Neural Organoids Research Articles

Application of neural organoids in studying neurodegenerative diseases

Neurodegenerative diseases are among the top causes of mortality and the aversion of DALYs (Disability-Adjusted Life Years) worldwide. Many attempts have been made to develop therapeutics to alleviate disease symptoms without much success. The preclinical models utilized in therapeutic testing are often inaccurate and cannot precisely translate into clinical studies. The introduction of neural organoids, a three-dimensional model grown from human-originated stem cells, was able to revolutionize the field of neurological drug development. Using induced pluripotent stem cell (iPSC), scientists are able to restore adult cells pluripotency and cultivate them into region specific brain organoids using a combination of growth factors, agonists, and inhibitors. These models have proven valuable in drug screen for myriad neurodegenerative disorders. To model such diseases, iPSCs are generated from patients with the respective diseases, and then cultivated in an environment that mimics the disease environment. For example, for Parkinsons disease, Wnt pathway inhibitors and the Sonic hedgehog agonist are used to induce midbrain neural progenitor cells from patients with risk factors. Despite neural organoids wide usage in screening for neurodegenerative disorders and drug testing, neural organoids present several limitations in their function, including a lack of complexity equivalent to that of the brain. This paper will discuss neural organoid technology and provide basic insight to its usage in drug screening and the field of neuroscience.

Enhanced cryopreservation efficiency of HIPSC-Derived neural organoids based on raman studies

Enhanced cryopreservation efficiency of HIPSC-Derived neural organoids based on raman studies

When is a brain organoid a sentience candidate?

It would be unwise to dismiss the possibility of human brain organoids developing sentience. However, scepticism about this idea is appropriate when considering current organoids. It is a point of consensus that a brainstem-dead human is not sentient, and current organoids lack a functioning brainstem. There are nonetheless troubling early warning signs, suggesting organoid research may create forms of sentience in the near future. To err on the side of caution, researchers with very different views about the neural basis of sentience should unite behind the “brainstem rule”: if a neural organoid develops or innervates a functioning brainstem that registers and prioritizes its needs, regulates arousal, and leads to sleep-wake cycles, then it is a sentience candidate. If organoid research leads to the creation of sentience candidates, a moratorium or indefinite ban on the creation of the relevant type of organoid may be appropriate. A different way forward, more consistent with existing approaches to animal research, would be to require ethical review and harm-benefit analysis for all research on sentience candidates.

Morphogenesis and development of human telencephalic organoids in the absence and presence of exogenous extracellular matrix.

The establishment and maintenance of apical-basal polarity is a fundamental step in brain development, instructing the organization of neural progenitor cells (NPCs) and the developing cerebral cortex. Particularly, basally located extracellular matrix (ECM) is crucial for this process. In vitro, epithelial polarization can be achieved via endogenous ECM production, or exogenous ECM supplementation. While neuroepithelial development is recapitulated in neural organoids, the effects of different ECM sources in tissue morphogenesis remain underexplored. Here, we show that exposure to a solubilized basement membrane matrix substrate, Matrigel, at early neuroepithelial stages causes rapid tissue polarization and rearrangement of neuroepithelial architecture. In cultures exposed to pure ECM components or unexposed to any exogenous ECM, polarity acquisition is slower and driven by endogenous ECM production. After the onset of neurogenesis, tissue architecture and neuronal differentiation are largely independent of the initial ECM source, but Matrigel exposure has long-lasting effects on tissue patterning. These results advance the knowledge on mechanisms of exogenously and endogenously guided morphogenesis, demonstrating the self-sustainability of neuroepithelial cultures by endogenous processes.

Engineering the Physical Microenvironment into Neural Organoids for Neurogenesis and Neurodevelopment.

Understanding the signals from the physical microenvironment is critical for deciphering the processes of neurogenesis and neurodevelopment. The discovery of how surrounding physical signals shape human developing neurons is hindered by the bottleneck of conventional cell culture and animal models. Notwithstanding neural organoids provide a promising platform for recapitulating human neurogenesis and neurodevelopment, building neuronal physical microenvironment that accurately mimics the native neurophysical features is largely ignored in current organoid technologies. Here, it is discussed how the physical microenvironment modulates critical events during the periods of neurogenesis and neurodevelopment, such as neural stem cell fates, neural tube closure, neuronal migration, axonal guidance, optic cup formation, and cortical folding. Although animal models are widely used to investigate the impacts of physical factors on neurodevelopment and neuropathy, the important roles of human stem cell-derived neural organoids in this field are particularly highlighted. Considering the great promise of human organoids, building neural organoid microenvironments with mechanical forces, electrophysiological microsystems, and light manipulation will help to fully understand the physical cues in neurodevelopmental processes. Neural organoids combined with cutting-edge techniques, such as advanced atomic force microscopes, microrobots, and structural color biomaterials might promote the development of neural organoid-based research and neuroscience.

