innovative cellular therapies in animal models of lung diseases.
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Affiliation(s)
- Katherina Katsirntaki
- 1 Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department for Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School , Hannover, Germany
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37
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Mong J, Panman L, Alekseenko Z, Kee N, Stanton LW, Ericson J, Perlmann T. Transcription factor-induced lineage programming of noradrenaline and motor neurons from embryonic stem cells. Stem Cells 2014; 32:609-22. [PMID: 24549637 DOI: 10.1002/stem.1585] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2013] [Accepted: 09/20/2013] [Indexed: 11/08/2022]
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.
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Affiliation(s)
- Jamie Mong
- Ludwig Institute for Cancer Research, Ltd., Stockholm, Sweden; Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden; Stem Cell and Developmental Biology, Genome Institute of Singapore, Singapore
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Hu Y, Liang J, Cui H, Wang X, Rong H, Shao B, Cui H. Wharton's jelly mesenchymal stem cells differentiate into retinal progenitor cells. Neural Regen Res 2014; 8:1783-92. [PMID: 25206475 PMCID: PMC4145957 DOI: 10.3969/j.issn.1673-5374.2013.19.006] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2013] [Accepted: 05/05/2013] [Indexed: 01/09/2023] Open
Abstract
Human Wharton's jelly mesenchymal stem cells were isolated from fetal umbilical cord. Cells were cultured in serum-free neural stem cell-conditioned medium or neural stem cell-conditioned medium supplemented with Dkk-1, a Wnt/β catenin pathway antagonist, and LeftyA, a Nodal signaling pathway antagonist to induce differentiation into retinal progenitor cells. Inverted microscopy showed that after induction, the spindle-shaped or fibroblast-like Wharton's jelly mesenchymal stem cells changed into bulbous cells with numerous processes. Immunofluorescent cytochemical ing and reverse-transcription PCR showed positive expression of retinal progenitor cell markers, Pax6 and Rx, as well as weakly down-regulated nestin expression. These results demonstrate that Wharton's jelly mesenchymal stem cells are capable of differentiating into retinal progenitor cells in vitro.
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Affiliation(s)
- Ying Hu
- Department of Ophthalmology, First Affiliated Hospital, Harbin Medical University, Harbin 200120, Heilongjiang Province, China ; Department of Ophthalmology, Shanghai East Hospital Affiliated to Tongji University, Shanghai 200120, China
| | - Jun Liang
- Department of Histology and Embryology, Mudanjiang Medical University, Mudanjiang 157011, Heilongjiang Province, China
| | - Hongping Cui
- Department of Ophthalmology, Shanghai East Hospital Affiliated to Tongji University, Shanghai 200120, China
| | - Xinmei Wang
- Department of Ophthalmology, Fourth Affiliated Hospital, Harbin Medical University, Harbin 200120, Heilongjiang Province, China
| | - Hua Rong
- Department of Ophthalmology, Shanghai East Hospital Affiliated to Tongji University, Shanghai 200120, China
| | - Bin Shao
- Department of Head-Neck and Breast Tumor, Mudanjiang Tumor Hospital, Mudanjiang 157009, Heilongjiang Province, China
| | - Hao Cui
- Department of Ophthalmology, First Affiliated Hospital, Harbin Medical University, Harbin 200120, Heilongjiang Province, China
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Wattanapanitch M, Klincumhom N, Potirat P, Amornpisutt R, Lorthongpanich C, U-pratya Y, Laowtammathron C, Kheolamai P, Poungvarin N, Issaragrisil S. Dual small-molecule targeting of SMAD signaling stimulates human induced pluripotent stem cells toward neural lineages. PLoS One 2014; 9:e106952. [PMID: 25207966 PMCID: PMC4160199 DOI: 10.1371/journal.pone.0106952] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2014] [Accepted: 08/01/2014] [Indexed: 01/10/2023] Open
Abstract
Incurable neurological disorders such as Parkinson's disease (PD), Huntington's disease (HD), and Alzheimer's disease (AD) are very common and can be life-threatening because of their progressive disease symptoms with limited treatment options. To provide an alternative renewable cell source for cell-based transplantation and as study models for neurological diseases, we generated induced pluripotent stem cells (iPSCs) from human dermal fibroblasts (HDFs) and then differentiated them into neural progenitor cells (NPCs) and mature neurons by dual SMAD signaling inhibitors. Reprogramming efficiency was improved by supplementing the histone deacethylase inhibitor, valproic acid (VPA), and inhibitor of p160-Rho associated coiled-coil kinase (ROCK), Y-27632, after retroviral transduction. We obtained a number of iPS colonies that shared similar characteristics with human embryonic stem cells in terms of their morphology, cell surface antigens, pluripotency-associated gene and protein expressions as well as their in vitro and in vivo differentiation potentials. After treatment with Noggin and SB431542, inhibitors of the SMAD signaling pathway, HDF-iPSCs demonstrated rapid and efficient differentiation into neural lineages. Six days after neural induction, neuroepithelial cells (NEPCs) were observed in the adherent monolayer culture, which had the ability to differentiate further into NPCs and neurons, as characterized by their morphology and the expression of neuron-specific transcripts and proteins. We propose that our study may be applied to generate neurological disease patient-specific iPSCs allowing better understanding of disease pathogenesis and drug sensitivity assays.
