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The Application of Ultrasmall Gold Nanoparticles (2 nm) Functionalized with Doxorubicin in Three-Dimensional Normal and Glioblastoma Organoid Models of the Blood-Brain Barrier.
Among brain tumors, glioblastoma (GBM) is very challenging to treat as chemotherapeutic drugs can only penetrate the brain to a limited extent due to the blood-brain barrier (BBB). Nanoparticles can be an attractive solution for the treatment of GBM as they can transport drugs across the BBB into the tumor. In this study, normal and GBM organoids comprising six brain cell types were developed and applied to study the uptake, BBB penetration, distribution, and efficacy of fluorescent, ultrasmall gold nanoparticles (AuTio-Dox-AF647s) conjugated with doxorubicin (Dox) and AlexaFluor-647-cadaverine (AF647) by confocal laser scanning microscopy (CLSM), using a mixture of dissolved doxorubicin and fluorescent AF647 molecules as a control. It was shown that the nanoparticles could easily penetrate the BBB and were found in normal and GBM organoids, while the dissolved Dox and AF647 molecules alone were unable to penetrate the BBB. Flow cytometry showed a reduction in glioblastoma cells after treatment with AuTio-Dox nanoparticles, as well as a higher uptake of these nanoparticles by GBM cells in the GBM model compared to astrocytes in the normal cell organoids. In summary, our results show that ultrasmall gold nanoparticles can serve as suitable carriers for the delivery of drugs into organoids to study BBB function.
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Brain Organoids: Tiny Mirrors of Human Neurodevelopment and Neurological Disorders.
Unravelling the complexity of the human brain is a challenging task. Nowadays, modern neurobiologists have developed 3D model systems called "brain organoids" to overcome the technical challenges in understanding human brain development and the limitations of animal models to study neurological diseases. Certainly like most model systems in neuroscience, brain organoids too have limitations, as these minuscule brains lack the complex neuronal circuitry required to begin the operational tasks of human brain. However, researchers are hopeful that future endeavors with these 3D brain tissues could provide mechanistic insights into the generation of circuit complexity as well as reproducible creation of different regions of the human brain. Herein, we have presented the contemporary state of brain organoids with special emphasis on their mode of generation and their utility in modelling neurological disorders, drug discovery, and clinical trials.
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Brain organoids: A revolutionary tool for modeling neurological disorders and development of therapeutics.
Brain organoids are self-organized, three-dimensional (3D) aggregates derived from pluripotent stem cells that have cell types and cellular architectures resembling those of the developing human brain. The current understanding of human brain developmental processes and neurological disorders has advanced significantly with the introduction of this in vitro model. Brain organoids serve as a translational link between two-dimensional (2D) cultures and in vivo models which imitate the neural tube formation at the early and late stages and the differentiation of neuroepithelium with whole-brain regionalization. In addition, the generation of region-specific brain organoids made it possible to investigate the pathogenic and etiological aspects of acquired and inherited brain disease along with drug discovery and drug toxicity testing. In this review article, we first summarize an overview of the existing methods and platforms used for generating brain organoids and their limitations and then discuss the recent advancement in brain organoid technology. In addition, we discuss how brain organoids have been used to model aspects of neurodevelopmental and neurodegenerative diseases, including autism spectrum disorder (ASD), Rett syndrome, Zika virus-related microcephaly, Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD).
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Generating and characterizing human telencephalic brain organoids from stem cell-derived single neural rosettes.