Dihydrofolate reductase activity controls neurogenic transitions in the developing neocortex.

One-carbon/folate (1C) metabolism supplies methyl groups required for DNA and histone methylation, and is involved in the maintenance of self-renewal in stem cells. Dihydrofolate reductase (DHFR), a key enzyme in 1C metabolism, is highly expressed in human and mouse neural progenitors at the early stages of neocortical development. Here, we have investigated the role of DHFR in the developing neocortex and report that reducing its activity in human neural organoids and mouse embryonic neocortex accelerates indirect neurogenesis, thereby affecting neuronal composition of the neocortex. Furthermore, we show that decreasing DHFR activity in neural progenitors leads to a reduction in one-carbon/folate metabolites and correlates with modifications of H3K4me3 levels. Our findings reveal an unanticipated role for DHFR in controlling specific steps of neocortex development and indicate that variations in 1C metabolic cues impact cell fate transitions.

Scalable mesh microelectrode arrays for neural spheroids and organoids

Abstract Introduction: Neural organoids promise to help understand the human brain and develop treatments for neurological diseases. Electrophysiological recordings are essential in neural models to evaluate the activity of neural circuits. Mesh microelectrode arrays (MEAs) have been demonstrated to be suitable for organoids and spheroids, and there is demand for easy-to-use devices that can be manufactured at scale. Methods: We present a new mesh MEA device with an easyto- use design. We produce mesh MEA chips on 100 mm carrier wafers and connect individual chips to PCBs by wirebonding. The devices are completed by assembly of a twopiece well and a glass cover slip. Results: Each device contains a suspended hammock-like mesh with 64 microelectrodes. The square grid’s pitch of 200 μm makes the mesh suitable for typical organoid sizes while spreading the electrodes across a 1.4 mm region. The well is designed for fluid handling by pipetting or pump systems. Impedance measurements indicate a high yield of functional microelectrodes, although further effort is needed to produce consistent low impedances. The devices are compatible with commercial amplifiers, while adaptation of the PCB to other formats will be straightforward. Conclusions: Using scalable production methods, we have developed a mesh MEA device design that offers improved ease-of-use. Next steps will include biological validation in collaboration with partners.

Targeted mechanical stimulation via magnetic nanoparticles guides in vitro tissue development

Tissues take shape through a series of morphogenetic movements guided by local cell-scale mechanical forces. Current in vitro approaches to recapitulate tissue mechanics rely on uncontrolled self-organization or on the imposition of extrinsic and homogenous forces using matrix or instrument-driven stimulation, thereby failing to recapitulate highly localized and spatially varying forces. Here we develop a method for targeted mechanical stimulation of organoids using embedded magnetic nanoparticles. We show that magnetic clusters within organoids can be produced by sequential aggregation of magnetically labeled and non-labeled human pluripotent stem cells. These clusters impose local mechanical forces on the surrounding cells in response to applied magnetic fields. We show that precise, spatially defined actuation provides short-term mechanical tissue perturbations as well as long-term cytoskeleton remodeling in these organoids, which we term “magnetoids”. We demonstrate that targeted magnetic nanoparticle-driven actuation guides asymmetric tissue growth and proliferation, leading to enhanced patterning in human neural magnetoids. This approach, enabled by nanoparticle technology, allows for precise and locally controllable mechanical actuation in human neural tube organoids, and could be widely applicable to interrogate the role of local mechanotransduction in developmental and disease model systems.

Spatially controlled construction of assembloids using bioprinting

The biofabrication of three-dimensional (3D) tissues that recapitulate organ-specific architecture and function would benefit from temporal and spatial control of cell-cell interactions. Bioprinting, while potentially capable of achieving such control, is poorly suited to organoids with conserved cytoarchitectures that are susceptible to plastic deformation. Here, we develop a platform, termed Spatially Patterned Organoid Transfer (SPOT), consisting of an iron-oxide nanoparticle laden hydrogel and magnetized 3D printer to enable the controlled lifting, transport, and deposition of organoids. We identify cellulose nanofibers as both an ideal biomaterial for encasing organoids with magnetic nanoparticles and a shear-thinning, self-healing support hydrogel for maintaining the spatial positioning of organoids to facilitate the generation of assembloids. We leverage SPOT to create precisely arranged assembloids composed of human pluripotent stem cell-derived neural organoids and patient-derived glioma organoids. In doing so, we demonstrate the potential for the SPOT platform to construct assembloids which recapitulate key developmental processes and disease etiologies.

