Modeling neurodegenerative diseases with brain organoids: from development to disease applications

The prevalence of dementia and other neurodegenerative diseases is rapidly increasing in aging nations. These relentless and progressive diseases remain largely without disease-modifying treatments despite decades of research and investments. It is becoming clear that traditional two-dimensional culture and animal model systems, while providing valuable insights on the major pathophysiological pathways associated with these diseases, have not translated well to patients' bedside. Fortunately, the advent of induced-pluripotent stem cells and three-dimensional cell culture now provide toolsthat are revolutionizing the study of human diseases by permitting analysis of patient-derived human tissue with non-invasive procedures. Specifically, brain organoids, self-organizing neural structures that can mimic human fetal brain development, have now been harnessed to develop alternative models of Alzheimer's disease, Parkinson's disease, motor neuron disease, and Frontotemporal dementia by recapitulating important neuropathological hallmarks found in these disorders. Despite these early breakthroughs, several limitations need to be vetted in brain organoid models in order to more faithfully match human tissue qualities, including relative tissue immaturity, lack of vascularization and incomplete cellular diversity found in this culture system. Here, we review current brain organoid protocols, the pathophysiology of neurodegenerative disorders, and early studies with brain organoid neurodegeneration models. We then discuss the multiple engineering and conceptual challenges surrounding their use and provide possible solutions and exciting avenues to be pursued. Altogether, we believe that brain organoids models, improved with classical and emerging molecular and analytic tools, have the potential to unravel the opaque pathophysiological mechanisms of neurodegeneration and devise novel treatments for an array of neurodegenerative disorders.

Toward improved understanding of cardiac development and congenital heart disease: The advent of cardiac organoids

Neurodegenerative diseases (NDs) such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) are progressive disorders characterized by complex, human-specific pathology that poses challenges to drug discovery efforts. Traditional models, including two-dimensional cell cultures and animal models, often fall short in replicating the intricate cellular interactions observed in human neurodegeneration. This review explores the potential of brain organoid technology to address these limitations and offer a model more relevant to humans. Recent advancements in induced pluripotent stem cell (iPSC) technology have enabled the generation of patient-derived brain organoids that differentiate into various neural cell types within 3-dimensional structures. These iPSC-derived brain organoids establish a physiologically relevant microenvironment that mimics human brain architecture and cellular diversity. This review synthesizes studies on the application of brain organoids in modeling PD and AD pathology, including approaches to improve model fidelity. Brain organoids replicate disease-specific features, including dopaminergic neuron degeneration in PD and amyloid plaque formation in AD, offering valuable insights into disease mechanisms and potential therapeutic targets. However, challenges remain, including incomplete maturation, batch variability, and the absence of vascularization and complete cortical layering. Bioengineering approaches, including CRISPR-based gene editing and organ-on-a-chip technologies, are being investigated to overcome these obstacles. Brain organoid technology presents a transformative platform for the study of NDs, facilitating detailed research into disease mechanisms and testing of therapeutics. Overcoming existing challenges is crucial for maximizing the translational value of brain organoids, advancing personalized medicine, and supporting the development of effective therapies for NDs.

Brain Organoids, the Path Forward?

Revealing Tissue-Specific SARS-CoV-2 Infection and Host Responses using Human Stem Cell-Derived Lung and Cerebral Organoids.

Retinal degenerative diseases (RDDs) comprise diverse genetic and phenotypic conditions that cause progressive retinal dysfunction and cell loss, leading to vision impairment or blindness. Most RDDs lack appropriate animal models for their study, which affects understanding their disease mechanisms and delays the progress of new treatment development. Recent advances in stem cell engineering, omics, and organoid technology are facilitating research into diseases for which there are no previously existing models. The development of retinal organoids produced from human stem cells has impacted the study of retinal development as well as the development of in vitro models of diseases, opening possibilities for applications in regenerative medicine, drug discovery, and precision medicine. In this review, we recapitulate research in the retinal organoid models for RDD, mentioning some of the main pathways underlying retinal neurodegeneration that can be studied in these new models, as well as their limitations and future challenges in this rapidly advancing field.

