Gene edited fluorescent cerebral organoids to study human brain function and disease

AbstractCerebral organoids are a promising model to study human brain function and disease, though the high inter-organoid variability of the mini-brains is still challenging. To overcome this limitation, we introduce the method of labeled mixed organoids generated from two different hiPSC lines, which enables the identification of cells from different origin within a single organoid. The method combines a gene editing workflow and subsequent organoid differentiation and offers a unique tool to study gene function in a complex human 3D tissue-like model. Using a CRISPR/Cas9 gene editing approach, different fluorescent proteins were fused to β-actin or lamin B1 in hiPSCs and subsequently used as a marker to identify each cell line. Mixtures of differently edited cells were seeded to induce embryoid body formation and cerebral organoid differentiation. As a consequence, the development of the 3D tissue was detectable by live confocal fluorescence microscopy and immunofluorescence staining in fixed samples. Analysis of mixed organoids allowed the identification and examination of specifically labeled cells in the organoid that belong to each of the two hiPSC donor lines. We demonstrate that a direct comparison of the individual cells is possible by having the edited and the control (or the two differentially labeled) cells within the same organoid, and thus the mixed organoids overcome the inter-organoid inhomogeneity limitations. The approach aims to pave the way for the reliable analysis of human genetic disorders by the use of organoids and to fundamentally understand the molecular mechanisms underlying pathological conditions.

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Gene Edited Fluorescent Cerebral Organoids to Study Human Brain Function and Disease

10.21203/rs.3.rs-248029/v1 ◽

2021 ◽

Author(s):

Thorsten Mueller ◽

Lisa Bachmann ◽

Lucia Gallego Villarejo ◽

Natalie Heinen ◽

David Marks

Keyword(s):

Human Brain ◽

Brain Function ◽

Gene Editing ◽

Molecular Mechanisms ◽

Embryoid Body ◽

Fluorescent Proteins ◽

Genetic Disorders ◽

Induced Pluripotent Stem Cell ◽

Induced Pluripotent ◽

The Individual

Abstract Cerebral organoids are a promising model to study human brain function and disease, though the high inter-organoid variability of the mini-brains is still challenging. To overcome this limitation, we introduce the method of labeled mixed organoids generated from two different human induced pluripotent stem cell (hiPSC) lines, which enables the identification of cells from different origin within a single organoid. The method combines a gene editing workflow and subsequent organoid differentiation and offers a unique tool to study gene function in a complex human 3D tissue-like model. Using a CRISPR/Cas9 gene editing approach, different fluorescent proteins were fused to β-actin or lamin B1 in hiPSCs and subsequently used as a marker to identify each cell line. Mixtures of differently edited cells were seeded to induce embryoid body formation and cerebral organoid differentiation. As a consequence, the development of the 3D tissue was detectable by live confocal fluorescence microscopy and immunofluorescence staining in fixed samples. Analysis of mixed organoids allowed the identification and examination of specifically labeled cells in the organoid that belong to each of the two hiPSC donor lines. We demonstrate that a direct comparison of the individual cells is possible by having the edited and the control (or the two differentially labeled) cells within the same organoid, and thus the mixed organoids overcome the inter-organoid inhomogeneity limitations. The approach aims to pave the way for the reliable analysis of human genetic disorders by the use of organoids and to fundamentally understand the molecular mechanisms underlying pathological conditions.

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Roles of Non-Coding RNAs in Normal Human Brain Development, Brain Tumor, and Neuropsychiatric Disorders

Non-Coding RNA ◽

10.3390/ncrna5020036 ◽

2019 ◽

Vol 5 (2) ◽

pp. 36 ◽

Cited By ~ 9

Author(s):

Nie ◽

Li ◽

Zhang ◽

Liu

Keyword(s):

Brain Tumor ◽

Human Brain ◽

Brain Function ◽

Molecular Mechanisms ◽

Normal Brain ◽

Normal Human Brain ◽

Neuropsychiatric Diseases ◽

Wide Range ◽

Normal Human ◽

Non Coding Rnas

One of modern biology’s great surprises is that the human genome encodes only ~20,000 protein-coding genes, which represents less than 2% of the total genome sequence, and the majority of them are transcribed into non-coding RNAs (ncRNAs). Increasing evidence has shown that ncRNAs, including miRNAs, long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), play important roles in regulating a wide range of biological processes of the human brain. They not only regulate the pathogenesis of brain tumors, but also the development of neuropsychiatric diseases. This review provides an integrated overview of the roles of ncRNAs in normal human brain function, brain tumor development, and neuropsychiatric disease. We discussed the functions and molecular mechanisms of miRNAs, lncRNAs, and circRNAs in normal brain function and glioma, respectively, including those in exosome vesicles that can act as a molecular information carrier. We also discussed the regulatory roles of ncRNAs in the development of neuropsychiatric diseases. Lastly, we summarized the currently available platforms and tools that can be used for ncRNA identification and functional exploration in human diseases. This study will provide comprehensive insights for the roles of ncRNAs in human brain function and disease.

