Cellular complexity in brain organoids: Current progress and unsolved issues

How well do brain organoids capture your brain?

<p indent="0mm">Brain organoids are <italic>in vitro</italic> three-dimensional neural cultures with similar cellular diversities, spatial organizations, and physiological functions of the human brain. Since its establishment, brain organoid technology has provided valuable tools for investigating the human brain development and functionality, disease etiology, drug discovery, and many other biological and medical questions. The progress of region-specific brain organoids has furthered the frontiers of brain organoid technology. In this article, we briefly review the development of brain organoid technology, including region-specific brain organoids. In particular, we focus on the technologies and applications of brain organoids in modeling human brain development. We also discuss the progress and challenges of various pivotal technologies in brain organoid studies, including vascularization, gene editing, single-cell sequencing, and other engineering approaches.

A brain organoid is a self-organizing three-dimensional tissue derived from human embryonic stem cells or pluripotent stem cells and is able to simulate the architecture and functionality of the human brain. Brain organoid generation methods are abundant and continue to improve, and now, an in vivo vascularized brain organoid has been encouragingly reported. The combination of brain organoids with immune-staining and single-cell sequencing technology facilitates our understanding of brain organoids, including the structural organization and the diversity of cell types. Recent publications have reported that brain organoids can mimic the dynamic spatiotemporal process of early brain development, model various human brain disorders, and serve as an effective preclinical platform to test and guide personalized treatment. In this review, we introduce the current state of brain organoid differentiation strategies, summarize current progress and applications in the medical domain, and discuss the challenges and prospects of this promising technology.

Chapter 18 - Cerebral and noncerebral organoids

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

One of the main goals of neurobiology is to understand the development and dysfunction of the human brain. Many of the tools and techniques that have informed our understanding of human brain development cannot fully capture the unique and dynamic features of human brain development. Recent advances in stem cell technologies that allow the generation of human brain organoids from pluripotent stem cells (PSCs) promise to profoundly change our understanding of human brain development and enable a detailed study of the pathogenesis of hereditary and acquired brain diseases. In this review, we will overview the development of brain organoid technology, its current progress and applications, and future prospects of this technology.

Brain organoids have become a valuable tool for studying human brain development, disease modeling, and drug testing. However, generating brain organoids with mature neurons is time-intensive and often incomplete, limiting their utility in studying age-related neurodegenerative diseases such as Alzheimer's disease (AD). Here, we report the generation of 3D brain organoids from human fibroblasts through direct reprogramming, with simplicity, efficiency, and reduced variability. We also demonstrate that induced brain organoids from APOE ε4 AD patient fibroblasts capture some disease-specific features and pathologies associated with APOE ε4 AD. Moreover, APOE ε4-induced brain organoids with mutant APP overexpression faithfully recapitulate the acceleration of AD-related pathologies, providing a more physiologically relevant and patient-specific model of familial AD. Importantly, transcriptome analysis reveals that gene sets specific to APOE ε4 patient-induced brain organoids are highly similar to those of APOE ε4 post-mortem AD brains. Overall, induced brain organoids from direct reprogramming offer a promising approach for more efficient and controlled studies of neurodegenerative disease modeling.

Microcephaly-associated mutations disrupt microtubule-dependent WDR62 translocation from the Golgi complex to the mitotic spindle poles, impair mitotic progression, and alter neurogenic trajectories in patient induced Pluripotent Stem Cell-derived 2D and 3D models of human neurodevelopment.

Human‐induced pluripotent stem cell (iPSC)‐derived brain organoids have attracted significant attention as promising models for drug screening owing to their remarkable resemblance to the human brain. The advent of disease model organoids, derived from patient's cells, has further elevated expectations for drug screening and personalized medicine. Nevertheless, the absence of a comprehensive platform for administering drugs and assessing their efficacy based on functional changes in brain organoids has remained a challenge. In this study, we introduce a one‐step drug screening system designed designed to investigate functional changes induced by diverse drug doses in multiple brain organoids, utilizing electrophysiological signal measurements. Our system comprises a specialized culture chamber with a microfluidic chip capable of accommodating 10 organoids and delivering varying doses of two drugs to each organoid. Additionally, we integrate a three dimentional microelectrode array (3D MEA) with ten shanks, enabling functional assessment of 10 brain organoids. This approach facilitates dose‐dependent drug screening across multiple organoids. We demonstrate the effectiveness of our system through real‐time analysis of neural activity changes triggered by different doses of pottassium chloride (KCl). Furthermore, sodium channel protein type 2 subunit alpha (SCN2A)‐epileptic organoids to demonstrate utility in disease‐model‐based drug screening. Our platform enables functional screening and personalized medicine using brain organoids.