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Affiliation(s)
- Methichit Wattanapanitch
- Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
| | - Nuttha Klincumhom
- Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
| | - Porntip Potirat
- Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
| | - Rattaya Amornpisutt
- Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
| | - Chanchao Lorthongpanich
- Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
| | - Yaowalak U-pratya
- Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
| | - Chuti Laowtammathron
- Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
| | - Pakpoom Kheolamai
- Division of Cell Biology, Department of Pre-clinical Sciences, Faculty of Medicine, Thammasat University, Pathumthani, Thailand
| | - Niphon Poungvarin
- Division of Neurology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
| | - Surapol Issaragrisil
- Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
- Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
- * E-mail:
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40
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Garita-Hernández M, Diaz-Corrales F, Lukovic D, González-Guede I, Diez-Lloret A, Valdés-Sánchez ML, Massalini S, Erceg S, Bhattacharya SS. Hypoxia increases the yield of photoreceptors differentiating from mouse embryonic stem cells and improves the modeling of retinogenesis in vitro. Stem Cells 2014; 31:966-78. [PMID: 23362204 DOI: 10.1002/stem.1339] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2012] [Accepted: 11/23/2012] [Indexed: 12/19/2022]
Abstract
Retinitis pigmentosa (RP), a genetically heterogeneous group of diseases together with age-related macular degeneration (AMD), are the leading causes of permanent blindness and are characterized by the progressive dysfunction and death of the light sensing photoreceptors of the retina. Due to the limited regeneration capacity of the mammalian retina, the scientific community has invested significantly in trying to obtain retinal progenitor cells from embryonic stem cells (ESC). These represent an unlimited source of retinal cells, but it has not yet been possible to achieve specific populations, such as photoreceptors, efficiently enough to allow them to be used safely in the future as cell therapy of RP or AMD. In this study, we generated a high yield of photoreceptors from directed differentiation of mouse ESC (mESC) by recapitulating crucial phases of retinal development. We present a new protocol of differentiation, involving hypoxia and taking into account extrinsic and intrinsic cues. These include niche-specific conditions as well as the manipulation of the signaling pathways involved in retinal development. Our results show that hypoxia promotes and improves the differentiation of mESC toward photoreceptors. Different populations of retinal cells are increased in number under the hypoxic conditions applied, such as Crx-positive cells, S-Opsin-positive cells, and double positive cells for Rhodopsin and Recoverin, as shown by immunofluorescence analysis. For the first time, this manuscript reports the high efficiency of differentiation in vivo and the expression of mature rod photoreceptor markers in a large number of differentiated cells, transplanted in the subretinal space of wild-type mice.
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Affiliation(s)
- Marcela Garita-Hernández
- CABIMER (Centro Andaluz de Biología Molecular y Medicina Regenerativa), Avda. Americo Vespucio s/n, Parque Científico y Tecnológico Cartuja, Sevilla, Spain
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41
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Kozhich OA, Hamilton RS, Mallon BS. Standardized generation and differentiation of neural precursor cells from human pluripotent stem cells. Stem Cell Rev Rep 2014; 9:531-6. [PMID: 22388559 DOI: 10.1007/s12015-012-9357-8] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Precise, robust and scalable directed differentiation of pluripotent stem cells is an important goal with respect to disease modeling or future therapies. Using the AggreWell™400 system we have standardized the differentiation of human embryonic and induced pluripotent stem cells to a neuronal fate using defined conditions. This allows reproducibility in replicate experiments and facilitates the direct comparison of cell lines. Since the starting point for EB formation is a single cell suspension, this protocol is suitable for standard and novel methods of pluripotent stem cell culture. Moreover, an intermediate population of neural precursor cells, which are routinely >95% NCAM(pos) and Tra-1-60(neg) by FACS analysis, may be expanded and frozen prior to differentiation allowing a convenient starting point for downstream experiments.
