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Long-term tracking of neural and oligodendroglial development in large-scale human cerebral organoids by noninvasive volumetric imaging.
Human cerebral organoids serve as a quintessential model for deciphering the complexities of brain development in a three-dimensional milieu. However, imaging these organoids, particularly when they exceed several millimeters in size, has been curtailed by the technical impediments such as phototoxicity, slow imaging speeds, and inadequate resolution and imaging depth. Addressing these pivotal challenges, our study has pioneered a high-speed scanning microscope, synergistically coupled with advanced computational image processing. This ensemble has empowered us to monitor the intricate dynamics of neuron and oligodendrocyte development within cerebral organoids across a trajectory of approximately two months. Line-shaped illumination mitigates photodamage and, alongside refined spatial gating, maximizes signal collection through integrating with computational processing. The integration of deconvolution and compressive sensing has improved image contrast by 6-fold, elucidating fine features of the neurites. Thus, noninvasive imaging enabled us to perform long-term tracking of neural and oligodendroglial development in the large-scale human cerebral organoid. Furthermore, our sophisticated volumetric segmentation algorithm has yielded a robust four-dimensional quantitative analysis, encapsulating both neuronal and oligodendroglial maturation. Collectively, these advances mark a significant advancement in the field of neurodevelopment, providing a powerful tool for in-depth study of complex brain organoid systems.
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Single-cell transcriptomics identifies perturbed molecular pathways in midbrain organoids using α-synuclein triplication Parkinson's disease patient-derived iPSCs.
Three-dimensional (3D) brain organoids provide a platform to study brain development, cellular coordination, and disease using human tissue. Here, we generate midbrain dopaminergic (mDA) organoids from induced pluripotent stem cells (iPSC) from healthy and Parkinson's Disease (PD) donors and assess them as a human PD model using single-cell RNAseq. We characterize cell types in our organoid cultures and analyze our model's Dopamine (DA) neurons using cytotoxic and genetic stressors. Our study provides the first in-depth, single-cell analysis of SNCA triplication and shows evidence for molecular dysfunction in oxidative phosphorylation, translation, and ER protein-folding in DA neurons. We perform an in-silico identification of rotenone-sensitive DA neurons and characterization of corresponding transcriptomic profiles associated with synaptic signalling and cholesterol biosynthesis. Finally, we show a novel chimera organoid model from healthy and PD iPSCs allowing the study of DA neurons from different individuals within the same tissue.
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Breeding brains? Patients' and laymen's perspectives on cerebral organoids.
Aim: To explore the perspectives of patients and laymen with regard to the development, use and storage of cerebral organoids, in order to contribute to the ethical debate about this technology. Materials & methods: In depth semi-structured interviews with 28 patients and laymen were conducted. A qualitative thematic analysis was undertaken using a constant comparative method. Results: Three interrelated themes emerged from the empirical material: moral value; willingness to donate; and elements of good governance. Conclusion: Patients and laymen are most concerned about cerebral organoids potentially developing consciousness and potential misuse. They support the use of cerebral organoids under the conditions that donors are adequately informed and that there will be good governance. Perspectives of patients and laymen are helpful to enable responsible development and use of cerebral organoids in practice.
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Development and Characterization of Human Cerebral Organoids: An Optimized Protocol.
Studies of human neurodevelopmental disorders and stem cell-based regenerative transplants have been hampered by the lack of a model of the developing human brain. Stem cell-derived neurons suffer major limitations, including the ability to recapitulate the 3-dimensional architecture of a brain tissue and the representation of multiple layers and cell types that contribute to the overall brain functions in vivo. Recently, cerebral organoid technology was introduced; however, such technology is still in its infancy, and its low reproducibility and limitations significantly reduce the reliability of such a model as it currently exists, especially considering the complexity of cerebral-organoid protocols. Here we have tested and compared multiple protocols and conditions for growth of organoids, and we describe an optimized methodology, and define the necessary and sufficient factors that support the development of optimal organoids. Our optimization criteria included organoids' overall growth and size, stratification and representation of the various cell types, inter-batch variability, analysis of neuronal maturation, and even the cost of the procedure. Importantly, this protocol encompasses a plethora of technical tips that allow researchers to easily reproduce it and obtain reliable organoids with the least variability, and showcases a robust array of approaches to characterize successful organoids. This optimized protocol provides a reliable system for genetic or pharmacological (drug development) screens and may enhance understanding and therapy of human neurodevelopmental disorders, including harnessing the therapeutic potential of stem cell-derived transplants.
