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Generation and long-term culture of human cerebellar organoids from pluripotent stem cells.
The advancement of research on human cerebellar development and diseases has been hindered by the lack of a cell-based system that mirrors the cellular diversity and functional characteristics of the human cerebellum. Here, we describe our protocol for a human pluripotent stem cell-derived human cerebellar organoid (hCerO) model, which successfully replicates the cellular diversity of the fetal cerebellum along with some of its distinct cytoarchitectural features. Our approach involves the patterning of human pluripotent stem cells, resulting in the generation of both cerebellar excitatory and inhibitory progenitor populations-specifically, the rhombic lip and ventricular zone progenitors, respectively. This patterning strategy leads to the reproducible differentiation of the major neurons of the cerebellum such as granule cells and Purkinje cells within just one month of culture. hCerOs serve as platforms for molecular, cellular and functional assays, including single-cell transcriptomics, immunohistochemistry and investigations into calcium dynamics and electrophysiological properties. Remarkably, the cultivation of hCerOs for up to 8 months enables the healthy survival and maturation of Purkinje cells, which exhibit molecular and electrophysiological features akin to their in vivo counterparts. Overall, our protocol generates and allows for the long-term culture of all major cell types within the cerebellum. Consequently, this significant advancement provides the developmental neurobiology field with a robust platform for exploring both cerebellar development and diseases within an all-human system. This protocol can be easily implemented by a technician with cell culture experience and takes 1-2 months to complete with an option for extended maturation over the course of several months.
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Modelling human brain development and disease with organoids.
Organoids are systems derived from pluripotent stem cells at the interface between traditional monolayer cultures and in vivo animal models. The structural and functional characteristics of organoids enable the modelling of early stages of brain development in a physiologically relevant 3D environment. Moreover, organoids constitute a tool with which to analyse how individual genetic variation contributes to the susceptibility and progression of neurodevelopmental disorders. This Roadmap article describes the features of brain organoids, focusing on the neocortex, and their advantages and limitations - in comparison with other model systems - for the study of brain development, evolution and disease. We highlight avenues for enhancing the physiological relevance of brain organoids by integrating bioengineering techniques and unbiased high-throughput analyses, and discuss future applications. As organoids advance in mimicking human brain functions, we address the ethical and societal implications of this technology.
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A Primer on Human Brain Organoids for the Neurosurgeon.
Human brain organoids emerged in 2013 as a technology that, unlike prior in Vitro neural models, recapitulates brain development with a high degree of spatial and temporal fidelity. As the platform matured with more accurate reproduction of cerebral architecture, brain organoids became increasingly valuable for studying both normal cortical neurogenesis and a variety of congenital human brain disorders. While the majority of research utilizing human brain organoids has been in the realm of basic science, clinical applications are forthcoming. These present and future translational efforts have the potential to make a considerable impact on the field of neurosurgery. For example, glioma organoids are already being used to study tumor biology and drug responses, and adaptation for the investigation of other neurosurgery-relevant diseases is underway. Moreover, organoids are being explored as a structured neural substrate for repairing brain circuitry. Thus, we believe it is important for our field to be aware and have an accurate understanding of this emerging technology. In this review, we describe the key characteristics of human brain organoids, review their relevant translational applications, and discuss the ethical implications of their use through a neurosurgical lens.
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Traumatic brain injury-on-a-chip: a microfluidic device for the compression of cortical spheroids.
Traumatic brain injury (TBI) affects more than 4.6 million people annually in the United States, with the true numbers of mild TBI (mTBI) likely much higher due to insufficient detection and diagnosis methods. Some of the current challenges for successfully detecting mTBIs include the lack of patient symptoms and the lack of robust biomarkers for its diagnosis. While the quest for mTBI biomarkers remains an active area of research, the inherent low concentrations and our lack of understanding of the temporal evolution of its parenchymal release pose a significant hurdle to the adoption of blood- or saliva-borne biomarkers for injury diagnosis. Three-dimensional neuroglia and organoid cell culture models offer an opportunity to model TBI and screen for injury biomarker expression profiles. Yet there remains a shortage of lab-on-a-chip platforms capable of monitoring the evolution of injury biomarker onset using both imaging- and extraction-based detection approaches in the same culture platform. In this study, we developed a novel lab-on-a-chip, three-layer microfluidic device capable of reproducibly injuring cortical spheroids to model TBI in vitro. The device can be operated stand-alone with a single pump or straightforwardly integrated with common laboratory microscopes to facilitate real-time imaging access. To demonstrate its applicability for TBI research, compression of cortical spheroids was performed at a strain of 25% and a fixed strain rate of 3.125 s-1 as it is comparable to rates of mild TBI. Assessment of cell viability after 24 hours revealed that the impacted samples, on average, exhibited up to 8% cell death. Additionally, by leveraging a simple push-pull perfusion method, non-impacted samples could be exposed to culture medium from injured organoids on the same chip, which also resulted in increased cell death 24 hours later. These findings demonstrated the capability of this device as a viable in vitro platform of TBI, which should lend itself to investigations of TBI and other diseases.