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Affiliation(s)
- O A Kozhich
- Laboratory of Molecular Biology, Division of Intramural Research, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA
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Fu X, Rong Z, Zhu S, Wang X, Xu Y, Lake BB. Genetic approach to track neural cell fate decisions using human embryonic stem cells. Protein Cell 2014; 5:69-79. [PMID: 24474203 PMCID: PMC3938840 DOI: 10.1007/s13238-013-0007-y] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2013] [Accepted: 11/15/2013] [Indexed: 11/25/2022] Open
Abstract
With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry(+) NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.
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Affiliation(s)
- Xuemei Fu
- Shenzhen Children’s Hospital, Shenzhen, 518026 China
- Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322 USA
| | - Zhili Rong
- Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322 USA
| | - Shengyun Zhu
- Shenzhen Children’s Hospital, Shenzhen, 518026 China
- Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322 USA
| | - Xiaocheng Wang
- Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322 USA
| | - Yang Xu
- Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322 USA
| | - Blue B. Lake
- Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322 USA
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Roubal I, Park SJ, Kim Y. Derivation of Neural Precursor Cells from Human Embryonic Stem Cells for DNA Methylomic Analysis. Methods Mol Biol 2014; 1341:345-57. [PMID: 25520282 DOI: 10.1007/7651_2014_152] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Embryonic stem cells are self-renewing pluripotent cells with competency to differentiate into all three-germ lineages. Many studies have demonstrated the importance of genetic and epigenetic molecular mechanisms in the maintenance of self-renewal and pluripotency. Stem cells are under unique molecular and cellular regulations different from somatic cells. Proper regulation should be ensured to maintain their unique self-renewal and undifferentiated characteristics. Understanding key mechanisms in stem cell biology will be important for the successful application of stem cells for regenerative therapeutic medicine. More importantly practical use of stem cells will require our knowledge on how to properly direct and differentiate stem cells into the necessary type of cells. Embryonic stem cells and adult stem cells have been used as study models to unveil molecular and cellular mechanisms in various signaling pathways. They are especially beneficial to developmental studies where in vivo molecular/cellular study models are not available. We have derived neural stem cells from human embryonic stem cells as a model to study the effect of teratogen in neural development. We have tested commercial neural differentiation system and successfully derived neural precursor cells exhibiting key molecular features of neural stem cells, which will be useful for experimental application.
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Affiliation(s)
- Ivan Roubal
- Laboratory of Stem Cell and Cancer Epigenetic Research, Division of Oral Biology and Medicine, UCLA School of Dentistry, Los Angeles, CA, USA
| | - Sun Joo Park
- Laboratory of Stem Cell and Cancer Epigenetic Research, Division of Oral Biology and Medicine, UCLA School of Dentistry, Los Angeles, CA, USA
| | - Yong Kim
- Laboratory of Stem Cell and Cancer Epigenetic Research, Division of Oral Biology and Medicine, UCLA School of Dentistry, Los Angeles, CA, USA. .,Center for Oral and Head/Neck Oncology Research Center, UCLA School of Dentistry, Los Angeles, CA, USA. .,UCLA's Jonsson Comprehensive Cancer Center, Los Angeles, CA, USA. .,UCLA Broad Stem Cell Research Center, Los Angeles, CA, USA.
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Rushton DJ, Mattis VB, Svendsen CN, Allen ND, Kemp PJ. Stimulation of GABA-induced Ca2+ influx enhances maturation of human induced pluripotent stem cell-derived neurons. PLoS One 2013; 8:e81031. [PMID: 24278369 PMCID: PMC3838360 DOI: 10.1371/journal.pone.0081031] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2013] [Accepted: 10/18/2013] [Indexed: 12/03/2022] Open
Abstract
Optimal use of patient-derived, induced pluripotent stem cells for modeling neuronal diseases is crucially dependent upon the proper physiological maturation of derived neurons. As a strategy to develop defined differentiation protocols that optimize electrophysiological function, we investigated the role of Ca2+ channel regulation by astrocyte conditioned medium in neuronal maturation, using whole-cell patch clamp and Ca2+ imaging. Standard control medium supported basic differentiation of induced pluripotent stem cell-derived neurons, as assayed by the ability to fire simple, single, induced action potentials. In contrast, treatment with astrocyte conditioned medium elicited complex and spontaneous neuronal activity, often with rhythmic and biphasic characteristics. Such augmented spontaneous activity correlated with astrocyte conditioned medium-evoked hyperpolarization and was dependent upon regulated function of L-, N- and R-type Ca2+ channels. The requirement for astrocyte conditioned medium could be substituted by simply supplementing control differentiation medium with high Ca2+ or γ-amino butyric acid (GABA). Importantly, even in the absence of GABA signalling, opening Ca2+ channels directly using Bay K8644 was able to hyperpolarise neurons and enhance excitability, producing fully functional neurons. These data provide mechanistic insight into how secreted astrocyte factors control differentiation and, importantly, suggest that pharmacological modulation of Ca2+ channel function leads to the development of a defined protocol for improved maturation of induced pluripotent stem cell-derived neurons.
