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From Cultured Rodent Neurons to Human Brain Tissue: Model Systems for Pharmacological and Translational Neuroscience.
To investigate the enormous complexity of the functional and pathological brain there are a number of possible experimental model systems to choose from. Depending on the research question choosing the appropriate model may not be a trivial task, and given the dynamic and intricate nature of an intact living brain several models might be needed to properly address certain questions. In this review, we aim to provide an overview of neural cell and tissue culture, reflecting on historic methodological milestones and providing a brief overview of the state-of-the-art. We additionally present an example of an effective model system pipeline, composed of dissociated mouse cultures, organotypics, acute mouse brain slices, and acute human brain slices, in that order. The sequential use of these four model systems allows a balance and progression from experimental control to human applicability, and provides a meta-model that can help validate basic research findings in a translational setting. We then conclude with a few remarks regarding the necessity of an integrated approach when performing translational and neuropharmacological studies.
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Highlight: Horizons in Neuroscience - Organoids, Optogenetics and Remote Control.
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Generation and validation of APOE knockout human iPSC-derived cerebral organoids.
Apolipoprotein E (apoE) is a major lipid carrier in the brain and closely associated with the pathogenesis of Alzheimer's disease (AD). Here, we describe a protocol for efficient knockout of APOE in human induced pluripotent stem cells (iPSCs) using the CRISPR-Cas9 system. We obtain homozygous APOE knockout (APOE-/- ) iPSCs and further validate the deficiency of apoE in iPSC-derived cerebral organoids. APOE-/- cerebral organoids can serve as a useful tool to study apoE functions and apoE-related pathogenic mechanisms in AD. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020).
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Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells.
The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain development, facilitating precise disease modeling, and advancing therapeutic research. This protocol outlines a method for generating midbrain-specific organoids using induced pluripotent stem cells (iPSCs), employing a strategic differentiation approach. Key techniques include dual-SMAD inhibition to suppress SMAD signaling, administration of fibroblast growth factor 8b (FGF-8b), and activation of the Sonic Hedgehog pathway using the agonist purmorphamine, guiding iPSCs towards a midbrain fate. The organoids produced by this method achieve diameters up to 2 mm and incorporate a diverse array of neuroepithelial cell types, reflecting the midbrain's inherent cellular diversity. Validation of these organoids as authentic midbrain structures involves the expression of midbrain-specific markers, confirming their identity. A notable outcome of this methodology is the effective differentiation of iPSCs into dopaminergic neurons, which are characteristic of the midbrain. The significance of this protocol lies in its ability to produce functionally mature, midbrain-specific organoids that closely replicate essential aspects of the midbrain, offering a valuable model for in-depth exploration of midbrain developmental processes and the pathophysiology of related disorders such as Parkinson's disease. Thus, this protocol serves as a crucial resource for researchers seeking to enhance our understanding of the human brain and develop new treatments for neurodegenerative diseases, making it an indispensable tool in the field of neurological research.
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Neural Differentiation in the Third Dimension: Generating a Human Midbrain.
In recent years, technological improvements in three-dimensional (3D) culture systems have enabled the generation of organoids or spheroids representing a variety of tissues, including the brain. In this issue of Cell Stem Cell, Jo et al. (2016) describe a 3D culture model of the human midbrain containing dopaminergic neurons and neuromelanin.
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Neuroblasts in cerebral tumors induced by ethylnitrosourea in rats. A fine structrual study.
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Generation of improved human cerebral organoids from single copy DYRK1A knockout induced pluripotent stem cells in trisomy 21: hypothetical solutions for neurodevelopmental models and therapeutic alternatives in down syndrome.
Dual-specificity thyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a strong therapeutic target to ameliorate cognitive functions of Down Syndrome (DS). Genetic normalization of Dyrk1a is sufficient to normalize early cortical developmental phenotypes in DS mouse models. Gyrencephalic human neocortical development is more complex than that in lissencephalic mice; hence, cerebral organoids (COs) can be used to model early neurodevelopmental defects of DS. Single copy DYRK1A knockout COs (scDYRK1AKO-COs) can be generated from manipulated DS derived (DS-) induced pluripotent stem cells (iPSCs) and genetic normalization of DYRK1A is expected to result in corrected neurodevelopmental phenotypes that can be reminiscent of normal COs. DYRK1A knock-in (DYRK1AKI) COs can be derived after genetic manipulations of normal iPSCs and would be valuable to evaluate impaired neocortical development as can be seen in DS-COs. DYRK1A mutations cause severe human primary microcephaly; hence, dose optimization studies of DYRK1A inhibitors will be critical for prenatal therapeutic applications in DS. Several doses of DYRK1A inhibitors can be tested in the neurodevelopment process of DS-COs and DS-scDYRK1AKO-COs would be used as optimum models for evaluating phenotypic ameliorations. Overdose drug exposure in DS-COs can be explained by similar defects present in DS-baDYRK1AKO-COs and DYRK1AKO-COs. There are several limitations in the current CO technology, which can be reduced by the generation of vascularized brain-like organoids giving opportunities to mimic late-stage corticogenesis and complete hippocampal development. In the future, improved DS-DYRK1AKO-COs can be efficient in studies that aim to generate efficiently transplantable and implantable neurons for tissue regeneration alternatives in DS individuals.
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Reintroduction of the archaic variant of NOVA1 in cortical organoids alters neurodevelopment.
The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 (NOVA1) plays a key role in neural development and function. NOVA1 also includes a protein-coding difference between the modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version of NOVA1 Moreover, levels of synaptic markers and synaptic protein coassociations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution in NOVA1, which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species' evolution.
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Moral Intuition Regarding the Possibility of Conscious Human Brain Organoids: An Experimental Ethics Study.
The moral status of human brain organoids (HBOs) has been debated in view of the future possibility that they may acquire phenomenal consciousness. This study empirically investigates the moral sensitivity in people's intuitive judgments about actions toward conscious HBOs. The results showed that the presence/absence of pain experience in HBOs affected the judgment about the moral permissibility of actions such as creating and destroying the HBOs; however, the presence/absence of visual experience in HBOs also affected the judgment. These findings suggest that people's intuitive judgments about the moral status of HBOs are sensitive to the valence-independent value of phenomenal consciousness. We discuss how these observations can have normative implications; particularly, we argue that they put pressure on the theoretical view that the moral status of conscious HBOs is grounded solely in the valence-dependent value of consciousness. We also discuss how our findings can be informative even when such a theoretical view is finally justified or when the future possibility of conscious HBOs is implausible.
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Constitutive Activity of Serotonin Receptor 6 Regulates Human Cerebral Organoids Formation and Depression-like Behaviors.
Serotonin receptor 6 (5-HT6R), a typical G protein-coupled receptor (GPCR) mainly expressed in the neurogenic area with constitutive activity, is of particular interest as a promising target for emotional impairment. Here, we found that 5-HT6R was highly expressed in human NSCs and activation of the receptor promoted self-renewal of human NSCs, and thus induced the expansion and folding of human cerebral organoids; dysfunction of receptor or inhibition of its constitutive activity resulted in the premature differentiation of NSCs, which ultimately depleted the NSC pool. The following mechanistic study revealed that EPAC-CREB signaling was involved in 5-HT6R regulation. Furthermore, we showed that mice with genetic deletion of 5-HT6R or knockin A268R mutant presented depression-like behaviors and impaired hippocampal neurogenesis for progressive decrease of the NSC pool. Thus, this study indicates that the modulation of 5-HT6R and its constitutive activity may provide a therapeutic alternative to alleviate depression.