Neural Organoids Research Articles

Generation of neural organoids for spinal-cord regeneration via the direct reprogramming of human astrocytes.

Organoids with region-specific architecture could facilitate the repair of injuries of the central nervous system. Here we show that human astrocytes can be directly reprogrammed into early neuroectodermal cells via the overexpression of OCT4, the suppression of p53 and the provision of the small molecules CHIR99021, SB431542, RepSox and Y27632. We also report that the activation of signalling mediated by fibroblast growth factor, sonic hedgehog and bone morphogenetic protein 4 in the reprogrammed cells induces them to form spinal-cord organoids with functional neurons specific to the dorsal and ventral domains. In mice with complete spinal-cord injury, organoids transplanted into the lesion differentiated into spinal-cord neurons, which migrated and formed synapses with host neurons. The direct reprogramming of human astrocytes into neurons may pave the way for in vivo neural organogenesis from endogenous astrocytes for the repair of injuries to the central nervous system.

Neural Organoids Making Connections, Getting Real

Neural Organoids Making Connections, Getting Real

Neural cortical organoids from self-assembling human iPSC as a model to investigate neurotoxicity in brain ischemia

Neural cortical organoids from self-assembling human iPSC as a model to investigate neurotoxicity in brain ischemia

Self-organizing in vitro mouse neural tube organoids mimic embryonic development.

The embryonic neural tube is the origin of the entire adult nervous system, and disturbances in its development cause life-threatening birth defects. However, the study of mammalian neural tube development is limited by the lack of physiologically realistic three-dimensional (3D) in vitro models. Here, we report a self-organizing 3D neural tube organoid model derived from single mouse embryonic stem cells that exhibits an in vivo-like tissue architecture, cell type composition and anterior-posterior (AP) patterning. Moreover, maturation of the neural tube organoids showed the emergence of multipotent neural crest cells and mature neurons. Single-cell transcriptome analyses revealed the sequence of transcriptional events in the emergence of neural crest cells and neural differentiation. Thanks to the accessibility of this model, phagocytosis of migrating neural crest cells could be observed in real time for the first time in a mammalian model. We thus introduce a tractable in vitro model to study some of the key morphogenetic and cell type derivation events during early neural development.

Approaches to investigating metabolism in human neurodevelopment using organoids: insights from intestinal and cancer studies.

Interrogating the impact of metabolism during development is important for understanding cellular and tissue formation, organ and systemic homeostasis, and dysregulation in disease states. To evaluate the vital functions metabolism coordinates during human brain development and disease, pluripotent stem cell-derived models, such as organoids, provide tractable access to neurodevelopmental processes. Despite many strengths of neural organoid models, the extent of their replication of endogenous metabolic programs is currently unclear and requires direct investigation. Studies in intestinal and cancer organoids that functionally evaluate dynamic bioenergetic changes provide a framework that can be adapted for the study of neural metabolism. Validation of in vitro models remains a significant challenge; investigation using in vivo models and primary tissue samples is required to improve our in vitro model systems and, concomitantly, improve our understanding of human development.

Maturation and circuit integration of transplanted human cortical organoids

Self-organizing neural organoids represent a promising in vitro platform with which to model human development and disease1–5. However, organoids lack the connectivity that exists in vivo, which limits maturation and makes integration with other circuits that control behaviour impossible. Here we show that human stem cell-derived cortical organoids transplanted into the somatosensory cortex of newborn athymic rats develop mature cell types that integrate into sensory and motivation-related circuits. MRI reveals post-transplantation organoid growth across multiple stem cell lines and animals, whereas single-nucleus profiling shows progression of corticogenesis and the emergence of activity-dependent transcriptional programs. Indeed, transplanted cortical neurons display more complex morphological, synaptic and intrinsic membrane properties than their in vitro counterparts, which enables the discovery of defects in neurons derived from individuals with Timothy syndrome. Anatomical and functional tracings show that transplanted organoids receive thalamocortical and corticocortical inputs, and in vivo recordings of neural activity demonstrate that these inputs can produce sensory responses in human cells. Finally, cortical organoids extend axons throughout the rat brain and their optogenetic activation can drive reward-seeking behaviour. Thus, transplanted human cortical neurons mature and engage host circuits that control behaviour. We anticipate that this approach will be useful for detecting circuit-level phenotypes in patient-derived cells that cannot otherwise be uncovered.