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Affiliation(s)
- David J. Rushton
- Divisions of Pathophysiology & Repair and Neuroscience, School of Biosciences, Cardiff University, Cardiff, United Kingdom
| | - Virginia B. Mattis
- Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States of America
| | - Clive N. Svendsen
- Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States of America
| | - Nicholas D. Allen
- Divisions of Pathophysiology & Repair and Neuroscience, School of Biosciences, Cardiff University, Cardiff, United Kingdom
- * E-mail: (PJK); (NDA)
| | - Paul J. Kemp
- Divisions of Pathophysiology & Repair and Neuroscience, School of Biosciences, Cardiff University, Cardiff, United Kingdom
- * E-mail: (PJK); (NDA)
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Hazeltine LB, Selekman JA, Palecek SP. Engineering the human pluripotent stem cell microenvironment to direct cell fate. Biotechnol Adv 2013; 31:1002-19. [PMID: 23510904 PMCID: PMC3758782 DOI: 10.1016/j.biotechadv.2013.03.002] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2012] [Revised: 02/20/2013] [Accepted: 03/11/2013] [Indexed: 01/31/2023]
Abstract
Human pluripotent stem cells (hPSCs), including both embryonic stem cells and induced pluripotent stem cells, offer a potential cell source for research, drug screening, and regenerative medicine applications due to their unique ability to self-renew or differentiate to any somatic cell type. Before the full potential of hPSCs can be realized, robust protocols must be developed to direct their fate. Cell fate decisions are based on components of the surrounding microenvironment, including soluble factors, substrate or extracellular matrix, cell-cell interactions, mechanical forces, and 2D or 3D architecture. Depending on their spatio-temporal context, these components can signal hPSCs to either self-renew or differentiate to cell types of the ectoderm, mesoderm, or endoderm. Researchers working at the interface of engineering and biology have identified various factors which can affect hPSC fate, often based on lessons from embryonic development, and they have utilized this information to design in vitro niches which can reproducibly direct hPSC fate. This review highlights culture systems that have been engineered to promote self-renewal or differentiation of hPSCs, with a focus on studies that have elucidated the contributions of specific microenvironmental cues in the context of those culture systems. We propose the use of microsystem technologies for high-throughput screening of spatial-temporal presentation of cues, as this has been demonstrated to be a powerful approach for differentiating hPSCs to desired cell types.
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Affiliation(s)
| | | | - Sean P. Palecek
- Department of Chemical and Biological Engineering, University of Wisconsin – Madison 1415 Engineering Drive, Madison, WI 53706 USA
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Biomedical and clinical promises of human pluripotent stem cells for neurological disorders. BIOMED RESEARCH INTERNATIONAL 2013; 2013:656531. [PMID: 24171168 PMCID: PMC3793324 DOI: 10.1155/2013/656531] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 07/13/2013] [Accepted: 08/13/2013] [Indexed: 01/25/2023]
Abstract
Neurological disorders are characterized by the chronic and progressive loss of neuronal structures and functions. There is a variability of the onsets and causes of clinical manifestations. Cell therapy has brought a new concept to overcome brain diseases, but the advancement of this therapy is limited by the demands of specialized neurons. Human pluripotent stem cells (hPSCs) have been promised as a renewable resource for generating human neurons for both laboratory and clinical purposes. By the modulations of appropriate signalling pathways, desired neuron subtypes can be obtained, and induced pluripotent stem cells (iPSCs) provide genetically matched neurons for treating patients. These hPSC-derived neurons can also be used for disease modeling and drug screening. Since the most urgent problem today in transplantation is the lack of suitable donor organs and tissues, the derivation of neural progenitor cells from hPSCs has opened a new avenue for regenerative medicine. In this review, we summarize the recent reports that show how to generate neural derivatives from hPSCs, and discuss the current evidence of using these cells in animal studies. We also highlight the possibilities and concerns of translating these hPSC-derived neurons for biomedical and clinical uses in order to fight against neurological disorders.