Chapter 6 - Brain organoids: models of cell type diversity, connectivity, and disease phenotypes

Dissecting human neurobiology at high resolution and with mechanistic precision requires a major leap in scalability, given the need for experimental designs that include multiple individuals and, prospectively, population cohorts. To lay the foundation for this, we have developed and benchmarked complementary strategies to multiplex brain organoids by pooling cells from different pluripotent stem cell (PSC) lines either during organoid generation (mosaic models) or before single-cell RNA sequencing (scRNA-seq) library preparation (downstream multiplexing). We have also developed a new computational method, SCanSNP, and a consensus call to deconvolve cell identities, overcoming current criticalities in doublets and low-quality cell identification. We validated both multiplexing methods for charting neurodevelopmental trajectories at high resolution, thus linking specific individuals’ trajectories to genetic variation. Finally, we modeled their scalability across different multiplexing combinations and showed that mosaic organoids represent an enabling method for high-throughput settings. Together, this multiplexing suite of experimental and computational methods provides a highly scalable resource for brain disease and neurodiversity modeling.

Upgrading the Physiological Relevance of Human Brain Organoids

The advent of human induced pluripotent stem cell (hiPSC)-derived brain organoids represents a significant advance in environmental neurotoxicology, propelling the discipline toward human-relevant, mechanistic, and predictive in vitro paradigms. This review explores the utility of brain organoids in environmental neurotoxicology, which uniquely address critical limitations of traditional models by recapitulating key aspects of human brain development, including three-dimensional (3D) cytoarchitecture, multilineage cellular heterogeneity, and functional network activity. This review systematically elaborates on their construction principles, unique advantages in neurotoxicological research, and the significant progress made in elucidating mechanisms of toxicity. Notably, brain organoids exhibit enhanced sensitivity in identifying the subtle adverse outcomes of chronic, low-dose exposures to environmental contaminants, often eluding conventional approaches. Their key advantage lies in the greater capacity to deconstruct complex toxicological pathways, enabling precise tracing of adverse outcome pathways (AOPs). Future development requires enhancing model complexity through vascularization, promoting automation and standardization, and integrating artificial intelligence (AI) for data analysis. Concurrently, establishing sustained ethical oversight and standardized frameworks is essential to ultimately advance the field toward more precise and efficient hazard identification and risk characterization.Graphical abstractHighlights1. Human brain organoids bridge species gaps for more human-relevant neurotoxicity assessment.2. Brain organoids effectively recapitulate chronic, low-dose and mixture environmental risks.3. Integrating brain organoid data into the AOP frameworks enhances mechanistic understanding.4. Coupling brain organoids with organ-on-a-chip and AI advances next-generation risk assessment.

Brain organoids are an exciting new technology with the potential to significantly change how diseases of the brain are understood and treated. These three-dimensional neural tissues are derived from the self-organization of pluripotent stem cells, and they recapitulate the developmental process of the human brain, including progenitor zones and rudimentary cortical layers. Brain organoids have been valuable in investigating different aspects of developmental neurobiology and comparative biology. Several characteristics of organoids also make them attractive as models of brain disorders. Data generated from human organoids are more generalizable to patients because of the match in species background. Personalized organoids also can be generated from patient-derived induced pluripotent stem cells. Furthermore, the three-dimensionality of brain organoids supports cellular, mechanical, and topographical cues that are lacking in planar systems. In this review, we discuss the translational potential of brain organoids, using the examples of Zika virus, autism-spectrum disorder, and glioblastoma multiforme to consider how they could contribute to disease modeling, personalized medicine, and testing of therapeutics. We then discuss areas of improvement in organoid technology that will enhance the translational potential of brain organoids, as well as the possibility of their use as substrates for repairing cerebral circuitry after injury. Developmental Dynamics 248:53-64, 2019. © 2018 Wiley Periodicals, Inc.

ABSTRACTIntroduction: Parkinson’s disease is a devastating neurodegenerative disorder preferentially involving loss of dopaminergic neurons in the substantia nigra, leading to typical motor symptoms. While there are still no therapeutics to modify disease course, recent work using induced pluripotent stem cell (iPSC) and 3D brain organoid models have provided further insight into Parkinson’s disease pathogenesis and potential therapeutic targets.Areas covered: This review highlights the generation of iPSC neurons and neural organoids as models for studying Parkinson’s disease. It further discusses the recent work using patient-derived neurons from both familial and sporadic forms of Parkinson’s to study disease pathogenic phenotypes and pathways. It additionally provides an evaluation of iPSC neurons and organoid models for therapeutic development in Parkinson’s.Expert opinion: The use of Parkinson’s disease patient-derived neurons and organoids provides us with the exciting opportunity to directly investigate pathogenic mechanisms and test drug compounds in human neurons. Future studies will involve generating more sophisticated models of brain organoids, studying neuronal pathways using larger patient cohorts, and routinely assessing therapeutics in these models.