Modeling human telencephalic development and autism-associated SHANK3 deficiency using organoids generated from single neural rosettes

Human telencephalon is an evolutionarily advanced brain structure associated with many uniquely human behaviors and disorders. However, cell lineages and molecular pathways implicated in human telencephalic development remain largely unknown. We produce human telencephalic organoids from stem cell-derived single neural rosettes and investigate telencephalic development under normal and pathological conditions. We show that single neural rosette-derived organoids contain pallial and subpallial neural progenitors, excitatory and inhibitory neurons, as well as macroglial and periendothelial cells, and exhibit predictable organization and cytoarchitecture. We comprehensively characterize the properties of neurons in SNR-derived organoids and identify transcriptional programs associated with the specification of excitatory and inhibitory neural lineages from a common pool of NPs early in telencephalic development. We also demonstrate that neurons in organoids with a hemizygous deletion of an autism- and intellectual disability-associated gene SHANK3 exhibit intrinsic and excitatory synaptic deficits and impaired expression of several clustered protocadherins. Collectively, this study validates SNR-derived organoids as a reliable model for studying human telencephalic cortico-striatal development and identifies intrinsic, synaptic, and clustered protocadherin expression deficits in human telencephalic tissue with SHANK3 hemizygosity.

Inferring and perturbing cell fate regulomes in human brain organoids.

Self-organizing neural organoids grown from pluripotent stem cells1-3 combined with single-cell genomic technologies provide opportunities to examine gene regulatory networks underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin data over a dense time course in human organoids covering neuroepithelial formation, patterning, brain regionalization and neurogenesis, and identify temporally dynamic and brain-region-specific regulatory regions. We developed Pando-a flexible framework that incorporates multi-omic data and predictions of transcription-factor-binding sites to infer a global gene regulatory network describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoids. We find that certain factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. We show that the transcription factor GLI3 is required for cortical fate establishment in humans, recapitulating previous research performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify two distinct GLI3 regulomes that are central to telencephalic fate decisions: one regulating dorsoventral patterning with HES4/5 as direct GLI3 targets, and one controlling ganglionic eminence diversification later in development. Together, we provide a framework for how human model systems and single-cell technologies can be leveraged to reconstruct human developmental biology.

Stretchable mesh microelectronics for the biointegration and stimulation of human neural organoids

Advances in tridimensional (3D) culture approaches have led to the generation of organoids that recapitulate cellular and physiological features of domains of the human nervous system. Although microelectrodes have been developed for long-term electrophysiological interfaces with neural tissue, studies of long-term interfaces between microelectrodes and free-floating organoids remain limited. In this study, we report a stretchable, soft mesh electrode system that establishes an intimate in vitro electrical interface with human neurons in 3D organoids. Our mesh is constructed with poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) based electrically conductive hydrogel electrode arrays and elastomeric poly(styrene-ethylene-butylene-styrene) (SEBS) as the substrate and encapsulation materials. This mesh electrode can maintain a stable electrochemical impedance in buffer solution under 50% compressive and 50% tensile strain. We have successfully cultured pluripotent stem cell-derived human cortical organoids (hCO) on this polymeric mesh for more than 3 months and demonstrated that organoids readily integrate with the mesh. Using simultaneous stimulation and calcium imaging, we show that electrical stimulation through the mesh can elicit intensity-dependent calcium signals comparable to stimulation from a bipolar stereotrode. This platform may serve as a tool for monitoring and modulating the electrical activity of in vitro models of neuropsychiatric diseases.

Optogenetic control of apical constriction induces synthetic morphogenesis in mammalian tissues

The emerging field of synthetic developmental biology proposes bottom-up approaches to examine the contribution of each cellular process to complex morphogenesis. However, the shortage of tools to manipulate three-dimensional (3D) shapes of mammalian tissues hinders the progress of the field. Here we report the development of OptoShroom3, an optogenetic tool that achieves fast spatiotemporal control of apical constriction in mammalian epithelia. Activation of OptoShroom3 through illumination in an epithelial Madin-Darby Canine Kidney (MDCK) cell sheet reduces the apical surface of the stimulated cells and causes displacements in the adjacent regions. Light-induced apical constriction provokes the folding of epithelial cell colonies on soft gels. Its application to murine and human neural organoids leads to thickening of neuroepithelia, apical lumen reduction in optic vesicles, and flattening in neuroectodermal tissues. These results show that spatiotemporal control of apical constriction can trigger several types of 3D deformation depending on the initial tissue context.

Rapid and robust directed differentiation of mouse epiblast stem cells into definitive endoderm and forebrain organoids.

Directed differentiation of pluripotent stem cells (PSCs) is a powerful model system for deconstructing embryonic development. Although mice are the most advanced mammalian model system for genetic studies of embryonic development, state-of-the-art protocols for directed differentiation of mouse PSCs into defined lineages require additional steps and generates target cell types with lower purity than analogous protocols for human PSCs, limiting their application as models for mechanistic studies of development. Here, we examine the potential of mouse epiblast stem cells cultured in media containing Wnt pathway inhibitors as a starting point for directed differentiation. As a proof of concept, we focused our efforts on two specific cell/tissue types that have proven difficult to generate efficiently and reproducibly from mouse embryonic stem cells: definitive endoderm and neural organoids. We present new protocols for rapid generation of nearly pure definitive endoderm and forebrain-patterned neural organoids that model the development of prethalamic and hippocampal neurons. These differentiation models present new possibilities for combining mouse genetic tools with in vitro differentiation to characterize molecular and cellular mechanisms of embryonic development.