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Ethical Aspects of Brain Organoid Research in News Reports: An Exploratory Descriptive Analysis.
Background and Objectives: Brain organoids are self-assembled, three-dimensional (3D) aggregates generated from pluripotent stem cells. These models are useful for experimental studies on human brain development and function and are therefore increasingly used for research worldwide. As their increasing use raises several ethical questions, we aimed to assess the current state of the press on brain organoid research using a cross-sectional database to understand the extent of discussion of this subject in the public. Materials and Methods: We conducted a descriptive analysis of news reports obtained from the Nexis Uni database, searched in April 2020. After extracting the news reports, the number of published reports in each year and the included terms were analyzed. Results: Up to April 2020, 332 news reports had been published, with over half of them published in the United States and the United Kingdom, with the numbers gradually increasing every year. In total, 113 (34.0%) news reports included ethics-related keywords, and the ratio of studies before and after the study-period midpoint was significantly increased (21.0% (2013-2016) vs. 38.2% (2017-2020); p = 0.0066, Chi-square test with Yates' continuity correction). Conclusions: Although news reports on the ethical aspects of brain organoid research have been increasing gradually, there was a bias in the region of publication. Additional studies focusing on the ethical aspects of brain organoid research should strive to assess the public perception on the subject in different parts of the world.
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Reproducible generation of human midbrain organoids for in vitro modeling of Parkinson's disease.
The study of human midbrain development and midbrain related diseases, like Parkinson's disease (PD), is limited by deficiencies in the currently available and validated laboratory models. Three dimensional midbrain organoids represent an innovative strategy to recapitulate some aspects of the complexity and physiology of the human midbrain. Nevertheless, also these novel organoid models exhibit some inherent weaknesses, including the presence of a necrotic core and batch-to-batch variability. Here we describe an optimized approach for the standardized generation of midbrain organoids that addresses these limitations, while maintaining key features of midbrain development like dopaminergic neuron and astrocyte differentiation. Moreover, we have established a novel time-efficient, fit for purpose analysis pipeline and provided proof of concept for its usage by investigating toxin induced PD.
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Making Sense of Patient-Derived iPSCs, Transdifferentiated Neurons, Olfactory Neuronal Cells, and Cerebral Organoids as Models for Psychiatric Disorders.
The improvement of experimental models for disorders requires a constant approximation towards the dysregulated tissue. In psychiatry, where an impairment of neuronal structure and function is assumed to play a major role in disease mechanisms and symptom development, this approximation is an ongoing process implicating various fields. These include genetic, animal, and post-mortem studies. To test hypotheses generated through these studies, in vitro models using non-neuronal cells such as fibroblasts and lymphocytes have been developed. For brain network disorders, cells with neuronal signatures would, however, represent a more adequate tissue. Considering the limited accessibility of brain tissue, research has thus turned towards neurons generated from induced pluripotent stem cells as well as directly induced neurons, cerebral organoids, and olfactory neuroepithelium. Regarding the increasing importance and amount of research using these neuronal cells, this review aims to provide an overview of all these models to make sense of the current literature. The development of each model system and its use as a model for the various psychiatric disorder categories will be laid out. Also, advantages and limitations of each model will be discussed, including a reflection on implications and future perspectives.
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Dynamic Characterization of Structural, Molecular, and Electrophysiological Phenotypes of Human-Induced Pluripotent Stem Cell-Derived Cerebral Organoids, and Comparison with Fetal and Adult Gene Profiles.
