Development of a In vitro 2-D transplantation system of human neurons and the effects of dynamic stimulation on the system

2020 ◽  
Vol 2020 (0) ◽  
pp. 405
Author(s):  
Keigo TOMIZAWA ◽  
Shuhei ISHIHARA ◽  
Tadashi KOSAWADA ◽  
Zhonggang Feng
Keyword(s):  
Human Neurons ◽  
Transplantation System ◽  
Dynamic Stimulation

scholarly journals Fragile X syndrome patient-derived neurons developing in the mouse brain show FMR1 -dependent phenotypes

2021 ◽  
Author(s):  
Marine A Krzisch ◽  
Hao A Wu ◽  
Bingbing Yuan ◽  
Troy W. Whitfield ◽  
X. Shawn Liu ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Neuronal Development ◽  
Fragile X ◽  
In Vitro Models ◽  
Synaptic Activity ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
And Function ◽  
The Brain

Abnormal neuronal development in Fragile X syndrome (FXS) is poorly understood. Data on FXS patients remain scarce and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. Here, we co-injected neural precursor cells (NPCs) from FXS patient-derived and corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Single-cell RNA sequencing of transplanted cells revealed upregulated excitatory synaptic transmission and neuronal differentiation pathways in FXS neurons. Immunofluorescence analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, increased percentages of Arc- and Egr1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons pointed to an increase in synaptic activity and synaptic strength as compared to control. This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3D context, and could be used to test new therapeutic compounds correcting neuronal development defects in FXS.

Aging is associated with a mild acidification in neocortical human neurons in vitro

2018 ◽  
Vol 125 (10) ◽  
pp. 1495-1501 ◽  
Author(s):  
Udo Bonnet ◽  
Dieter Bingmann ◽  
Erwin-Josef Speckmann ◽  
Martin Wiemann
Keyword(s):  
Human Neurons

scholarly journals Human in vitro systems for examining synaptic function and plasticity in the brain

2020 ◽  
Vol 123 (3) ◽  
pp. 945-965 ◽  
Author(s):  
Kevin Lee ◽  
Thomas I.-H. Park ◽  
Peter Heppner ◽  
Patrick Schweder ◽  
Edward W. Mee ◽  
...  
Keyword(s):  
Animal Models ◽  
Human Brain ◽  
Brain Tissue ◽  
Human Brain Tissue ◽  
Adult Human Brain ◽  
Adult Human ◽  
Human Neurons ◽  
In Vitro Systems ◽  
Human Neuron

The human brain shows remarkable complexity in its cellular makeup and function, which are distinct from nonhuman species, signifying the need for human-based research platforms for the study of human cellular neurophysiology and neuropathology. However, the use of adult human brain tissue for research purposes is hampered by technical, methodological, and accessibility challenges. One of the major problems is the limited number of in vitro systems that, in contrast, are readily available from rodent brain tissue. With recent advances in the optimization of protocols for adult human brain preparations, there is a significant opportunity for neuroscientists to validate their findings in human-based systems. This review addresses the methodological aspects, advantages, and disadvantages of human neuron in vitro systems, focusing on the unique properties of human neurons and synapses in neocortical microcircuits. These in vitro models provide the incomparable advantage of being a direct representation of the neurons that have formed part of the human brain until the point of recording, which cannot be replicated by animal models nor human stem-cell systems. Important distinct cellular mechanisms are observed in human neurons that may underlie the higher order cognitive abilities of the human brain. The use of human brain tissue in neuroscience research also raises important ethical, diversity, and control tissue limitations that need to be considered. Undoubtedly however, these human neuron systems provide critical information to increase the potential of translation of treatments from the laboratory to the clinic in a way animal models are failing to provide.

scholarly journals Presynaptic dysfunction in CASK-related neurodevelopmental disorders

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Martin Becker ◽  
Francesca Mastropasqua ◽  
Jan Philipp Reising ◽  
Simon Maier ◽  
Mai-Lan Ho ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Splice Site Mutation ◽  
Autism Spectrum ◽  
Resonance Spectroscopy ◽  
Limited Information ◽  
Site Mutation ◽  
Mutation Carriers ◽  
Human Neurons