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Alsanie WF, Niclis JC, Petratos S. Human embryonic stem cell-derived oligodendrocytes: protocols and perspectives. Stem Cells Dev 2013; 22:2459-76. [PMID: 23621561 PMCID: PMC3760471 DOI: 10.1089/scd.2012.0520] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2012] [Accepted: 04/26/2013] [Indexed: 12/19/2022] Open
Abstract
Oligodendrocytes play a fundamental supportive role in the mammalian central nervous system (CNS) as the myelinating-glial cells. Disruption of fast axonal transport mechanisms can occur as a consequence of mature oligodendrocyte loss following spinal cord injury, stroke, or due to neuroinflammatory conditions, such as multiple sclerosis. As a result of the limited remyelination ability in the CNS after injury or disease, human embryonic stem cells (hESCs) may prove to be a promising option for the generation and replacement of mature oligodendrocytes. Moreover, hESC-derived oligodendrocytes may be experimentally utilized to unravel fundamental questions of oligodendrocyte development, along with their therapeutic potential through growth factor support of axons and neurons. However, an intensive characterization and examination of hESC-derived oligodendrocytes prior to preclinical or clinical trials is required to facilitate greater success in their integration following cellular replacement therapy (CRT). Currently, the protocols utilized to derive oligodendrocytes from hESCs consist of significant variations in culture style, time-length of differentiation, and the provision of growth factors in culture. Further, these differing protocols also report disparate patterns in the expression of oligodendroglial markers by these derived oligodendrocytes, throughout their differentiation in culture. We have comprehensively reviewed the published protocols describing the derivation of oligodendrocytes from hESCs and the studies that examine their efficacy to remyelinate, along with the fundamental issues of their safety as a viable CRT. Additionally, this review will highlight particular issues of concern and suggestions for troubleshooting to provide investigators critical information for the future improvement of establishing in vitro hESC-derived oligodendrocytes.
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Affiliation(s)
- Walaa F Alsanie
- Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia.
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Drury-Stewart D, Song M, Mohamad O, Guo Y, Gu X, Chen D, Wei L. Highly efficient differentiation of neural precursors from human embryonic stem cells and benefits of transplantation after ischemic stroke in mice. Stem Cell Res Ther 2013; 4:93. [PMID: 23928330 PMCID: PMC3854684 DOI: 10.1186/scrt292] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2013] [Accepted: 07/26/2013] [Indexed: 02/07/2023] Open
Abstract
Introduction Ischemic stroke is a leading cause of death and disability, but treatment options are severely limited. Cell therapy offers an attractive strategy for regenerating lost tissues and enhancing the endogenous healing process. In this study, we investigated the use of human embryonic stem cell-derived neural precursors as a cell therapy in a murine stroke model. Methods Neural precursors were derived from human embryonic stem cells by using a fully adherent SMAD inhibition protocol employing small molecules. The efficiency of neural induction and the ability of these cells to further differentiate into neurons were assessed by using immunocytochemistry. Whole-cell patch-clamp recording was used to demonstrate the electrophysiological activity of human embryonic stem cell-derived neurons. Neural precursors were transplanted into the core and penumbra regions of a focal ischemic stroke in the barrel cortex of mice. Animals received injections of bromodeoxyuridine to track regeneration. Neural differentiation of the transplanted cells and regenerative markers were measured by using immunohistochemistry. The adhesive removal test was used to determine functional improvement after stroke and intervention. Results After 11 days of neural induction by using the small-molecule protocol, over 95% of human embryonic stem-derived cells expressed at least one neural marker. Further in vitro differentiation yielded cells that stained for mature neuronal markers and exhibited high-amplitude, repetitive action potentials in response to depolarization. Neuronal differentiation also occurred after transplantation into the ischemic cortex. A greater level of bromodeoxyuridine co-localization with neurons was observed in the penumbra region of animals receiving cell transplantation. Transplantation also improved sensory recovery in transplant animals over that in control animals. Conclusions Human embryonic stem cell-derived neural precursors derived by using a highly efficient small-molecule SMAD inhibition protocol can differentiate into electrophysiologically functional neurons in vitro. These cells also differentiate into neurons in vivo, enhance regenerative activities, and improve sensory recovery after ischemic stroke.