Mycobacterium tuberculosisinfection of human neural progenitor cells alters their functions

Abstract Central nervous system tuberculosis (CNS-TB) is the most dangerous form of extrapulmonary tuberculosis. However, the current understanding of infectious mechanisms and anti-mycobacterial immunity within the brain parenchyma is limited due to the absence of an ideal human CNS-TB model. In this study, we developed a novel human in vitro model for the study of CNS-TB by infecting 3D neural organoids and 2D neural progenitor cells (NPC) with Mycobacterium tuberculosis (Mtb). Here we report that the subset of the NPC population can be phagocytized by the apoptotic cell binding receptors. Internalized Mtb bacilli, co-localized within late endosomal and phagolysosomal compartments as well as in the cytoplasm of the NPCs. Mtb infection of NPC induced an elevated cell death and limited proliferation of the infected NPCs Additionally a strong type-1 interferon (IFN) responses were detected in Mtb-infected NPCs by the RNA-sequencing and by immunohistochemistry. We demonstrated that the treatments with anti-IFNα neutralization antibodies could partially rescue Mtb-induced NPC death and proliferation defects. Our findings suggest that these novel neural organoids and NPC-based in vitro platforms provide a new platform for elucidating CNS-TB pathogenesis and can be ultimately used for testing novel anti-mycobacterial treatment strategies for the human CNS-TB. "Supported by grants from NIH (R56AI162164-01)

Understanding neural development and diseases using CRISPR screens in human pluripotent stem cell-derived cultures.

The brain is arguably the most complex part of the human body in form and function. Much remains unclear about the molecular mechanisms that regulate its normal and pathological physiology. This lack of knowledge largely stems from the inaccessible nature of the human brain, and the limitation of animal models. As a result, brain disorders are difficult to understand and even more difficult to treat. Recent advances in generating human pluripotent stem cells (hPSCs)-derived 2-dimensional (2D) and 3-dimensional (3D) neural cultures have provided an accessible system to model the human brain. Breakthroughs in gene editing technologies such as CRISPR/Cas9 further elevate the hPSCs into a genetically tractable experimental system. Powerful genetic screens, previously reserved for model organisms and transformed cell lines, can now be performed in human neural cells. Combined with the rapidly expanding single-cell genomics toolkit, these technological advances culminate to create an unprecedented opportunity to study the human brain using functional genomics. This review will summarize the current progress of applying CRISPR-based genetic screens in hPSCs-derived 2D neural cultures and 3D brain organoids. We will also evaluate the key technologies involved and discuss their related experimental considerations and future applications.

A mesh microelectrode array for non-invasive electrophysiology within neural organoids

Organoids are emerging in vitro models of human physiology. Neural models require the evaluation of functional activity of single cells and networks, which is commonly measured by microelectrode arrays. The characteristics of organoids clash with existing in vitro or in vivo microelectrode arrays. With inspiration from implantable mesh electronics and growth of organoids on polymer scaffolds, we fabricated suspended hammock-like mesh microelectrode arrays for neural organoids. We have demonstrated the growth of organoids enveloping these meshes and the culture of organoids on meshes for up to one year. Furthermore, we present proof-of-principle recordings of spontaneous electrical activity across the volume of an organoid. Our concept enables a new class of microelectrode arrays for in vitro models of three-dimensional electrically active tissue.

Neural Organoids and the Quest to Understand and Treat Psychiatric Disease

Neural Organoids and the Quest to Understand and Treat Psychiatric Disease

Generation of self-organized autonomic ganglion organoids from fibroblasts

Neural organoids have been shown to serve as powerful tools for studying the mechanism of neural development and diseases as well as for screening drugs and developing cell-based therapeutics. Somatic cells have previously been reprogrammed into scattered autonomic ganglion (AG) neurons but not AG organoids. Here we have identified a combination of triple transcription factors (TFs) Ascl1, Phox2a/b, and Hand2 (APH) capable of efficiently reprogramming mouse fibroblasts into self-organized and networked induced AG (iAG) organoids, and characterized them by immunostaining, qRT-PCR, patch-clamping, and scRNA-seq approaches. The iAG neurons exhibit molecular properties, subtype diversity, and electrophysiological characteristics of autonomic neurons. Moreover, they can integrate into the superior cervical ganglia following transplantation and innervate and control the beating rate of co-cultured ventricular myocytes. Thus, iAG organoids may provide a valuable tool to study the pathogenesis of autonomic nervous system diseases and screen for drugs, as well as a source for cell-based therapies.