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Gene Expression Manipulation Via Acute Electroporation in Human Brain Organoids.
Brain organoids are in vitro 3D cultures generated in the lab from human induced pluripotent stem cells or embryonic stem cells and can mimic the human brain structure and function. Specifically, they reproduce to some extent in vivo developmental events as they consist of diverse cell types, such as apical radial glial cells, intermediate progenitors, basal radial glial cells, and neurons forming stratified cortical layers similar to what is observed in the human brain in vivo. Due to cytoarchitecture similarities between the human brain and brain organoids, the latter have been proposed as excellent models for studying human brain development and disease. Thus, genome manipulation in brain organoids is crucial for investigating the functions of specific genes and mutations that have been associated with brain-related disorders. For this reason, gene manipulation has been implemented in brain organoids in the last few years. Here, we describe a step-by-step protocol for gene expression manipulation and analyses in brain organoids via acute electroporation that we have optimized based on the in vivo electroporation that has been widely used in animal models. This easy-to-apply protocol is fast and robust and facilitates the precise spatiotemporal manipulation of the expression of any gene of interest.
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Immuno-Regulation of Brain Region-Specific Organoids Containing Isogenic Microglia-Like Cells.
Most brain organoids derived from human induced pluripotent stem cells (iPSCs) lack microglia and thus immune function. Microglia-like cells (MGCs) can be differentiated from iPSCs, while the characteristics of isogenic MGC-containing brain organoids in modeling neurodegeneration and cell-cell communications have not been well investigated. In this study, iPSC-derived MGCs are co-cultured with isogenic forebrain cortical organoids (iFCo), which are stimulated with extracellular vesicles (EVs) of brain organoids differentiated from Alzheimer's disease (AD) patient-derived iPSCs (APOE ε4/ε4 and presenilin 1). The AD EV-stimulated co-culture organoids are treated with EVs from healthy MGCs or co-culture. Differential responses of the co-cultured organoids and the MGCs to AD EVs are demonstrated. The co-cultured organoids mitigated pro-inflammatory gene expressions. EVs from healthy MGCs or co-culture reduced the expression of IL-12β, iNOS, TREM2, and CASS4, which are associated with neural inflammation and degeneration, as well as showed regulation on genes involved in microglial activation and carbon metabolism. AD EV cargo analysis by proteomics and microRNA-sequencing revealed APOE and APP proteins and microRNAs regulated pathways such as mitophagy. This study paves the way for understanding the role of microglia and brain organoids in modeling neural degeneration and the development of EV-based cell-free therapeutics for AD treatment.
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Mechanisms of hyperexcitability in Alzheimer's disease hiPSC-derived neurons and cerebral organoids vs isogenic controls.
Human Alzheimer's disease (AD) brains and transgenic AD mouse models manifest hyperexcitability. This aberrant electrical activity is caused by synaptic dysfunction that represents the major pathophysiological correlate of cognitive decline. However, the underlying mechanism for this excessive excitability remains incompletely understood. To investigate the basis for the hyperactivity, we performed electrophysiological and immunofluorescence studies on hiPSC-derived cerebrocortical neuronal cultures and cerebral organoids bearing AD-related mutations in presenilin-1 or amyloid precursor protein vs. isogenic gene corrected controls. In the AD hiPSC-derived neurons/organoids, we found increased excitatory bursting activity, which could be explained in part by a decrease in neurite length. AD hiPSC-derived neurons also displayed increased sodium current density and increased excitatory and decreased inhibitory synaptic activity. Our findings establish hiPSC-derived AD neuronal cultures and organoids as a relevant model of early AD pathophysiology and provide mechanistic insight into the observed hyperexcitability.
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Stem cells: The developing human brain--modeled in a dish.
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The use of brain organoids to investigate neural development and disease.
Understanding the development and dysfunction of the human brain is a major goal of neurobiology. Much of our current understanding of human brain development has been derived from the examination of post-mortem and pathological specimens, bolstered by observations of developing non-human primates and experimental studies focused largely on mouse models. However, these tissue specimens and model systems cannot fully capture the unique and dynamic features of human brain development. Recent advances in stem cell technologies that enable the generation of human brain organoids from pluripotent stem cells (PSCs) promise to profoundly change our understanding of the development of the human brain and enable a detailed study of the pathogenesis of inherited and acquired brain diseases.
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Protocol for controlled cortical impact in human cerebral organoids to model traumatic brain injury.
Modeling traumatic brain injury (TBI) has been a challenge. Rodent and cellular models have provided relevant contributions despite their limitations. Here, we present a protocol for a TBI model based on the controlled cortical impact (CCI) performed on human cerebral organoids (COs), self-assembled 3D cultures that recapitulate features of the human brain. Here, we generate COs from iPSCs obtained from reprogrammed fibroblasts. For complete details on the use and execution of this protocol, please refer to Ramirez et al. (2021).