Brain organoids encompass a large collection of in vitro stem cell-derived 3D culture systems that aim to recapitulate multiple aspects of in vivo brain development and function. First, this review provides a brief introduction to the current state-of-the-art for neuro-ectoderm brain organoid development, emphasizing their biggest advantages in comparison with classical two-dimensional cell cultures and animal models. However, despite their usefulness for developmental studies, a major limitation for most brain organoid models is the absence of contributing cell types from endodermal and mesodermal origin. As such, current research is highly investing towards the incorporation of a functional vasculature and the microglial immune component. In this review, we will specifically focus on the development of immune-competent brain organoids. By summarizing the different approaches applied to incorporate microglia, it is highlighted that immune-competent brain organoids are not only important for studying neuronal network formation, but also offer a clear future as a new tool to study inflammatory responses in vitro in 3D in a brain-like environment. Therefore, our main focus here is to provide a comprehensive overview of assays to measure microglial phenotype and function within brain organoids, with an outlook on how these findings could better understand neuronal network development or restoration, as well as the influence of physical stress on microglia-containing brain organoids. Finally, we would like to stress that even though the development of immune-competent brain organoids has largely evolved over the past decade, their full potential as a pre-clinical tool to study novel therapeutic approaches to halt or reduce inflammation-mediated neurodegeneration still needs to be explored and validated.

Mitochondrial diseases are a group of severe pathologies that cause complex neurodegenerative disorders for which, in most cases, no therapy or treatment is available. These organelles are critical regulators of both neurogenesis and homeostasis of the neurological system. Consequently, mitochondrial damage or dysfunction can occur as a cause or consequence of neurodevelopmental or neurodegenerative diseases. As genetic knowledge of neurodevelopmental disorders advances, associations have been identified between genes that encode mitochondrial proteins and neurological symptoms, such as neuropathy, encephalomyopathy, ataxia, seizures, and developmental delays, among others. Understanding how mitochondrial dysfunction can alter these processes is essential in researching rare diseases. Three-dimensional (3D) cell cultures, which self-assemble to form specialized structures composed of different cell types, represent an accessible manner to model organogenesis and neurodevelopmental disorders. In particular, brain organoids are revolutionizing the study of mitochondrial-based neurological diseases since they are organ-specific and model-generated from a patient's cell, thereby overcoming some of the limitations of traditional animal and cell models. In this review, we have collected which neurological structures and functions recapitulate in the different types of reported brain organoids, focusing on those generated as models of mitochondrial diseases. In addition to advancements in the generation of brain organoids, techniques, and approaches for studying neuronal structures and physiology, drug screening and drug repositioning studies performed in brain organoids with mitochondrial damage and neurodevelopmental disorders have also been reviewed. This scope review will summarize the evidence on limitations in studying the function and dynamics of mitochondria in brain organoids.

How well do brain organoids capture your brain?

Recently, research is undergoing a drastic change in the application of the animal model as a unique investigation strategy, considering an alternative approach for the development of science for the future. Although conventional monolayer cell cultures represent an established and widely used in vitro method, the lack of tissue architecture and the complexity of such a model fails to inform true biological processes in vivo. Recent advances in cell culture techniques have revolutionized in vitro culture tools for biomedical research by creating powerful three-dimensional (3D) models to recapitulate cell heterogeneity, structure and functions of primary tissues. These models also bridge the gap between traditional two-dimensional (2D) single-layer cultures and animal models. 3D culture systems allow researchers to recreate human organs and diseases in one dish and thus holds great promise for many applications such as regenerative medicine, drug discovery, precision medicine, and cancer research, and gene expression studies. Bioengineering has made an important contribution in the context of 3D systems using scaffolds that help mimic the microenvironments in which cells naturally reside, supporting the mechanical, physical and biochemical requirements for cellular growth and function. We therefore speak of models based on organoids, bioreactors, organ-on-a-chip up to bioprinting and each of these systems provides its own advantages and applications. All of these techniques prove to be excellent candidates for the development of alternative methods for animal testing, as well as revolutionizing cell culture technology. 3D systems will therefore be able to provide new ideas for the study of cellular interactions both in basic and more specialized research, in compliance with the 3R principle. In this review, we provide a comparison of 2D cell culture with 3D cell culture, provide details of some of the different 3D culture techniques currently available by discussing their strengths as well as their potential applications.

