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Human cerebral organoids recapitulate gene expression programs of fetal neocortex development.
Cerebral organoids-3D cultures of human cerebral tissue derived from pluripotent stem cells-have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.
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Establishment and characterization of human pluripotent stem cells-derived brain organoids to model cerebellar diseases.
The establishment of robust human brain organoids to model cerebellar diseases is essential to study new therapeutic strategies for cerebellum-associated disorders. Machado-Joseph disease (MJD) is a cerebellar hereditary neurodegenerative disease, without therapeutic options able to prevent the disease progression. In the present work, control and MJD induced-pluripotent stem cells were used to establish human brain organoids. These organoids were characterized regarding brain development, cell type composition, and MJD-associated neuropathology markers, to evaluate their value for cerebellar diseases modeling. Our data indicate that the organoids recapitulated, to some extent, aspects of brain development, such as astroglia emerging after neurons and the presence of ventricular-like zones surrounded by glia and neurons that are found only in primate brains. Moreover, the brain organoids presented markers of neural progenitors proliferation, neuronal differentiation, inhibitory and excitatory synapses, and firing neurons. The established brain organoids also exhibited markers of cerebellar neurons progenitors and mature cerebellar neurons. Finally, MJD brain organoids showed higher ventricular-like zone numbers, an indication of lower maturation, and an increased number of ataxin-3-positive aggregates, compared with control organoids. Altogether, our data indicate that the established organoids recapitulate important characteristics of human brain development and exhibit cerebellar features, constituting a resourceful tool for testing therapeutic approaches for cerebellar diseases.
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Cell diversity in the human cerebral cortex: from the embryo to brain organoids.
The development and wiring of the central nervous system is a remarkable biological process that starts with the generation of and interaction between a large diversity of cell types. Our understanding of the developmental logic that drives cellular diversification in the mammalian brain comes, to a large extent, from studies in rodents. However, identifying the unique cellular processes underlying primate corticogenesis has been slow, due to the challenges associated with directly observing and manipulating brain tissue from these species. Recent technological advances in two areas hold promise to accelerate discovery of the mechanisms that govern human brain development, evolution, and pathophysiology of disease. Molecular profiling of large numbers of single cells can now capture cell identity and cell states within a complex tissue. Furthermore, modeling aspects of human organogenesis in vitro, even for tissues as complex as the brain, has been advanced by the use of three-dimensional organoid systems. Here, we describe how these approaches have been applied to date and how they promise to uncover the principles of cell diversification in the developing human brain.
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Deciphering neuronal deficit and protein profile changes in human brain organoids from patients with creatine transporter deficiency.
Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the SLC6A8 gene. The impaired creatine uptake in the brain results in intellectual disability, behavioral disorders, language delay, and seizures. In this work, we generated human brain organoids from induced pluripotent stem cells of healthy subjects and CTD patients. Brain organoids from CTD donors had reduced creatine uptake compared with those from healthy donors. The expression of neural progenitor cell markers SOX2 and PAX6 was reduced in CTD-derived organoids, while GSK3β, a key regulator of neurogenesis, was up-regulated. Shotgun proteomics combined with integrative bioinformatic and statistical analysis identified changes in the abundance of proteins associated with intellectual disability, epilepsy, and autism. Re-establishment of the expression of a functional SLC6A8 in CTD-derived organoids restored creatine uptake and normalized the expression of SOX2, GSK3β, and other key proteins associated with clinical features of CTD patients. Our brain organoid model opens new avenues for further characterizing the CTD pathophysiology and supports the concept that reinstating creatine levels in patients with CTD could result in therapeutic efficacy.
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Conversion of silent synapses to AMPA receptor-mediated functional synapses in human cortical organoids.
Despite the crucial role of synaptic connections and neural activity in the development and organization of cortical circuits, the mechanisms underlying the formation of functional synaptic connections in the developing human cerebral cortex remain unclear. We investigated the development of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission using human cortical organoids (hCOs) derived from induced pluripotent stem cells. Two-photon Ca2⁺ imaging revealed an increase in the frequency and amplitude of spontaneous activity in hCOs on day 80 compared to day 50. Additionally, spontaneous neural activity in late-stage hCOs, but not in early-stage hCOs, was blocked by N-methyl-D-aspartate receptor (NMDAR) and AMPAR antagonists. However, transsynaptic circuit tracing with G-deleted rabies viral vectors indicated a similar number of synaptic connections in early- and late-stage hCOs. Notably, chemical labeling demonstrated a significant increase in AMPAR expression on the postsynaptic membrane and colocalization with NMDARs in late-stage hCOs. These results suggest that hCOs progressively organize excitatory synaptic transmission, concurrent with the transition from silent synapses lacking AMPARs to functional synapses containing NMDARs and AMPARs. This in vitro model of human cortical circuits derived from induced pluripotent stem cells reflects the developmental programs underlying physiological transitions, providing valuable insights into human corticogenesis and neurodevelopmental disorders.
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Perspectives in the investigation of Cockayne syndrome group B neurological disease: the utility of patient-derived brain organoid models.
