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Organoids for modeling prion diseases.
Human cerebral organoids are an exciting and novel model system emerging in the field of neurobiology. Cerebral organoids are spheres of self-organizing, neuronal lineage tissue that can be differentiated from human pluripotent stem cells and that present the possibility of on-demand human neuronal cultures that can be used for non-invasively investigating diseases affecting the brain. Compared with existing humanized cell models, they provide a more comprehensive replication of the human cerebral environment. The potential of the human cerebral organoid model is only just beginning to be elucidated, but initial studies have indicated that they could prove to be a valuable model for neurodegenerative diseases such as prion disease. The application of the cerebral organoid model to prion disease, what has been learned so far and the future potential of this model are discussed in this review.
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Temporal morphogen gradient-driven neural induction shapes single expanded neuroepithelium brain organoids with enhanced cortical identity.
Pluripotent stem cell (PSC)-derived human brain organoids enable the study of human brain development in vitro. Typically, the fate of PSCs is guided into subsequent specification steps through static medium switches. In vivo, morphogen gradients are critical for proper brain development and determine cell specification, and associated defects result in neurodevelopmental disorders. Here, we show that initiating neural induction in a temporal stepwise gradient guides the generation of brain organoids composed of a single, self-organized apical-out neuroepithelium, termed ENOs (expanded neuroepithelium organoids). This is at odds with standard brain organoid protocols in which multiple and independent neuroepithelium units (rosettes) are formed. We find that a prolonged, decreasing gradient of TGF-β signaling is a determining factor in ENO formation and allows for an extended phase of neuroepithelium expansion. In-depth characterization reveals that ENOs display improved cellular morphology and tissue architectural features that resemble in vivo human brain development, including expanded germinal zones. Consequently, cortical specification is enhanced in ENOs. ENOs constitute a platform to study the early events of human cortical development and allow interrogation of the complex relationship between tissue architecture and cellular states in shaping the developing human brain.
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Cerebral Organoids Recapitulate Epigenomic Signatures of the Human Fetal Brain.
Organoids derived from human pluripotent stem cells recapitulate the early three-dimensional organization of the human brain, but whether they establish the epigenomic and transcriptional programs essential for brain development is unknown. We compared epigenomic and regulatory features in cerebral organoids and human fetal brain, using genome-wide, base resolution DNA methylome and transcriptome sequencing. Transcriptomic dynamics in organoids faithfully modeled gene expression trajectories in early-to-mid human fetal brains. We found that early non-CG methylation accumulation at super-enhancers in both fetal brain and organoids marks forthcoming transcriptional repression in the fully developed brain. Demethylated regions (74% of 35,627) identified during organoid differentiation overlapped with fetal brain regulatory elements. Interestingly, pericentromeric repeats showed widespread demethylation in multiple types of in vitro human neural differentiation models but not in fetal brain. Our study reveals that organoids recapitulate many epigenomic features of mid-fetal human brain and also identified novel non-CG methylation signatures of brain development.
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APOE3ch alleviates Aβ and tau pathology and neurodegeneration in the human APPNL-G-F cerebral organoid model of Alzheimer's disease.
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Modeling Human Cytomegalovirus-Induced Microcephaly in Human iPSC-Derived Brain Organoids.
Although congenital infection by human cytomegalovirus (HCMV) is well recognized as a leading cause of neurodevelopmental defects, HCMV neuropathogenesis remains poorly understood. A major challenge for investigating HCMV-induced abnormal brain development is the strict CMV species specificity, which prevents the use of animal models to directly study brain defects caused by HCMV. We show that infection of human-induced pluripotent-stem-cell-derived brain organoids by a "clinical-like" HCMV strain results in reduced brain organoid growth, impaired formation of cortical layers, and abnormal calcium signaling and neural network activity. Moreover, we show that the impeded brain organoid development caused by HCMV can be prevented by neutralizing antibodies (NAbs) that recognize the HCMV pentamer complex. These results demonstrate in a three-dimensional cellular biosystem that HCMV can impair the development and function of the human brain and provide insights into the potential capacity of NAbs to mitigate brain defects resulted from HCMV infection.