We have developed a method for generating human telencephalic organoids from stem cell-derived isolated single neural rosettes. The use of single neural rosettes for generating organoids offers several important advantages. First, it mimics the development of neural tissue from a singular neural tube in vivo. Second, single neural rosette-derived organoids exhibit a relatively consistent and reproducible composition of telencephalic neural cells. Finally, single neural rosette-derived organoids demonstrate predictable organization of the identified neural cells around a single neural rosette-derived lumen and contain a large proportion of functionally mature neurons that generate action potentials and receive both excitatory and inhibitory synaptic inputs. These unique features of our protocol enable the study of the specification and organization of different neural cells in the developing human telencephalon, as well as modeling of neurodevelopmental disorders associated with disrupted neural networks. Here, we describe our protocols for generating CRISPR-Cas9-engineered human stem cells with a hemizygous SHANK3 deletion, stem cell-derived single neural rosettes and telencephalic brain organoids. We also offer insights on how to conduct single-cell RNA sequencing, immunohistochemistry and slice patch-clamp electrophysiology on these organoids. Completion of the protocols takes 5-6 months and requires experience working with cultured cells. We expect this protocol will prove useful for studies of human brain development and disease, as well as for advancing the development of new organoid-based biocomputers.
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Inkjet-Printed 3D Sensor Arrays with FIB-Induced Electrode Refinement for Low-Noise Amperometric Recordings in hiPSC-Derived Brain Organoids.
Understanding the functional connectivity and behavior of 3D cell cultures and organoids requires monitoring electrical activity across multiple planes. However, traditional planar microelectrode arrays (MEAs) are limited to surface recordings and struggle to capture signals from deeper layers. Additionally, current fabrication methods face challenges such as prolonged production times and limited design flexibility, which hinder the development of high-precision 3D electrode arrays and affect the quality of cell-electrode coupling. To overcome these obstacles, we introduce a new approach that integrates inkjet printing with focused ion beam (FIB) milling and electrodeposition, resulting in highly customizable 3D MEAs. The FIB milling enables the creation of precise electrode openings at predetermined locations, which is essential for selective recordings within the tissue. The MEAs, fabricated on glass substrates, incorporate high-aspect-ratio (up to 44:1) electrode structures with heights up to 1 mm, a pitch of 500 μm, and electrode openings of 3 and 6 μm, providing the necessary resolution for targeted measurements. Impedance and noise characteristics (down to a root-mean-square of (RMS) noise of 0.2 pA) for amperometric measurements were assessed in dependence on the electrode size. We demonstrate the effectiveness of these 3D MEAs by recording electrophysiological activity from hiPSC-derived cortical organoids (age: 24 month) both in situ and after 10 days of cultivation of the organoid directly on the MEA. This approach facilitates in vitro studies of neural activity in organoids and holds promise for high-throughput, selective amperometric analyses in normal and pathologically altered conditions.
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Vascularized Brain Assembloids With Enhanced Cellular Complexity Provide Insights Into the Cellular Deficits of Tauopathy.
Advanced technologies have enabled the engineering of self-organized 3-dimensional (3D) cellular structures from human induced pluripotent stem cells (hiPSCs), namely organoids, which recapitulate some key features of tissue development and functions of the human central nervous system (CNS). While hiPSC-derived 3D CNS organoids hold promise in providing a human-specific platform for studying CNS development and diseases, most of them do not incorporate the full range of implicated cell types, including vascular cell components and microglia, limiting their ability to accurately recreate the CNS environment and their utility in the study of certain aspects of the disease. Here we have developed a novel approach, called vascularized brain assembloids, for constructing hiPSC-derived 3D CNS structures with a higher level of cellular complexity. This is achieved by integrating forebrain organoids with common myeloid progenitors and phenotypically stabilized human umbilical vein endothelial cells (VeraVecs), which can be cultured and expanded in serum-free conditions. Compared with organoids, these assembloids exhibited enhanced neuroepithelial proliferation, advanced astrocytic maturation, and increased synapse numbers. Strikingly, the assembloids derived from hiPSCs harboring the tauP301S mutation exhibited increased levels of total tau and phosphorylated tau, along with a higher proportion of rod-like microglia-like cells and enhanced astrocytic activation, when compared to the assembloids derived from isogenic hiPSCs. Additionally, the tauP301S assembloids showed an altered profile of neuroinflammatory cytokines. This innovative assembloid technology serves as a compelling proof-of-concept model, opening new avenues for unraveling the intricate complexities of the human brain and accelerating progress in the development of effective treatments for neurological disorders.
