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Proteomics profile of astrocytes derived from hESC-cerebral organoids and human adult surgical specimens. (A) Venn diagram comparing the number of proteins identified by shotgun mass spectrometry of astrocytes derived from hESC cell lines, BR1 and H9-cerebral organoids, and human adult astrocytes. (B) Spearman correlation between global relative protein expression of hESC-cerebral organoids-astrocytes and human adult astrocytes. p represents the spearman correlation coefficient. (C) Plot of selected proteins related to astrocytes; the comparison was normalized to the abundance of proteins from hESC-derived and human adult astrocytes.
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Astrocytes play a critical role in the development and homeostasis of the central nervous system (CNS). Astrocyte dysfunction results in several neurological and degenerative diseases. However, a major challenge to our understanding of astrocyte physiology and pathology is the restriction of studies to animal models, human post-mortem brain tissues...
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Background
Cardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule–mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line‐to‐line variation....
Citations
... In addition to this approach, iPSC-induced astrocytes can also be obtained with alternative 9780367468873_C013.indd 312 17/04/24 11:11 AM methods for example by isolating them from cerebral organoids derived from human pluripotent stem cells [129,130] or by intrinsic activation of transcription factors critical for astrocyte differentiation [131]. ...
... microglia oligodendrocytes. Mono-cultures of glial cells showed altered gene expression, morphology, and physical features(Dezonne et al. 2017;Bohlen et al. 2017). ...
Adult neurotoxicity (ANT) and developmental neurotoxicity (DNT) assessments aim to understand the adverse effects and underlying mechanisms of toxicants on the human nervous system. In recent years, there has been an increasing focus on the so-called new approach methodologies (NAMs). The Organization for Economic Co-operation and Development (OECD), together with European and American regulatory agencies, promote the use of validated alternative test systems, but to date, guidelines for regulatory DNT and ANT assessment rely primarily on classical animal testing. Alternative methods include both non-animal approaches and test systems on non-vertebrates (e.g., nematodes) or non-mammals (e.g., fish). Therefore, this review summarizes the recent advances of NAMs focusing on ANT and DNT and highlights the potential and current critical issues for the full implementation of these methods in the future. The status of the DNT in vitro battery (DNT IVB) is also reviewed as a first step of NAMs for the assessment of neurotoxicity in the regulatory context. Critical issues such as (i) the need for test batteries and method integration (from in silico and in vitro to in vivo alternatives, e.g., zebrafish, C. elegans) requiring interdisciplinarity to manage complexity, (ii) interlaboratory transferability, and (iii) the urgent need for method validation are discussed.
... buffer and distilled water and incubated in 1% uranyl acetate overnight. After gradual dehydration with acetone series (30,50,70,90, and 100%), bMPS were embedded in Poly/Bed 812 resin (Ted Pella Inc, USA) and polymerized at 60°C for 48 h. The ultrathin cross-sections (70 nm) were obtained with an RMC ultramicrotome. ...
... While no particular marker has been associated with selective molecular signatures that could distinguish astrocytic phenotypes, the intermediate filament protein, glial fibrillary acidic protein (GFAP) is a canonical marker used to identify astrocytes throughout the CNS (48). Aligned with our results, other groups have found that iPSC-derived astrocytes resemble human morphology (49,50). The structural and functional plasticity of neuronal networks is partly governed by astrocytes, and as astrocytes age, they lose their ability to modulate and promote neurite outgrowth (35)(36)(37). ...
Brain microphysiological systems (bMPS), which recapitulate human brain cellular architecture and functionality more closely than traditional monolayer cultures, have become a practical, non-invasive, and increasingly relevant platform for the study of neurological function in health and disease. These models include 3D spheroids and organoids as well as organ-on-chip models. Currently, however, existing 3D brain models vary in reflecting the relative populations of the different cell types present in the human brain. Most of the models consist mainly of neurons, while glial cells represent a smaller portion of the cell populations. Here, by means of a chemically defined glial-enriched medium (GEM), we present an improved method to expand the population of astrocytes and oligodendrocytes without compromising neuronal differentiation in bMPS. An important finding is that astrocytes not only increased in number but also changed in morphology when cultured in GEM, more closely recapitulating primary culture astrocytes. We demonstrate oligodendrocyte and astrocyte enrichment in GEM bMPS using a variety of complementary methods. We found that GEM bMPS are electro-chemically active and showed different patterns of Ca +2 staining and flux. Synaptic vesicles and terminals observed by electron microscopy were also present. No significant changes in neuronal differentiation were observed by gene expression, however, GEM enhanced neurite outgrowth and cell migration, and differentially modulated neuronal maturation in two different iPSC lines. Our results have the potential to significantly improve in vivo-like functionality of bMPS for the study of neurological diseases and drug discovery, contributing to the unmet need for safe human models.
