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Protocol to encapsulate cerebral organoids with alginate hydrogel shell to induce volumetric compression.
In vitro organoids, including cerebral organoids, are usually developed without mechanical compression, which may contribute to a delay in maturation. Here, we present a protocol for encapsulating cerebral organoids with a thin shell of low-concentration alginate hydrogel. We describe steps for organoid generation, microfluidic chip culture, Matrigel coating, expansion culture, and alginate encapsulation. We then detail procedures for maturation culture and organoid characterization. The moderate compressive stimulation that the shell provides promotes cell proliferation and neuronal maturation. For complete details on the use and execution of this protocol, please refer to Tang et al.1.
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Axonal Extensions along Corticospinal Tracts from Transplanted Human Cerebral Organoids.
The reconstruction of lost neural circuits by cell replacement is a possible treatment for neurological deficits after cerebral cortex injury. Cerebral organoids can be a novel source for cell transplantation, but because the cellular composition of the organoids changes along the time course of the development, it remains unclear which developmental stage of the organoids is most suitable for reconstructing the corticospinal tract. Here, we transplanted human embryonic stem cell-derived cerebral organoids at 6 or 10 weeks after differentiation (6w- or 10w-organoids) into mouse cerebral cortices. 6w-organoids extended more axons along the corticospinal tract but caused graft overgrowth with a higher percentage of proliferative cells. Axonal extensions from 10w-organoids were smaller in number but were enhanced when the organoids were grafted 1 week after brain injury. Finally, 10w-organoids extended axons in cynomolgus monkey brains. These results contribute to the development of a cell-replacement therapy for brain injury and stroke.
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Microfabricated dynamic brain organoid cocultures to assess the effects of surface geometry on assembloid formation.
Organoids have emerged as valuable tools for the study of development and disease. Assembloids are formed by integrating multiple organoid types to create more complex models. However, the process by which organoids integrate to form assembloids remains unclear and may play an important role in the resulting organoid structure. Here, a microfluidic platform is developed that allows separate culture of distinct organoid types and provides the capacity to partially control the geometry of the resulting organoid surfaces. Removal of a microfabricated barrier then allows the shaped and positioned organoids to interact and form an assembloid. When midbrain and unguided brain organoids were allowed to assemble with a defined spacing between them, axonal projections from midbrain organoids and cell migration out of unguided organoids were observed and quantitatively measured as the two types of organoids fused together. Axonal projection directions were statistically biased toward other midbrain organoids, and unguided organoid surface geometry was found to affect cell invasion. This platform provides a tool to observe cellular interactions between organoid surfaces that are spaced apart in a controlled manner, and may ultimately have value in exploring neuronal migration, axon targeting, and assembloid formation mechanisms.
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Establishment of In Vitro Brain Models for AON Delivery.
Progress in stem cell biology has made it possible to generate human-induced pluripotent stem cells (hiPSC) that can be differentiated into complex, three-dimensional structures, where the cells are spatially organized. To study brain development, Lancaster and colleagues developed an hiPSC-derived three-dimensional organoid culture system, termed cerebral organoids, that develop various discrete, although interdependent, brain regions. Here we describe in detail the generation of cerebral organoids using a modified version of the culture protocol.
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Progress in Modeling Neural Tube Development and Defects by Organoid Reconstruction.
It is clear that organoids are useful for studying the structure as well as the functions of organs and tissues; they are able to simulate cell-to-cell interactions, symmetrical and asymmetric division, proliferation, and migration of different cell groups. Some progress has been made using brain organoids to elucidate the genetic basis of certain neurodevelopmental disorders. Such as Parkinson's disease and Alzheimer's disease. However, research on organoids in early neural development has received insufficient attention, especially that focusing on neural tube precursors. In this review, we focus on the recent research progress on neural tube organoids and discuss both their challenges and potential solutions.
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Galangin Rescues Alzheimer's Amyloid-β Induced Mitophagy and Brain Organoid Growth Impairment.