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In vitro models of the choroid plexus and the blood-cerebrospinal fluid barrier: advances, applications, and perspectives.
The choroid plexus (CP), a highly vascularized endothelial-epithelial convolute, is placed in the ventricular system of the brain and produces a large part of the cerebrospinal fluid (CSF). Additionally, the CP is the location of a blood-CSF barrier (BCSFB) that separates the CSF from the blood stream in the CP endothelium. In vitro models of the CP and the BCSFB are of high importance to investigate the biological functions of the CP and the BCSFB. Since the CP is involved in several serious diseases, these in vitro models promise help in researching the processes contributing to the diseases and during the development of treatment options. In this review, we provide an overview on the available models and the advances that have been made toward more sophisticated and "in vivo near" systems as organoids and microfluidic lab-on-a-chip approaches. We go into the applications and research objectives for which the various modeling systems can be used and discuss the possible future prospects and perspectives.
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Epitope imprinting of alpha-synuclein for sensing in Parkinson's brain organoid culture medium.
Parkinson's disease (PD) is a progressive nervous system disorder that affects movement, whose early signs may be mild and unnoticed. α-Synuclein has been identified as the major component of Lewy bodies and Lewy neurites, which are the characteristic proteinaceous deposits that are the hallmarks of PD. In this work, three alpha-synuclein peptides were synthesized as templates for the molecular imprinting of conductive polymers to enable recognition of alpha-synuclein via ultrasensitive electrochemical measurements. The peptide sequences encompassed specific residues where mutations are known to accelerate PD (though the target sequences, in this study, were wild-type.) The different peptide targets were all successfully imprinted, but with differing imprinting effectiveness, probably owing to differences in target carboxylic acids (which can bind to the aniline (AN) m-aminobenzenesulfonic acid (MSAN) MIP polymers.) Composition of the imprinted polymer, (the mole proportions of AN and MSAN), and the concentrations and sequences of imprinted peptide templates were optimized by measuring the electrochemical responses to target peptides. The imprinted electrode can detect alpha-synuclein at fg/mL levels, and was therefore used to measure alpha-synuclein in the culture medium of human brain organoids generated from normal and idiopathic PD patients.
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When to create embryos or organoids for research.
The development of brain organoids and use of human embryonic neural structures for research each raise distinct ethical considerations that require careful analysis. We propose that rather than attempting to resolve longstanding debates about embryonic moral status, a more productive approach is to examine how different positions on this fundamental question lead to distinct conclusions about appropriate research strategies. For those who ground moral status in species membership or developmental potential, even early-stage embryo research may be ethically impermissible, suggesting focus on carefully bounded organoid development. Conversely, for those who ground moral status in current capacities, embryonic neural tissue studied before the emergence of consciousness may offer significant advantages over organoids while raising fewer novel ethical concerns. Our analysis reveals inadequacies in current policies, particularly the 14-day rule, which appears difficult to justify under either ethical framework. We demonstrate how careful attention to the relationship between ethical premises and research implications can advance both scientific progress and ethical oversight, while suggesting specific policy reforms including capacity-based research guidelines and sophisticated monitoring protocols.
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Human microglia states are conserved across experimental models and regulate neural stem cell responses in chimeric organoids.
Microglia are resident macrophages in the brain that emerge in early development and respond to the local environment by altering their molecular and phenotypic states. Fundamental questions about microglia diversity and function during development remain unanswered because we lack experimental strategies to interrogate their interactions with other cell types and responses to perturbations ex vivo. We compared human microglia states across culture models, including cultured primary and pluripotent stem cell-derived microglia. We developed a "report card" of gene expression signatures across these distinct models to facilitate characterization of their responses across experimental models, perturbations, and disease conditions. Xenotransplantation of human microglia into cerebral organoids allowed us to characterize key transcriptional programs of developing microglia in vitro and reveal that microglia induce transcriptional changes in neural stem cells and decrease interferon signaling response genes. Microglia additionally accelerate the emergence of synchronized oscillatory network activity in brain organoids by modulating synaptic density.