Deep-learning analysis of micropattern-based organoids enables high-throughput drug screening of Huntington’s disease models

SummaryOrganoids are carrying the promise of modeling complex disease phenotypes and serving as a powerful basis for unbiased drug screens, potentially offering a more efficient drug-discovery route. However, unsolved technical bottlenecks of reproducibility and scalability have prevented the use of current organoids for high-throughput screening. Here, we present a method that overcomes these limitations by using deep-learning-driven analysis for phenotypic drug screens based on highly standardized micropattern-based neural organoids. This allows us to distinguish between disease and wild-type phenotypes in complex tissues with extremely high accuracy as well as quantify two predictors of drug success: efficacy and adverse effects. We applied our approach to Huntington’s disease (HD) and discovered that bromodomain inhibitors revert complex phenotypes induced by the HD mutation. This work demonstrates the power of combining machine learning with phenotypic drug screening and its successful application to reveal a potentially new druggable target for HD.

Toward the next generation of vascularized human neural organoids.

Thanks to progress in the development of three-dimensional (3D) culture technologies, human central nervous system (CNS) development and diseases have been gradually deciphered by using organoids derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). Selforganized neural organoids (NOs) have been used to mimic morphogenesis and functions of specific organs in vitro. Many NOs have been reproduced in vitro, such as those mimicking the human brain, retina, and spinal cord. However, NOs fail to capitulate to the maturation and complexity of in vivo neural tissues. The persistent issues with current NO cultivation protocols are inadequate oxygen supply and nutrient diffusion due to the absence of vascular networks. In vivo, the developing CNS is interpenetrated by vasculature that not only supplies oxygen and nutrients but also provides a structural template for neuronal growth. To address these deficiencies, recent studies have begun to couple NO culture with bioengineering techniques and methodologies, including genetic engineering, coculture, multidifferentiation, microfluidics and 3D bioprinting, and transplantation, which might promote NO maturation and create more functional NOs. These cutting-edge methods could generate an ever more reliable NO model in vitro for deciphering the codes of human CNS development, disease progression, and translational application. In this review, we will summarize recent technological advances in culture strategies to generate vascularized NOs (vNOs), with a special focus on cerebral- and retinal-organoid models.

Progress in Modeling Neural Tube Development and Defects by Organoid Reconstruction.

It is clear that organoids are useful for studying the structure as well as the functions of organs and tissues; they are able to simulate cell-to-cell interactions, symmetrical and asymmetric division, proliferation, and migration of different cell groups. Some progress has been made using brain organoids to elucidate the genetic basis of certain neurodevelopmental disorders. Such as Parkinson's disease and Alzheimer's disease. However, research on organoids in early neural development has received insufficient attention, especially that focusing on neural tube precursors. In this review, we focus on the recent research progress on neural tube organoids and discuss both their challenges and potential solutions.

A multiparametric calcium signal screening platform using iPSC-derived cortical neural spheroids.

Discovery of therapeutics for neurological diseases is hampered by the lack of predictive in vitro and in vivo models. Traditionally, in vitro assays rely on engineered cell lines grown two-dimensionally (2D) outside a physiological tissue context, which makes them very amenable for large scale drug screening but reduces their relevance to in vivo neurophysiology. In recent years, three-dimensional (3D) neural cell culture models derived from human induced pluripotent stem cells (iPSCs) have been developed as an in vitro assay platform to investigate brain development, neurological diseases, and for drug screening. iPSC-derived neural spheroids or organoids can be developed to include complex neuronal and glial cell populations and display spontaneous, synchronous activity, which is a hallmark of in vivo neural communication. In this report we present a proof-of-concept study evaluating 3D iPSC-derived cortical neural spheroids as a physiologically- and pharmacologically-relevant high-throughput screening (HTS) platform and investigate their potential for use for therapeutic development. To this end, a library of 687 neuroactive compounds were tested in a phenotypic screening paradigm which measured calcium activity as a functional biomarker for neural modulation through fluctuations in calcium fluorescence. Pharmacological responses of cortical neural spheroids were analyzed using a multi-parametric approach, whereby seven peak characteristics from the calcium activity in each well were quantified and incorporated into principal component analysis and Sammon mapping to measure compound response. Here, we describe the implementation of the 687-compound library screen and data analysis demonstrating that iPSC-derived cortical spheroids are a robust and information-rich assay platform for HTS.

Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies.