Forecasting Approach to Investigate Dynamic Growth of Organoid within 3D Matrix for Distinct Perspective

Organoid as a 3D structured model in vitro has difficulty in controlling its size. This issue becomes problematic when it is applied in a microfluidic source and sink-based because different dimension leads to different exposure to morphogen resulting in different cell fate. As a model used for biomedical purposes, this problem could lead to a discrepancy. This research is imposed to implement the forecasting method to study the dynamic of organoid growth profile. This approach could help a better understanding via spatiotemporal perspective complemented with a mathematical formula. The forecasting approach that clarifies the trend of this organoid growth by assessing whether the decided trend fits in every (or particular) stage (or not) has not been informed yet. Neural tube organoids have four different mechanical stiffness (0,5 kPa, 2 kPa, 4 kPa, 8kPa) which are documented in three days by time-lapse microscopy used in this experiment. These objects are mapped in a spatiotemporal fashion investigated in the profile and assessed by exponential trend. The actual phenomenon and forecasted result are evaluated by Mean Absolute Percentage Error (MAPE). Based on the result, the profile of organoid growth indicates that the organoid develops mostly following an exponential profile with the highest R2 value of 0,9868 and the lowest being 0,8734. Based on the MAPE value calculation it could be confirmed that the MAPE value on day 3 is the highest among the others indicating that the extended time of growth tends to have a different profile rather than the exponential trend after day 2. It should be noted that on the lowest stiffness (0,5 kPa) the mechanical properties do not significantly affect the organoid size during the development. Almost all (11 by 12 data or 91,6%) of the MAPE value is in excellent criteria (the value is less than 10%). Only one data does not belong to that classification which is in 8 kPa on day 3. Indicating that the higher stiffness the stronger effect on the system. From the axis development perspective, the organoid does not follow any specific pattern. This research could be a reference for a better understanding of the organoid growth profile in the 3D matrix environment which is nowadays become a hot topic in biomedical applications.

Abstract TMP81: Immunocompetent Neural Organoids Respond To Blood Stimulation: A Novel Model Of Intracerebral Hemorrhage

Introduction: The lack of treatments for intracerebral hemorrhage (ICH) suggests the need for novel preclinical models of this disease. To that end, this study aimed to explore the utility of an ICH neural organoid model. Methods: Human neural organoids consisting of several cell types, including microglia, were provided by Stem Pharm, Inc. On DIV 21, organoids were treated with human whole blood (n=3 organoids in each condition) for 24 hours. To serve as positive and negative controls, one organoid was treated with lipopolysaccharide (LPS) for 24 hours and three remained untreated. Human IL-6 concentrations were measured in the culture media prior to and after treatments using an enzyme-linked immunosorbent assay (ELISA) and the fold-change from baseline was normalized to the negative control condition. A one-way ANOVA with Dunnett’s test was then performed to compare IL-6 secretion between the control and treatment conditions. Results: LPS and blood stimulation both induced IL-6 secretion after 24 hours. The greatest fold increase in IL-6 concentration relative to the control group was observed with 10% blood stimulation (29.76 +/- 7.91, p<0.0001) followed by LPS (23.43 +/- 11.18, p<0.001) and 5% blood (22.56 +/- 7.91, p<0.0001). Significant fold increases in IL-6 secretion were also observed in the lower concentration conditions of 2.5% (15.75 +/- 7.91, p<0.001), 1.25% (11.46 +/- 7.91, p<0.01), and 0.6% blood (11.60 +/- 7.91, p<0.01). Conclusion: This preliminary study is the first to explore a neural organoid model of ICH. Comparable to LPS, the observed trends in IL-6 secretion suggest that whole blood is a potent, dose-dependent inducer of organoid inflammation. Given the blood-induced neuroinflammation that occurs during ICH, these initial results support a potential role for neural organoids in ICH modeling. Future work will aim to further characterize the organoid inflammatory response and validate this ICH model system via gene expression analyses.