Dysfunctional mitochondria and mitophagy are hallmarks of Alzheimer's disease (AD). It is widely accepted that restoration of mitophagy helps to maintain cellular homeostasis and ameliorates the pathogenesis of AD. It is imperative to create appropriate preclinical models to study the role of mitophagy in AD and to assess potential mitophagy-targeting therapies. Here, by using a novel 3D human brain organoid culturing system, we found that amyloid-β (Aβ1-42,10 μM) decreased the growth level of organoids, indicating that the neurogenesis of organoids may be impaired. Moreover, Aβ treatment inhibited neural progenitor cell (NPC) growth and induced mitochondrial dysfunction. Further analysis revealed that mitophagy levels were reduced in the brain organoids and NPCs. Notably, galangin (10 μM) treatment restored mitophagy and organoid growth, which was inhibited by Aβ. The effect of galangin was blocked by the mitophagy inhibitor, suggesting that galangin possibly acted as a mitophagy enhancer to ameliorate Aβ-induced pathology. Together, these results supported the important role of mitophagy in AD pathogenesis and suggested that galangin may be used as a novel mitophagy enhancer to treat AD.
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Microfabricated disk technology: Rapid scale up in midbrain organoid generation.
By providing a three-dimensional in vitro culture system with key features of the substantia nigra region in the brain, 3D neuronal organoids derived from human induced pluripotent stem cells (iPSCs) provide living neuronal tissue resembling the midbrain region of the brain. However, a major limitation of conventional brain organoid culture is that it is often labor-intensive, requiring highly specialized personnel for moderate throughput. Additionally, the methods published for long-term cultures require time-consuming maintenance to generate brain organoids in large numbers. With the increasing need for human midbrain organoids (hMOs) to better understand and model Parkinson's disease (PD) in a dish, there is a need to implement new workflows and methods to both generate and maintain hMOs, while minimizing batch to batch variation. In this study, we developed a method with microfabricated disks to scale up the generation of hMOs. This opens up the possibility to generate larger numbers of hMOs, in a manner that minimizes the amount of labor required, while decreasing variability and maintaining the viability of these hMOs over time. Taken together, producing hMOs in this manner opens up the potential for these to be used to further PD studies.
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Isolation and Characterization of Cell-Free DNA from Cerebral Organoids.
Early detection of neurological conditions is critical for timely diagnosis and treatment. Identifying cellular-level changes is essential for implementing therapeutic interventions prior to symptomatic disease onset. However, monitoring brain tissue directly through biopsies is invasive and poses a high risk. Bodily fluids such as blood or cerebrospinal fluid contain information in many forms, including proteins and nucleic acids. In particular, cell-free DNA (cfDNA) has potential as a versatile neurological biomarker. Yet, our knowledge of cfDNA released by brain tissue and how cfDNA changes in response to deleterious events within the brain is incomplete. Mapping changes in cfDNA to specific cellular events is difficult in vivo, wherein many tissues contribute to circulating cfDNA. Organoids are tractable systems for examining specific changes consistently in a human background. However, few studies have investigated cfDNA released from organoids. Here, we examined cfDNA isolated from cerebral organoids. We found that cerebral organoids release quantities of cfDNA sufficient for downstream analysis with droplet-digital PCR and whole-genome sequencing. Further, gene ontology analysis of genes aligning with sequenced cfDNA fragments revealed associations with terms related to neurodevelopment and autism spectrum disorder. We conclude that cerebral organoids hold promise as tools for the discovery of cfDNA biomarkers related to neurodevelopmental and neurological disorders.
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Cerebral Organoid Models for Neurotropic Viruses.
Across zoonotic pathogens, RNA viruses are responsible for disproportionate levels of human disease, suffering, and death. Neurotropic RNA viruses (e.g., rabies, Japanese and Eastern Equine Encephalitis, Ebola, West Nile, Powassan) infect the brain and spinal cord, causing meningitis, encephalitis, microcephaly, and Guillain-Barré syndrome. Mechanistic data explaining the molecular mechanisms of these diseases are lacking, and the enclosure of the central nervous system and the associated meninges in bone complicates access for diagnosis, clinical treatment, and research. Here, we discuss new tissue models, imaging methods, and molecular techniques that are changing research aimed at understanding the pathogenesis of neurotropic RNA viruses.
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Injection and electroporation of plasmid DNA into human cortical organoids.
Pluripotent stem cell-derived human cortical organoids allow for the analysis of stem cell behavior and neurogenesis in neural tissues. Delivery of plasmid DNA into organoids permits visualization of individual cells, characterization of cellular components, and manipulation of gene expression. We describe a protocol to transfect cells inside organoids with plasmid DNA using micro-injection and electroporation, allowing for DNA delivery to cells within cortical units. This protocol was optimized for cortical organoids; however, it may be adapted to other organoid models. For complete details on the use and execution of this protocol, please refer to Denoth-Lippuner et al. (2021).