Cockayne syndrome (CS) group B (CSB), which results from mutations in the excision repair cross-complementation group 6 (ERCC6) genes, which produce CSB protein, is an autosomal recessive disease characterized by multiple progressive disorders including growth failure, microcephaly, skin photosensitivity, and premature aging. Clinical data show that brain atrophy, demyelination, and calcification are the main neurological manifestations of CS, which progress with time. Neuronal loss and calcification occur in various brain areas, particularly the cerebellum and basal ganglia, resulting in dyskinesia, ataxia, and limb tremors in CSB patients. However, the understanding of neurodevelopmental defects in CS has been constrained by the lack of significant neurodevelopmental and functional abnormalities observed in CSB-deficient mice. In this review, we focus on elucidating the protein structure and distribution of CSB and delve into the impact of CSB mutations on the development and function of the nervous system. In addition, we provide an overview of research models that have been instrumental in exploring CS disorders, with a forward-looking perspective on the substantial contributions that brain organoids are poised to further advance this field.
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Matrix rigidity-modulated cardiovascular organoid formation from embryoid bodies.
Stem cell clusters, such as embryoid bodies (EBs) derived from embryonic stem cells, are extensively studied for creation of multicellular clusters and complex functional tissues. It is common to control phenotypes of ES cells with varying molecular compounds; however, there is still a need to improve the controllability of cell differentiation, and thus, the quality of created tissue. This study demonstrates a simple but effective strategy to promote formation of vascularized cardiac muscle-like tissue in EBs and form contracting cardiovascular organoids by modulating the stiffness of a cell adherent hydrogel. Using collagen-conjugated polyacrylamide hydrogels with controlled elastic moduli, we discovered that cellular organization in a form of vascularized cardiac muscle sheet was maximal on the gel with the stiffness similar to cardiac muscle. We envisage that the results of this study will greatly contribute to better understanding of emergent behavior of stem cells in developmental and regeneration process and will also expedite translation of EB studies to drug-screening device assembly and clinical treatments.
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SARS-CoV-2 promotes microglial synapse elimination in human brain organoids.
Neuropsychiatric manifestations are common in both the acute and post-acute phase of SARS-CoV-2 infection, but the mechanisms of these effects are unknown. In a newly established brain organoid model with innately developing microglia, we demonstrate that SARS-CoV-2 infection initiate neuronal cell death and cause a loss of post-synaptic termini. Despite limited neurotropism and a decelerating viral replication, we observe a threefold increase in microglial engulfment of postsynaptic termini after SARS-CoV-2 exposure. We define the microglial responses to SARS-CoV-2 infection by single cell transcriptomic profiling and observe an upregulation of interferon-responsive genes as well as genes promoting migration and synapse engulfment. To a large extent, SARS-CoV-2 exposed microglia adopt a transcriptomic profile overlapping with neurodegenerative disorders that display an early synapse loss as well as an increased incident risk after a SARS-CoV-2 infection. Our results reveal that brain organoids infected with SARS-CoV-2 display disruption in circuit integrity via microglia-mediated synapse elimination and identifies a potential novel mechanism contributing to cognitive impairments in patients recovering from COVID-19.
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Brain organoids and insights on human evolution.
Human brain organoids, generated from pluripotent stem cells, have emerged as a promising technique for modeling early stages of human neurodevelopment in controlled laboratory conditions. Although the applications for disease modeling in a dish have become routine, the use of these brain organoids as evolutionary tools is only now getting momentum. Here, we will review the current state of the art on the use of brain organoids from different species and the molecular and cellular insights generated from these studies. Besides, we will discuss how this model might be beneficial for human health and the limitations and future perspectives of this technology.
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Benchmarking brain organoid recapitulation of fetal corticogenesis.
Brain organoids are becoming increasingly relevant to dissect the molecular mechanisms underlying psychiatric and neurological conditions. The in vitro recapitulation of key features of human brain development affords the unique opportunity of investigating the developmental antecedents of neuropsychiatric conditions in the context of the actual patients' genetic backgrounds. Specifically, multiple strategies of brain organoid (BO) differentiation have enabled the investigation of human cerebral corticogenesis in vitro with increasing accuracy. However, the field lacks a systematic investigation of how closely the gene co-expression patterns seen in cultured BO from different protocols match those observed in fetal cortex, a paramount information for ensuring the sensitivity and accuracy of modeling disease trajectories. Here we benchmark BO against fetal corticogenesis by integrating transcriptomes from in-house differentiated cortical BO (CBO), other BO systems, human fetal brain samples processed in-house, and prenatal cortices from the BrainSpan Atlas. We identified co-expression patterns and prioritized hubs of human corticogenesis and CBO differentiation, highlighting both well-preserved and discordant trends across BO protocols. We evaluated the relevance of identified gene modules for neurodevelopmental disorders and psychiatric conditions finding significant enrichment of disease risk genes especially in modules related to neuronal maturation and synapsis development. The longitudinal transcriptomic analysis of CBO revealed a two-step differentiation composed of a fast-evolving phase, corresponding to the appearance of the main cell populations of the cortex, followed by a slow-evolving one characterized by milder transcriptional changes. Finally, we observed heterochronicity of differentiation across BO models compared to fetal cortex. Our approach provides a framework to directly compare the extent of in vivo/in vitro alignment of neurodevelopmentally relevant processes and their attending temporalities, structured as a resource to query for modeling human corticogenesis and the neuropsychiatric outcomes of its alterations.