BACKGROUND: The development of 3D cerebral organoid technology using human-induced pluripotent stem cells (iPSCs) provides a promising platform to study how brain diseases are appropriately modeled and treated. So far, understanding of the characteristics of organoids is still in its infancy. The current study profiled, for the first time, the electrophysiological properties of organoids at molecular and cellular levels and dissected the potential age equivalency of 2-month-old organoids to human ones by a comparison of gene expression profiles among cerebral organoids, human fetal and adult brains. RESULTS: Cerebral organoids exhibit heterogeneous gene and protein markers of various brain cells, such as neurons, astrocytes, and vascular cells (endothelial cells and smooth muscle cells) at 2 months, and increases in neural, glial, vascular, and channel-related gene expression over a 2-month differentiation course. Two-month organoids exhibited action potentials, multiple channel activities, and functional electrophysiological responses to the anesthetic agent propofol. A bioinformatics analysis of 20,723 gene expression profiles showed the similar distance of gene profiles in cerebral organoids to fetal and adult brain tissues. The subsequent Ingenuity Pathway Analysis (IPA) of select canonical pathways related to neural development, network formation, and electrophysiological signaling, revealed that only calcium signaling, cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) signaling in neurons, glutamate receptor signaling, and synaptogenesis signaling were predicted to be downregulated in cerebral organoids relative to fetal samples. Nearly all cerebral organoid and fetal pathway phenotypes were predicted to be downregulated compared with adult tissue. CONCLUSIONS: This novel study highlights dynamic development, cellular heterogeneity and electrophysiological activity. In particular, for the first time, electrophysiological drug response recapitulates what occurs in vivo, and neural characteristics are predicted to be highly similar to the human brain, further supporting the promising application of the cerebral organoid system for the modeling of the human brain in health and disease. Additionally, the studies from these characterizations of cerebral organoids in multiple levels and the findings from gene comparisons between cerebral organoids and humans (fetuses and adults) help us better understand this cerebral organoid-based cutting-edge platform and its wide uses in modeling human brain in terms of health and disease, development, and testing drug efficacy and toxicity.
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Facilitating Cerebral Organoid Culture via Lateral Soft Light Illumination.
At present, cerebral organoid culture technology is still complicated to operate and difficult to apply on a large scale. It is necessary to find a simple and practical solution. Therefore, a more feasible cerebral organoid protocol is proposed in the present study. To solve the unavoidable inconvenience in medium change and organoid transfer in the early stage, the current research optimizes the operation technology by applying the engineering principle. A soft light lamp was adopted to laterally illuminate the embryoid body (EB) samples, allowing the EBs to be seen by the naked eye through the enhanced diffuse reflection effect. Using the principle of secondary flow generated by rotation, the organoids gather toward the center of the well, which facilitates the operation of medium change or organoid transfer. Compared to the dispersed cell, the embryoid body settles faster in the pipette. Using this phenomenon, most of the free cells and dead cell fragments can be effectively removed in a simple way, preventing EBs from incurring damage from centrifugation. This study facilitates the operation of cerebral organoid culture and helps to promote the application of brain organoids.
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Human Cerebral Organoid Implantation Alleviated the Neurological Deficits of Traumatic Brain Injury in Mice.
Traumatic brain injury (TBI) causes a high rate of mortality and disability, and its treatment is still limited. Loss of neurons in damaged area is hardly rescued by relative molecular therapies. Based on its disease characteristics, we transplanted human embryonic stem cell- (hESC-) derived cerebral organoids in the brain lesions of controlled cortical impact- (CCI-) modeled severe combined immunodeficient (SCID) mice. Grafted organoids survived and differentiated in CCI-induced lesion pools in mouse cortical tissue. Implanted cerebral organoids differentiated into various types of neuronal cells, extended long projections, and showed spontaneous action, as indicated by electromyographic activity in the grafts. Induced vascularization and reduced glial scar were also found after organoid implantation, suggesting grafting could improve local situation and promote neural repair. More importantly, the CCI mice's spatial learning and memory improved after organoid grafting. These findings suggest that cerebral organoid implanted in lesion sites differentiates into cortical neurons, forms long projections, and reverses deficits in spatial learning and memory, a potential therapeutic avenue for TBI.