... Using, for example, Frontiers in Cell and Developmental Biology frontiersin.org human cerebral organoids (Dezonne et al., 2017) would be a valuable strategy to test how human astrocytes are affected by DREADDs and help in the transition from the bench to the clinic. It is interesting to note that very few studies so far have demonstrated inhibition of astrocytic Ca 2+ waves following DREADD activation. ...
Astrocytes are the major glial cell type in the central nervous system (CNS). Initially regarded as supportive cells, it is now recognized that this highly heterogeneous cell population is an indispensable modulator of brain development and function. Astrocytes secrete neuroactive molecules that regulate synapse formation and maturation. They also express hundreds of G protein-coupled receptors (GPCRs) that, once activated by neurotransmitters, trigger intracellular signalling pathways that can trigger the release of gliotransmitters which, in turn, modulate synaptic transmission and neuroplasticity. Considering this, it is not surprising that astrocytic dysfunction, leading to synaptic impairment, is consistently described as a factor in brain diseases, whether they emerge early or late in life due to genetic or environmental factors. Here, we provide an overview of the literature showing that activation of genetically engineered GPCRs, known as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), to specifically modulate astrocyte activity partially mimics endogenous signalling pathways in astrocytes and improves neuronal function and behavior in normal animals and disease models. Therefore, we propose that expressing these genetically engineered GPCRs in astrocytes could be a promising strategy to explore (new) signalling pathways which can be used to manage brain disorders. The precise molecular, functional and behavioral effects of this type of manipulation, however, differ depending on the DREADD receptor used, targeted brain region and timing of the intervention, between healthy and disease conditions. This is likely a reflection of regional and disease/disease progression-associated astrocyte heterogeneity. Therefore, a thorough investigation of the effects of such astrocyte manipulation(s) must be conducted considering the specific cellular and molecular environment characteristic of each disease and disease stage before this has therapeutic applicability.
... Subsequent studies refined these aspects of normal neurodevelopment in region-specific organoids, resulting in rudimentary segregation of superficial and deep cortical layers (Paş ca et al., 2015;Qian et al., 2016), generation of a distinct layer of outer radial glial cells (Qian et al., 2016), and expansion of cortical folds . Glial populations were observed at later time points, including astrocytes (Dezonne et al., 2017;Paş ca et al., 2015;Qian et al., 2016;Sloan et al., 2017) and oligodendrocytes (Matsui et al., 2018). Integration of interneurons into these cortical organoids has been studied through fusion of dorsalized glutamatergic organoids with ventralized organoids containing GABAergic neurons (Bagley et al., 2017;Birey et al., 2017;Xiang et al., 2017). ...
Brain organoids are self-organizing, three-dimensional tissues derived from pluripotent stem cells that recapitulate many aspects of the cellular diversity and architectural features of the developing brain. Recently, there has been growing interest in using human brain organoid transplantation in animal models as a means of addressing the limitations of in vitro culture, such as the lack of vascularization, and to explore the potential of organoids for neural repair. While there has been substantial debate on the ethical implications of brain organoid research, particularly the potential for organoids to exhibit higher-order brain functions such as consciousness, the impact of human organoid grafts on animal hosts has been less extensively discussed. Enhancement of host animal brain function may not be technically feasible at this time, but it is imperative to carefully consider the moral significance of these potential outcomes. Here, we discuss the ethical implications of enhancing somatosensation, motor processes, memory, and basic socialization in small-animal models. We consider the moral implications of such outcomes and if safeguards are needed to accommodate any increased moral status of animals transplanted with human brain organoids.
... During the developmental stages of brain organoids, it is common to observe a progressive organization with immature neurons predominantly concentrated in the outer region and fewer in the inner region, accompanied by a gradual thickening of the TUBB3+ cell layer [109], particularly in the cortical plate [110]. Our data showed positive cells for TUBB3, which aligns with the previously reported findings of a gradual thickening of the TUBB3+ cell layer in the outer layer of the organoids. ...