Neural cortical organoids from self-assembling human iPSC as a model to investigate neurotoxicity in brain ischemia

Brain ischemia is a common acute injury resulting from impaired blood flow to the brain. Translation of effective drug candidates from experimental models to patients has systematically failed. The use of human induced pluripotent stem cells (iPSC) offers new opportunities to gain translational insights into diseases including brain ischemia. We used a human 3D self-assembling iPSC-derived model (human cortical organoids, hCO) to characterize the effects of ischemia caused by oxygen-glucose deprivation (OGD). hCO exposed to 2 h or 8 h of OGD had neuronal death and impaired neuronal network complexity, measured in whole-mounting microtubule-associated protein 2 (MAP-2) immunostaining. Neuronal vulnerability was reflected by a reduction in MAP-2 mRNA levels, and increased release of neurofilament light chain (NfL) in culture media, proportional to OGD severity. Glial fibrillary acidic protein (GFAP) gene or protein levels did not change in hCO, but their release in medium increased after prolonged OGD. In conclusion, this human 3D iPSC-based in vitro model of brain ischemic injury is characterized by marked neuronal injury reflected by the release of the translational biomarker NfL which is relevant for testing neuroprotective strategies.

Modelling Koolen-de Vries syndrome in neural organoids

Modelling Koolen-de Vries syndrome in neural organoids

Message in a Scaffold: Natural Biomaterials for Three-Dimensional (3D) Bioprinting of Human Brain Organoids

Brain organoids are invaluable tools for pathophysiological studies or drug screening, but there are still challenges to overcome in making them more reproducible and relevant. Recent advances in three-dimensional (3D) bioprinting of human neural organoids is an emerging approach that may overcome the limitations of self-organized organoids. It requires the development of optimal hydrogels, and a wealth of research has improved our knowledge about biomaterials both in terms of their intrinsic properties and their relevance on 3D culture of brain cells and tissue. Although biomaterials are rarely biologically neutral, few articles have reviewed their roles on neural cells. We here review the current knowledge on unmodified biomaterials amenable to support 3D bioprinting of neural organoids with a particular interest in their impact on cell homeostasis. Alginate is a particularly suitable bioink base for cell encapsulation. Gelatine is a valuable helper agent for 3D bioprinting due to its viscosity. Collagen, fibrin, hyaluronic acid and laminin provide biological support to adhesion, motility, differentiation or synaptogenesis and optimize the 3D culture of neural cells. Optimization of specialized hydrogels to direct differentiation of stem cells together with an increased resolution in phenotype analysis will further extend the spectrum of possible bioprinted brain disease models.

Human Forebrain Organoid-Derived Extracellular Vesicle Labeling with Iron Oxides for In Vitro Magnetic Resonance Imaging.

The significant roles of extracellular vesicles (EVs) as intracellular mediators, disease biomarkers, and therapeutic agents, make them a scientific hotspot. In particular, EVs secreted by human stem cells show significance in treating neurological disorders, such as Alzheimer’s disease and ischemic stroke. However, the clinical applications of EVs are limited due to their poor targeting capabilities and low therapeutic efficacies after intravenous administration. Superparamagnetic iron oxide (SPIO) nanoparticles are biocompatible and have been shown to improve the targeting ability of EVs. In particular, ultrasmall SPIO (USPIO, <50 nm) are more suitable for labeling nanoscale EVs due to their small size. In this study, induced forebrain neural progenitor cortical organoids (iNPCo) were differentiated from human induced pluripotent stem cells (iPSCs), and the iNPCo expressed FOXG1, Nkx2.1, α-catenin, as well as β-tubulin III. EVs were isolated from iNPCo media, then loaded with USPIOs by sonication. Size and concentration of EV particles were measured by nanoparticle tracking analysis, and no significant changes were observed in size distribution before and after sonication, but the concentration decreased after labeling. miR-21 and miR-133b decreased after sonication. Magnetic resonance imaging (MRI) demonstrated contrast visualized for the USPIO labeled EVs embedded in agarose gel phantoms. Upon calculation, USPIO labeled EVs exhibited considerably shorter relaxation times, quantified as T2 and T2* values, reducing the signal intensity and generating higher MRI contrast compared to unlabeled EVs and gel only. Our study demonstrated that USPIO labeling was a feasible approach for in vitro tracking of brain organoid-derived EVs, which paves the way for further in vivo examination.