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A New Generation of Functional Tagged Proteins for HIV Fluorescence Imaging

Viruses ◽

10.3390/v13030386 ◽

2021 ◽

Vol 13 (3) ◽

pp. 386

Author(s):

João I. Mamede ◽

Joseph Griffin ◽

Stéphanie Gambut ◽

Thomas J. Hope

Keyword(s):

Molecular Mechanisms ◽

Fluorescent Proteins ◽

Functional Characterization ◽

Hiv Replication ◽

Virus Particles ◽

Wild Type Phenotype ◽

Viral Particles ◽

The Individual ◽

Tools And Techniques ◽

New Generation

During the last decade, there was a marked increase in the development of tools and techniques to study the molecular mechanisms of the HIV replication cycle by using fluorescence microscopy. Researchers often apply the fusion of tags and fluorophores to viral proteins, surrogate proteins, or dyes to follow individual virus particles while they progress throughout infection. The inclusion of such fusion motifs or surrogates frequently disrupts viral infectivity or results in a change of the wild-type phenotype. Here, we detail the construction and functional characterization of two new constructs where we fused fluorescent proteins to the N-terminus of HIV-1 Integrase. In the first, IN is recruited into assembling particles via a codon optimized Gag to complement other viral constructs, while the second is fused to a Gag-Pol expression vector fully capable of integration. Our data shows that N-terminal tagged IN is functional for integration by both recovery of integration of catalytically inactive IN and by the successful infectivity of viruses carrying only labeled IN. These tools will be important to study the individual behavior of viral particles and associate such behavior to infectivity.

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Toward a neuroscope: An application of high-performance computing for real-time evaluation of brain function using MRI

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100172346 ◽

1994 ◽

Vol 52 ◽

pp. 922-923

Author(s):

C. S. Potter ◽

C. D. Gregory ◽

H. D. Morris ◽

Z.-P. Liang ◽

P. C. Lauterbur

Keyword(s):

Human Brain ◽

Brain Function ◽

High Performance ◽

Complex Signal ◽

Time Step ◽

Brain Organization ◽

Cognitive Studies ◽

Positron Emission ◽

Imaging And Spectroscopy ◽

3D Anatomy

Over the past few years, several laboratories have demonstrated that changes in local neuronal activity associated with human brain function can be detected by magnetic resonance imaging and spectroscopy. Using these methods, the effects of sensory and motor stimulation have been observed and cognitive studies have begun. These new methods promise to make possible even more rapid and extensive studies of brain organization and responses than those now in use, such as positron emission tomography.Human brain studies are enormously complex. Signal changes on the order of a few percent must be detected against the background of the complex 3D anatomy of the human brain. Today, most functional MR experiments are performed using several 2D slice images acquired at each time step or stimulation condition of the experimental protocol. It is generally believed that true 3D experiments must be performed for many cognitive experiments. To provide adequate resolution, this requires that data must be acquired faster and/or more efficiently to support 3D functional analysis.

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Frontier concept of rabi chip for intelligent humanoid

Journal of Analytical Chromatography and Spectroscopy ◽

10.24294/jacs.v1i2.908 ◽

2018 ◽

Vol 1 (2) ◽

Author(s):

Preecha Yupapin ◽

Amiri I. S. ◽

Ali J. ◽

Ponsuwancharoen N. ◽

Youplao P.

Keyword(s):

Human Brain ◽

Brain Function ◽

Rabi Oscillation ◽

Liquid Core ◽

Brain Surface ◽

Dipole Oscillation ◽

On Chip ◽

The Brain ◽

Robotic Applications ◽

Atom System

The sequence of the human brain can be configured by the originated strongly coupling fields to a pair of the ionic substances(bio-cells) within the microtubules. From which the dipole oscillation begins and transports by the strong trapped force, which is known as a tweezer. The tweezers are the trapped polaritons, which are the electrical charges with information. They will be collected on the brain surface and transport via the liquid core guide wave, which is the mixture of blood content and water. The oscillation frequency is called the Rabi frequency, is formed by the two-level atom system. Our aim will manipulate the Rabi oscillation by an on-chip device, where the quantum outputs may help to form the realistic human brain function for humanoid robotic applications.