Neural injuries in cerebral malaria patients are a significant cause of morbidity and mortality. Nevertheless, a comprehensive research approach to study this issue is lacking, so herein we propose an in vitro system to study human cerebral malaria using cellular approaches. Our first goal was to establish a cellular system to identify the molecular alterations in human brain vasculature cells that resemble the blood–brain barrier (BBB) in cerebral malaria (CM). Through transcriptomic analysis, we characterized specific gene expression profiles in human brain microvascular endothelial cells (HBMEC) activated by the Plasmodium falciparum parasites. We also suggest potential new genes related to parasitic activation. Then, we studied its impact at brain level after Plasmodium falciparum endothelial activation to gain a deeper understanding of the physiological mechanisms underlying CM. For that, the impact of HBMEC-P. falciparum-activated secretomes was evaluated in human brain organoids. Our results support the reliability of in vitro cellular models developed to mimic CM in several aspects. These systems can be of extreme importance to investigate the factors (parasitological and host) influencing CM, contributing to a molecular understanding of pathogenesis, brain injury, and dysfunction.
... As we have claimed above, and in several publications, astrocytes embody the hydro-ionic waves of sentience, the capacity of feeling, which is necessary for the emergence of consciousness. These waves also play a key role in the development of brain organoids (where they can develop [28]) and possible applications in Regenerative Neuromedicine. ...
The new science called Sentiomics aims to identify the dynamic patterns that endow living systems with the capacity to feel and become conscious. One of the most promising fields of investigation in Sentiomics is the development and ‘education’ of human brain organoids to become sentient and useful for the promotion of human health in the (also new) field of Regenerative Neuromedicine. Here, we discuss the type of informational-rich input necessary to make a brain organoid sentient in experimental settings. Combining this research with the ecological preoccupation of preserving ways of sentience in the Amazon Rainforest, we also envisage the development of a new generation of biosensors to capture dynamic patterns from the forest, and use them in the ‘education’ of brain organoids to afford them a ‘mental health’ quality that is likely to be important in future advances in ‘post-humanist’ procedures in regenerative medicine. This study is closely related to the psychophysical approach to human mental health therapy, in which we have proposed the use of dynamic patterns in electric and magnetic brain stimulation protocols, addressing electrochemical waves in neuro-astroglial networks.
... iPSC-derived astrocytes express 212 proteins not detected in primary astrocyte cultures, whereas primary astrocytes express 93 unique proteins. Protein expression levels between these astrocyte cultures are correlated (Spearman correlation coefficient = 0.71) Dezonne et al. (2017) Primary rat P1 astrocytes isolated by immunopanning and cultured for 7 days compared to freshly isolated astrocytes Expression of Areg, Atf3,Axud1,Cbs,Dio2,Dusp1,Grm3,Klf2,Klf4,Maff,Nfkbiz,Nptx3,Npy1r,Nr4a1,Nr4a2,Nr4a3,Pla2g3,Sloco1c1, and Zfp36 is down-regulated at least 14-fold Expression of Gsta3, Scn7a, and Pdgfra is up-regulated at least 13-fold Foo et al. (2011) Primary rat P7 astrocytes isolated by immunopanning and cultured for 7 days compared to freshly isolated astrocytes Expression of Axud1, Areg,Cbs,Dio2,Gabbr2,Grm3,Hapln1,Hspa1a,Hspa1b,Ier2,Junb,Kcnd2,Maff,Nptx2,Nr4a1,Nr4a2,Nr4a3,Pla2g3,Slc13a5,Sloco1c1, and Tgfpi2 is down-regulated at least 13-fold Expression of Spta1 is up-regulated 66-fold Primary rat microglia cultured in the presence and absence of primary rat neurons Fibrillar amyloidβ reduces the viability of neurons in monoculture The toxicity of fibrillar amyloidβ toward neurons is halved when cocultured with microglia A combination of fibrillar amyloidβ plus IFNγ reduces the viability of neurons when co-cultured with microglia. This combination of molecules does not have an affect on neuron viability unless microglia and neurons directly contacted one another Li et al. (2004) Eight-week-old C57BL/6J mice receiving hippocampal or cortical injections of L-AAA to ablate astrocytes compared to mice injected with saline ...
... For example, data from Dezonne et al. (2017) show that the same iPSC differentiation protocol can result in cells that express different proteins depending on genetic background of the iPSC line. ...