Abstract CASK-related disorders are genetically defined neurodevelopmental syndromes. There is limited information about the effects of CASK mutations in human neurons. Therefore, we sought to delineate CASK-mutation consequences and neuronal effects using induced pluripotent stem cell-derived neurons from two mutation carriers. One male case with autism spectrum disorder carried a novel splice-site mutation and a female case with intellectual disability carried an intragenic tandem duplication. We show reduction of CASK protein in maturing neurons from the mutation carriers, which leads to significant downregulation of genes involved in presynaptic development and of CASK protein interactors. Furthermore, CASK-deficient neurons showed decreased inhibitory presynapse size as indicated by VGAT staining, which may alter the excitatory–inhibitory (E/I) balance in developing neural circuitries. Using in vivo magnetic resonance spectroscopy quantification of GABA in the male mutation carrier, we further highlight the possibility to validate in vitro cellular data in the brain. Our data show that future pharmacological and clinical studies on targeting presynapses and E/I imbalance could lead to specific treatments for CASK-related disorders.

scholarly journals Human neuroblastoma cells acquire regulated secretory properties and different sensitivity to Ca2+ and alpha-latrotoxin after exposure to differentiating agents.

1989 ◽  
Vol 108 (6) ◽  
pp. 2291-2300 ◽  
Author(s):  
E Sher ◽  
S Denis-Donini ◽  
A Zanini ◽  
C Bisiani ◽  
F Clementi
Keyword(s):  
Electron Microscopy ◽  
De Novo ◽  
Neuroblastoma Cells ◽  
Calcium Ionophore ◽  
Membrane Receptors ◽  
Human Neuroblastoma Cells ◽  
Human Neuroblastoma ◽  
Adequate Model ◽  
Human Neurons

IMR-32 human neuroblastoma cells are unable to release [3H]dopamine in response to secretagogues. However, they express a normal complement of membrane receptors and ion channels which are efficiently coupled to second messenger production. In the present study we took advantage of the ability of this cell line to differentiate in vitro in the presence of either dibutyrryl-cAMP or 5-bromodeoxyuridine, to analyze any developmentally regulated changes in its secretory properties. Uptake, storage, and release of [3H]dopamine were studied biochemically and by autoradiography. The calcium ionophore ionomycin, phorbol 12-myristate 13-acetate and the presynaptic acting neurotoxin alpha-latrotoxin were used in both control and differentiated cells as secretagogue agents. The presence of secretory organelles was investigated by electron microscopy; the expression of secretory organelle markers, such as chromogranin/secretogranin proteins (secretory proteins) and synaptophysin (membrane protein), was detected by Western blotting and immunofluorescence. The results obtained indicate that IMR-32 cells acquire regulated secretory properties after in vitro drug-induced differentiation: (a) they assemble "de novo" secretory organelles, as revealed by electron microscopy and detection of secretory organelle markers, and (b) they are able to store [3H]dopamine and to release the neurotransmitter in response to secretagogue stimuli. Furthermore, secretagogue sensitivity was found to be different, depending on the differentiating agent. In fact, dibutyrryl-cAMP treated cells release [3H]dopamine in response to alpha-latrotoxin, but not in response to ionomycin, whereas 5-bromodeoxyuridine treated cells release the neurotransmitter in response to both secretagogues. All together these results suggest that IMR-32 cells represent an adequate model for studying the development of the secretory apparatus in cultured human neurons.

scholarly journals Neuroprotective Effects of a Variety of Pomegranate Juice Extracts against MPTP-Induced Cytotoxicity and Oxidative Stress in Human Primary Neurons

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Nady Braidy ◽  
Subash Selvaraju ◽  
Musthafa Mohamed Essa ◽  
Ragini Vaishnav ◽  
Samir Al-Adawi ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Pomegranate Juice ◽  
Neuroprotective Effects ◽  
Age Related ◽  
Therapeutic Value ◽  
Human Neurons ◽  
Environmental Toxin ◽  
Endogenous Antioxidant ◽  
Therapeutic Compounds