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Kmet M, Guo C, Edmondson C, Chen B. Directed differentiation of human embryonic stem cells into corticofugal neurons uncovers heterogeneous Fezf2-expressing subpopulations. PLoS One 2013; 8:e67292. [PMID: 23826257 PMCID: PMC3691138 DOI: 10.1371/journal.pone.0067292] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2013] [Accepted: 05/17/2013] [Indexed: 12/24/2022] Open
Abstract
Understanding how neuronal diversity is achieved within the cerebral cortex remains a major challenge in neuroscience. The advent of human embryonic stem cells (hESCs) as a model system provides a unique opportunity to study human corticogenesis in vitro and to identify the mechanisms that promote neuronal differentiation to achieve neuronal diversity in human brain. The transcription factor Fezf2 is necessary and sufficient for the specification of subcerebral projection neurons in mouse. However, its function during human corticogenesis is poorly understood. This study reports the differentiation of a hFezf2-YFP hESC reporter line into corticofugal projection neurons capable of extending axons toward the spinal cord upon transplantation into neonatal mouse brains. Additionally, we show that triple inhibition of the TGFß/BMP/Wnt-Shh pathway promotes the generation of hFezf2-expressing cells in vitro. Finally, this study unveils the isolation of two novel and distinct populations of hFezf2-YFP expressing cells reminiscent of the distinct Fezf2-expressing neuronal subtypes in the developing mouse brain. Overall our data suggest that the directed differentiation of hESCs into corticofugal neurons provides a useful model to identify the molecular mechanisms regulating human corticofugal differentiation and survival.
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Affiliation(s)
- Muriel Kmet
- Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America
| | - Chao Guo
- Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America
| | - Carina Edmondson
- Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America
| | - Bin Chen
- Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America
- * E-mail:
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Fazeli AS, Nasrabadi D, Pouya A, Mirshavaladi S, Sanati MH, Baharvand H, Salekdeh GH. Proteome analysis of post-transplantation recovery mechanisms of an EAE model of multiple sclerosis treated with embryonic stem cell-derived neural precursors. J Proteomics 2013; 94:437-50. [PMID: 23791935 DOI: 10.1016/j.jprot.2013.06.008] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2012] [Revised: 06/02/2013] [Accepted: 06/09/2013] [Indexed: 12/21/2022]
Abstract
UNLABELLED Multiple sclerosis (MS) is a chronic inflammatory and progressive disorder of the central nervous system (CNS), which ultimately causes demyelination and subsequent axonal injury. Experimental autoimmune encephalomyelitis (EAE) is a well-characterized animal model to study the etiology and pathogenesis of MS. This model can also be used to investigate various therapeutic approaches for MS. Herein; we have treated a score 3 EAE mouse model with an embryonic stem cell-derived neural precursor. Clinical analysis showed recovery of the EAE model of MS following transplantation. We analyzed the proteome of spinal cords of healthy and EAE samples before and after transplantation. Proteome analysis revealed that expressions of 86 spinal cord protein spots changed in the EAE or transplanted mouse compared to controls. Mass spectrometry resulted in identification of 72 proteins. Of these, the amounts of 27 differentially expressed proteins in EAE samples returned to sham levels after transplantation, suggesting a possible correlation between changes at the proteome level and clinical signs of EAE in transplanted mice. The recovered proteins belonged to various functional groups that included disturbances in ionic and neurotransmitter release, apoptosis, iron hemostasis, and signal transduction. Our results provided a proteomic view of the molecular mechanisms of EAE recovery after stem cell transplantation. BIOLOGICAL SIGNIFICANCE In this study, we applied proteomics to analyze the changes in proteome pattern of EAE mouse model after embryonic stem cell-derived neural precursor transplantation. Our proteome results clearly showed that the expression levels of several differentially expressed proteins in EAE samples returned to sham levels after transplantation, which suggested a possible correlation between changes at the proteome level and decreased clinical signs of EAE in transplanted mice. These results will serve as a basis to address new questions and design new experiments to elucidate the biology of EAE and recovery after transplantation. A thorough understanding of stem cell-mediated therapeutic mechanisms might result in the development of more efficacious therapies for MS than are currently available.
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Affiliation(s)
- Abolhassan Shahzadeh Fazeli
- Department of Molecular Systems Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran; Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
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