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Molecular Mechanisms of Neural Circuit Development and Regeneration

International Journal of Molecular Sciences ◽

10.3390/ijms22094593 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4593

Author(s):

Lieve Moons ◽

Lies De Groef

Keyword(s):

Human Brain ◽

Molecular Mechanisms ◽

Neural Circuit ◽

Circuit Development

The human brain contains 86 billion neurons [...]

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Integration of fMRI, NIROT and ERP for studies of human brain function

Magnetic Resonance Imaging ◽

10.1016/j.mri.2005.12.039 ◽

2006 ◽

Vol 24 (4) ◽

pp. 507-513 ◽

Cited By ~ 12

Author(s):

John C. Gore ◽

Silvina G. Horovitz ◽

Christopher J. Cannistraci ◽

Pavel Skudlarski

Keyword(s):

Human Brain ◽

Brain Function

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Physical Activity and Redox Balance in the Elderly: Signal Transduction Mechanisms

Applied Sciences ◽

10.3390/app11052228 ◽

2021 ◽

Vol 11 (5) ◽

pp. 2228

Author(s):

Daniela Galli ◽

Cecilia Carubbi ◽

Elena Masselli ◽

Mauro Vaccarezza ◽

Valentina Presta ◽

...

Keyword(s):

Physical Activity ◽

Molecular Mechanisms ◽

Vascular Tissue ◽

The Elderly ◽

Redox Balance ◽

Antioxidant Systems ◽

Targeted Prevention ◽

Individual Subject ◽

Aged People ◽

The Individual

Reactive Oxygen Species (ROS) are molecules naturally produced by cells. If their levels are too high, the cellular antioxidant machinery intervenes to bring back their quantity to physiological conditions. Since aging often induces malfunctioning in this machinery, ROS are considered an effective cause of age-associated diseases. Exercise stimulates ROS production on one side, and the antioxidant systems on the other side. The effects of exercise on oxidative stress markers have been shown in blood, vascular tissue, brain, cardiac and skeletal muscle, both in young and aged people. However, the intensity and volume of exercise and the individual subject characteristics are important to envisage future strategies to adequately personalize the balance of the oxidant/antioxidant environment. Here, we reviewed the literature that deals with the effects of physical activity on redox balance in young and aged people, with insights into the molecular mechanisms involved. Although many molecular pathways are involved, we are still far from a comprehensive view of the mechanisms that stand behind the effects of physical activity during aging. Although we believe that future precision medicine will be able to transform exercise administration from wellness to targeted prevention, as yet we admit that the topic is still in its infancy.

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The brain timewise: how timing shapes and supports brain function

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2014.0170 ◽

2015 ◽

Vol 370 (1668) ◽

pp. 20140170 ◽

Cited By ~ 39

Author(s):

Riitta Hari ◽

Lauri Parkkonen

Keyword(s):

Human Brain ◽

Brain Imaging ◽

Brain Function ◽

Temporal Dynamics ◽

Generative Models ◽

Network Activity ◽

Brain Diseases ◽

Specific Information ◽

Non Invasive ◽

The Brain

We discuss the importance of timing in brain function: how temporal dynamics of the world has left its traces in the brain during evolution and how we can monitor the dynamics of the human brain with non-invasive measurements. Accurate timing is important for the interplay of neurons, neuronal circuitries, brain areas and human individuals. In the human brain, multiple temporal integration windows are hierarchically organized, with temporal scales ranging from microseconds to tens and hundreds of milliseconds for perceptual, motor and cognitive functions, and up to minutes, hours and even months for hormonal and mood changes. Accurate timing is impaired in several brain diseases. From the current repertoire of non-invasive brain imaging methods, only magnetoencephalography (MEG) and scalp electroencephalography (EEG) provide millisecond time-resolution; our focus in this paper is on MEG. Since the introduction of high-density whole-scalp MEG/EEG coverage in the 1990s, the instrumentation has not changed drastically; yet, novel data analyses are advancing the field rapidly by shifting the focus from the mere pinpointing of activity hotspots to seeking stimulus- or task-specific information and to characterizing functional networks. During the next decades, we can expect increased spatial resolution and accuracy of the time-resolved brain imaging and better understanding of brain function, especially its temporal constraints, with the development of novel instrumentation and finer-grained, physiologically inspired generative models of local and network activity. Merging both spatial and temporal information with increasing accuracy and carrying out recordings in naturalistic conditions, including social interaction, will bring much new information about human brain function.

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