The recent development of human cerebral organoids provides an invaluable in vitro model of human brain development to assess the toxicity of natural or man-made toxic substances. By recapitulating key aspects of early human neurodevelopment, investigators can evaluate with this three-dimensional (3D) model the effect of certain compounds on the formation of neuronal networks and their electrophysiological properties with more physiological relevance than neurons grown in monolayers and in cultures composed of a unique cell type. This promising potential has contributed to the development of a large number of diverse protocols to generate human cerebral organoids, making interlaboratory comparisons of results difficult. Based on a previously published protocol to generate human cortical organoids (herein called cerebral organoids), we detail several approaches to evaluate the effect of chemicals on neurogenesis, apoptosis, and neuronal function when exogenously applied to cultured specimens. Here, we take as an example 4-aminopyridine, a potassium channel blocker that modulates the activity of neurons and neurogenesis, and describe a simple and cost-effective way to test the impact of this agent on cerebral organoids derived from human induced pluripotent stem cells. We also provide tested protocols to evaluate neurogenesis in cerebral organoids with ethynyl deoxyuridine labeling and neuronal activity with live calcium imaging and microelectrode arrays. Together, these protocols should facilitate the implementation of cerebral organoid technologies in laboratories wishing to evaluate the effects of specific compounds or conditions on the development and function of human neurons with only basic cell culture equipment. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of human cerebral organoids from pluripotent stem cells Support Protocol 1: Human pluripotent stem cell culture Basic Protocol 2: Evaluation of neurogenesis in cerebral organoids with ethynyl deoxyuridine labeling Basic Protocol 3: Calcium imaging in cerebral organoids Basic Protocol 4: Electrophysiological evaluation of cerebral organoids with microelectrode arrays Support Protocol 2: Immunostaining of cerebral organoids.

Heart disease remains a leading cause of mortality and a major worldwide healthcare burden. Recent advances in stem cell biology have made it feasible to derive large quantities of cardiomyocytes for disease modeling, drug development, and regenerative medicine. The discoveries of reprogramming and transdifferentiation as novel biological processes have significantly contributed to this paradigm. This review surveys the means by which reprogramming and transdifferentiation can be employed to generate induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and induced cardiomyocytes (iCMs). The application of these patient-specific cardiomyocytes for both in vitro disease modeling and in vivo therapies for various cardiovascular diseases will also be discussed. We propose that, with additional refinement, human disease-specific cardiomyocytes will allow us to significantly advance the understanding of cardiovascular disease mechanisms and accelerate the development of novel therapeutic options.

Kidney organoids derived from human pluripotent stem cells (hPSCs) have emerged as powerful platforms for translational nephrology, enabling complex renal pathophysiology modeling in physiologically relevant three-dimensional contexts. This review synthesizes recent advances in kidney organoid applications for disease modeling and drug discovery, highlighting their translational potential beyond developmental biology. These organoids recapitulate key human kidney architectural features, including nephron-like structures with glomeruli and tubules, while exhibiting greater cellular heterogeneity than traditional two-dimensional cultures. They effectively model monogenic renal disorders including autosomal dominant polycystic kidney disease (ADPKD), congenital nephrotic syndrome, and Alport syndrome, as well as acquired conditions like acute kidney injury and drug-induced nephrotoxicity. Kidney organoids serve as predictive nephrotoxicity screening platforms, demonstrating dose- and time-dependent responses to cisplatin, tenofovir, and aristolochic acid. However, significant challenges persist, including insufficient vascularization, developmental immaturity, segmental bias, absent urinary drainage systems, and reproducibility variability. Emerging bioengineering strategies-including endothelial co-culture, microfluidic integration, and 3D bioprinting-aim to address these limitations. Integrating stem cell biology with engineering innovations and multi-omics technologies will be crucial for refining kidney organoids into scalable, reproducible models that faithfully recapitulate human kidney physiology and disease, ultimately enabling their translation into precision medicine applications.

Gastric Organoids: Progress and Remaining Challenges.

The International Space Station (ISS) is a collaborative platform for advanced research in microgravity, significantly contributing to cancer studies, drug development, and tissue engineering. Since its assembly in 1998, the ISS has become an invaluable environment for experiments not possible on Earth. A key focus is organoid development – miniature, 3D human organ models grown in space, offering insights into disease mechanisms, drug responses, and regenerative medicine. Microgravity promotes scaffold-free tissue formation, advancing neurodegenerative disease research and therapeutic discovery. Cancer research on the ISS has expanded since the 2010s, using microgravity to grow 3D tumor models that mimic human tumors more accurately than Earth-based systems. These studies have improved our understanding of cancer cell behavior, chemotherapy resistance, and immune responses, driving new drug development. NASA’s collaboration with initiatives like the Cancer Moonshot accelerates cancer research in space. Emerging biomarker studies in space are enhancing early cancer detection and environmental monitoring. Additionally, ISS research on radiation-induced DNA damage has led to improved diagnostic tools, such as microsatellite instability (MSI) tests, which aid in cancer detection and treatment planning. Overall, the ISS continues to push boundaries in biomedical research, offering novel insights that are transforming cancer biology, regenerative medicine, and precision health, with far-reaching implications for human health on Earth and beyond.