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Organoids: a systematic review of ethical issues.
Organoids are 3D structures grown from pluripotent stem cells derived from human tissue and serve as in vitro miniature models of human organs. Organoids are expected to revolutionize biomedical research and clinical care. However, organoids are not seen as morally neutral. For instance, tissue donors may perceive enduring personal connections with their organoids, setting higher bars for informed consent and patient participation. Also, several organoid sub-types, e.g., brain organoids and human-animal chimeric organoids, have raised controversy. This systematic review provides an overview of ethical discussions as conducted in the scientific literature on organoids. The review covers both research and clinical applications of organoid technology and discusses the topics informed consent, commercialization, personalized medicine, transplantation, brain organoids, chimeras, and gastruloids. It shows that further ethical research is needed especially on organoid transplantation, to help ensure the responsible development and clinical implementation of this technology in this field.
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Electrophysiological and morphological characterization of single neurons in intact human brain organoids.
Brain organoids represent a new model system for studying developmental human neurophysiology. Methods for studying the electrophysiology and morphology of single neurons in organoids require acute slices or dissociated cultures. While these methods have advantages (e.g., visual access, ease of experimentation), they risk damaging cells and circuits present in the intact organoid. To access single cells within intact organoid circuits, we have demonstrated a method for fixturing and performing whole cell patch clamp recording from intact brain organoids using both manual and automated tools. We demonstrate applied electrophysiology methods development followed by an integration of electrophysiology with reconstructing the morphology of the neurons within the brain organoid using dye filling and tissue clearing. We found that whole cell patch clamp recordings could be achieved both on the surface and within the interior of intact human brain organoids using both manual and automated methods. Manual experiments were higher yield (53 % whole cell success rate manual, 9 % whole cell success rate automated), but automated experiments were more efficient (30 patch attempts per day automated, 10 patch attempts per day manual). Using these methods, we performed an unbiased survey of cells within human brain organoids between 90 and 120 days in vitro (DIV) and present preliminary data on morphological and electrical diversity in human brain organoids. The further development of intact brain organoid patch clamp methods could be broadly applicable to studies of cellular, synaptic, and circuit-level function in the developing human brain.
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Protocol for generating and using human iPSC-derived microglia-containing air-liquid-interface cortical organoid cultures.
Here, we present a protocol for generating long-term microglia-containing air-liquid-interface cortical organoid (MG-ALI-CO) cultures. This approach minimizes necrotic core formation, a common limitation of extended organoid cultures, favoring microglia survival and homeostasis. We describe steps for generating air-liquid-interface cortical organoids (ALI-COs), integrating macrophage precursors, and maintaining MG-ALI-COs. Additionally, we outline several experimental analyses of MG-ALI-COs, including immunostaining, imaging, and patch-clamp electrophysiological recordings. This model provides a physiologically relevant system to investigate human neuroimmune interactions in a 3D brain-like environment.
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Synaptic plasticity in human thalamocortical assembloids.
Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA sequencing revealed that >80% of cells in thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits.
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Modeling HIV-1 Infection in CNS via Infected Monocytes Using Immunocompetent Brain Organoids.
The development of 3D-organoid models has revolutionized the way diseases are studied. Recently, our brain organoid model has been shown to recapitulate in in vitro the human brain cytoarchitecture originally encountered in HIV-1 neuropathogenesis, allowing downstream applications. Infected monocytes, macrophages, and microglia are critically important immune cells for infection and dissemination of HIV-1 throughout brain during acute and chronic phase of the disease. Once in the brain parenchyma, long-lived infected monocytes/macrophages along with resident microglia contribute to the establishment of CNS latency in people with HIV (PWH). Hence, it is important to better understand how HIV-1 enters and establishes infection and latency in CNS to further develop cure strategies. Here we detailed an accessible protocol to incorporate monocytes (infected and/or labeled) as a model of transmigration of peripheral monocytes into brain organoids that can be applied to characterize HIV-1 neuroinvasion and virus dissemination.