The use of organoids has become increasingly popular recently due to their self-organizing abilities, which facilitate developmental and disease modeling. Various methods have been described to create embryoid bodies (EBs) generated from embryonic or pluripotent stem cells but with varying levels of differentiation success and producing organoids of variable size. Commercial ultra-low attachment (ULA) V-bottom well plates are frequently used to generate EBs. These plates are relatively expensive and not as widely available as standard concave well plates. Here, we describe a cost-effective and low labor-intensive method that creates homogeneous EBs at high yield in standard V- and U-bottom well plates by applying an anti-adherence solution to reduce surface attachment, followed by centrifugation to enhance cellular aggregation. We also explore the effect of different seeding densities, in the range of 1 to 11 ×103 cells per well, for the fabrication of neuroepithelial EBs. Our results show that the use of V-bottom well plates briefly treated with anti-adherent solution (for 5 min at room temperature) consistently yields functional neural EBs in the range of seeding densities from 5 to 11×103 cells per well. A brief post-seeding centrifugation step further enhances EB establishment. EBs fabricated using centrifugation exhibited lower variability in their final size than their non-centrifuged counterparts, and centrifugation also improved EB yield. The span of conditions for reliable EB production is narrower in U-bottom wells than in V-bottom wells (i.e., seeding densities between 7×103 and 11×103 and using a centrifugation step). We show that EBs generated by the protocols introduced here successfully developed into neural organoids and expressed the relevant markers associated with their lineages. We anticipate that the cost-effective and easily implemented protocols presented here will greatly facilitate the generation of EBs, thereby further democratizing the worldwide ability to conduct organoid-based research.

Physiological Electric Field: A Potential Construction Regulator of Human Brain Organoids.

Brain organoids can reproduce the regional three-dimensional (3D) tissue structure of human brains, following the in vivo developmental trajectory at the cellular level; therefore, they are considered to present one of the best brain simulation model systems. By briefly summarizing the latest research concerning brain organoid construction methods, the basic principles, and challenges, this review intends to identify the potential role of the physiological electric field (EF) in the construction of brain organoids because of its important regulatory function in neurogenesis. EFs could initiate neural tissue formation, inducing the neuronal differentiation of NSCs, both of which capabilities make it an important element of the in vitro construction of brain organoids. More importantly, by adjusting the stimulation protocol and special/temporal distributions of EFs, neural organoids might be created following a predesigned 3D framework, particularly a specific neural network, because this promotes the orderly growth of neural processes, coordinate neuronal migration and maturation, and stimulate synapse and myelin sheath formation. Thus, the application of EF for constructing brain organoids in a3D matrix could be a promising future direction in neural tissue engineering.

Tweaking neural organoids to model human reactive astrocytes.

The study of human reactive astrocytes has been limited by resource availability. In this issue, Cvetkovic et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202107135) develop multicellular organoid systems containing mature astrocytes to study the dynamics of human astrocytes reactivity and its downstream effects on neuronal activity.

Re-defining and tackling the emerging challenges in stem cell research and translation: A report of the 10th CSSCR annual meeting.

The 10th congress of the Chinese Society for Stem Cell Research was held from 11 to 13 October 2020 at Guiyang International Eco‐Conference Center in Guizhou (Southwest China) with 700 offline participants (and 500 online participants). With a theme adopted from a poem by Mao Zedong during the long march, ‘However difficult it might appear; the challenges will be overcome’, the congress provided opportunities for the fast‐growing field to exchange ideas among people from academia, industry and regulatory authorities, to help accelerate translation. Eight plenary lectures and six concurrent sessions on cell fate decision, stem cell metabolism, neural stem cells and neural regeneration, organoids and disease models, tissue and organ regeneration and clinical translation of stem cell research were covered, including 77 oral presentations and 75 poster presentations. The congress also included special programmes of a youth forum, a lecture award programme, a flagship journal forum and a dedicated networking session. This 3‐day event will significantly boost the stem cell research in an era closing to application.

Assessing Gq-GPCR-induced human astrocyte reactivity using bioengineered neural organoids.

Astrocyte reactivity can directly modulate nervous system function and immune responses during disease and injury. However, the consequence of human astrocyte reactivity in response to specific contexts and within neural networks is obscure. Here, we devised a straightforward bioengineered neural organoid culture approach entailing transcription factor-driven direct differentiation of neurons and astrocytes from human pluripotent stem cells combined with genetically encoded tools for dual cell-selective activation. This strategy revealed that Gq-GPCR activation via chemogenetics in astrocytes promotes a rise in intracellular calcium followed by induction of immediate early genes and thrombospondin 1. However, astrocytes also undergo NF-κB nuclear translocation and secretion of inflammatory proteins, correlating with a decreased evoked firing rate of cocultured optogenetic neurons in suboptimal conditions, without overt neurotoxicity. Altogether, this study clarifies the intrinsic reactivity of human astrocytes in response to targeting GPCRs and delivers a bioengineered approach for organoid-based disease modeling and preclinical drug testing.