Brain organoids have the potential to improve clinical translation, with the added benefit of reducing any extraneous use of experimental animals. As brain organoids are three‐dimensional in vitro constructs that emulate the human brain, they bridge in vitro and in vivo studies more appropriately than monocultures. Although many factors contribute to the failure of extrapolating monoculture‐based information to animal‐based experiments and clinical trials, for the purpose of this review, we will focus on glia (non‐neuronal brain cells), whose functions and transcriptome are particularly abnormal in monocultures. As discussed herein, glia require signals from—and contact with—other cell types to exist in their homeostatic state, which likely contributes to some of the differences between data derived from monocultures and data derived from brain organoids and even two‐dimensional co‐cultures. Furthermore, we highlight transcriptomic differences between humans and mice in regard to aging and Alzheimer's disease, emphasizing need for a model using the human genome—again, a benefit of brain organoids—to complement data derived from animals. We also identify an urgency for guidelines to improve the reporting and transparency of research using organoids. The lack of reporting standards creates challenges for the comparison and discussion of data from different articles. Importantly, brain organoids mark the first human model enabling the study of brain cytoarchitecture and development. image
... Specific procedures, so-called "guided" procedures, have been reported to drive organoid patterning towards distinctive lineages [19]. For instance, organoids with high density in hippocampal [20], cortical [21], dopaminergic [22], glutaminergic [23], gammaaminobutyric acid (GABA)-ergic [23] neurons, as well as retinal ganglionic neurons [24], astrocytes [25], microglia [26,27] or oligodendrocytes [28], have been produced, to mention a few ground-breaking examples. Furthermore, organoids which recapitulated bilateral optic vesicles [29] and vasculature-like structures [30,31] have been reported. ...
Brain organoids are invaluable tools for pathophysiological studies or drug screening, but there are still challenges to overcome in making them more reproducible and relevant. Recent advances in three-dimensional (3D) bioprinting of human neural organoids is an emerging approach that may overcome the limitations of self-organized organoids. It requires the development of optimal hydrogels, and a wealth of research has improved our knowledge about biomaterials both in terms of their intrinsic properties and their relevance on 3D culture of brain cells and tissue. Although biomaterials are rarely biologically neutral, few articles have reviewed their roles on neural cells. We here review the current knowledge on unmodified biomaterials amenable to support 3D bioprinting of neural organoids with a particular interest in their impact on cell homeostasis. Alginate is a particularly suitable bioink base for cell encapsulation. Gelatine is a valuable helper agent for 3D bioprinting due to its viscosity. Collagen, fibrin, hyaluronic acid and laminin provide biological support to adhesion, motility, differentiation or synaptogenesis and optimize the 3D culture of neural cells. Optimization of specialized hydrogels to direct differentiation of stem cells together with an increased resolution in phenotype analysis will further extend the spectrum of possible bioprinted brain disease models.
... Human induced pluripotent stem cells (hiPSCs) offer the unique ability to generate large numbers of patient-specific differentiated cells. However, many standard protocols for differentiating astrocytes from pluripotent stem cells require an extensive culture time (up to 6 months) (Dezonne et al., 2017;Krencik et al., 2011;Palm et al., 2015;Sloan et al., 2017) and/or shorter protocols that rely on the isolation of intermediate cells, such as neural and oligodendrocyte progenitor cells (Barbar et al., 2020;Jiang et al., 2013), or repeated cell passages (Byun et al., 2020;Jovanovic et al., 2021;Leng et al., 2021;Lundin et al., 2018;Peteri et al., 2021;Santos et al., 2017;Soubannier et al., 2020;Tcw et al., 2017). To successfully develop valuable preclinical models for highthroughput screening of astrocytes, it would be preferable to have differentiation protocols that can rapidly and reproducibly generate large numbers of those cells. ...
In the brain, the complement system plays a crucial role in the immune response and in synaptic elimination during normal development and disease. Here, we sought to identify pathways that modulate the production of complement component 4 (C4), recently associated with an increased risk of schizophrenia. To design a disease-relevant assay, we first developed a rapid and robust 3D protocol capable of producing large numbers of astrocytes from pluripotent cells. Transcriptional profiling of these astrocytes confirmed the homogeneity of this population of dorsal fetal-like astrocytes. Using a novel ELISA-based small-molecule screen, we identified epigenetic regulators, as well as inhibitors of intracellular signaling pathways, able to modulate C4 secretion from astrocytes. We then built a connectivity map to predict and validate additional key regulatory pathways, including one involving c-Jun-kinase. This work provides a foundation for developing therapies for CNS diseases involving the complement cascade.