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is an environmental toxin which selectively induces oxidative damage and mitochondrial and proteasomal dysfunctions to dopaminergic neurons in the substantia nigra leading to Parkinsonian syndrome in animal models and humans. MPTP is one of the most widely usedin vitromodels to investigate the pathophysiology of Parkinson's disease (PD) and, screen for novel therapeutic compounds that can slow down or ameliorate this progressive degenerative disease. We investigated the therapeutic effect of pomegranate juice extracts (PJE), Helow, Malasi, Qusum, and Hamadh against MPTP-induced neurotoxicity in primary human neurons by examining extracellular LDH activity, intracellular NAD+and ATP levels, and endogenous antioxidant levels including lipid peroxidation products, catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, and reduced glutathione (GSH) levels. MPTP induced a reduction in SOD and GPx activities and intracellular NAD+, ATP, and GSH levels parallel to an increase in extracellular LDH and CAT activities, although lipid peroxidation was not altered. We report that helow and malasi can ameliorate MPTP-induced neurotoxicity by attenuating the observed changes in redox function to a greater extent than qusum and hamedh. Selected PJE varieties may exhibit properties which may be of therapeutic value to slow down age-related degeneration and neurodegeneration in particular.

scholarly journals From “Directed Differentiation” to “Neuronal Induction”: Modeling Neuropsychiatric Disease

2015 ◽  
Vol 10s1 ◽  
pp. BMI.S20066 ◽  
Author(s):  
Seok-Man Ho ◽  
Aaron Topol ◽  
Kristen J. Brennand
Keyword(s):  
Psychiatric Disorders ◽  
Neurological Diseases ◽  
Disease Onset ◽  
Induced Pluripotent Stem Cell ◽  
Directed Differentiation ◽  
Neuropsychiatric Disease ◽  
Somatic Cell Reprogramming ◽  
Human Neurons ◽  
Induced Pluripotent

Aberrant behavior and function of neurons are believed to be the primary causes of most neurological diseases and psychiatric disorders. Human postmortem samples have limited availability and, while they provide clues to the state of the brain after a prolonged illness, they offer limited insight into the factors contributing to disease onset. Conversely, animal models cannot recapitulate the polygenic origins of neuropsychiatric disease. Novel methods, such as somatic cell reprogramming, deliver nearly limitless numbers of pathogenic human neurons for the study of the mechanism of neuropsychiatric disease initiation and progression. First, this article reviews the advent of human induced pluripotent stem cell (hiPSC) technology and introduces two major methods, “directed differentiation” and “neuronal induction,” by which it is now possible to generate neurons for modeling neuropsychiatric disease. Second, it discusses the recent applications, and the limitations, of these technologies to in vitro studies of psychiatric disorders.

scholarly journals Sodium channel Nav1.2-L1342P variant displaying complex biophysical properties renders hyperexcitability of cortical neurons derived from human iPSCs

2021 ◽  
Author(s):  
Zhefu Que ◽  
Maria I. Olivero-Acosta ◽  
Jingliang Zhang ◽  
Muriel Eaton ◽  
William C. Skarnes ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Cortical Neurons ◽  
Sodium Current ◽  
Electrode Array ◽  
Induced Pluripotent Stem Cell ◽  
Epileptic Seizures ◽  
Action Potential Firing ◽  
Human Neurons ◽  
Human Ipscs

AbstractWith the wide adoption of whole-exome sequencing in children having seizures, an increasing number of SCN2A variants has been revealed as possible genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is strongly expressed in the pyramidal excitatory neurons and supports action potential firing. One recurrent SCN2A variant is L1342P, which was identified in multiple patients with early-onset encephalopathy and intractable seizures. Our biophysical analysis and computational modeling predicted gain-of-function features of this epilepsy-associated Nav1.2 variant. However, the mechanism underlying L1342P mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human induced pluripotent stem cell (hiPSC) line, in which L1342P was engineered by CRISPR/Cas9 mediated genome-editing. Using patch-clamping and micro-electrode array (MEA) recording, we found that the cortical neurons derived from hiPSCs carrying heterozygous L1342P variant presented significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, showing clear hyperexcitability phenotypes. Interestingly, the L1342P neuronal culture displayed a degree of resistance to the anti-seizure medication (phenytoin), which likely recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, was able to potently alleviate spontaneous and chemical-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures, and demonstrate the utility of genome-edited hiPSCs as an in vitro platform to advance personalized phenotyping and drug discovery.

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