Human Brain Organoids Research Articles

Application of Flow Cytometric Analysis for Measuring Multiple Mitochondrial Parameters in 3D Brain Organoids.

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, mitochondrial respiratory chain (MRC) complexes, and mitochondrial DNA (mtDNA) copy number often feature in these processes. Human brain organoids derived from human induced pluripotent stem cells (iPSCs) recapitulate the brain's three-dimensional (3D) cytoarchitectural arrangement and offer the possibility to study disease mechanisms and screen new therapeutics in a complex human system. Here, we report a unique flow cytometry-based approach to measure multiple mitochondrial parameters in iPSC-derived cortical organoids. This report details a protocol for generating cortical brain organoids from iPSCs, single-cell dissociation of generated organoids, fixation, staining, and subsequent flow cytometric analysis to assess multiple mitochondrial parameters. Double staining with antibodies against the MRC complex subunit NADH: Ubiquinone Oxidoreductase Subunit B10 (NDUFB10) or mitochondrial transcription factor A (TFAM) together with voltage-dependent anion-selective channel 1 (VDAC 1) permits assessment of the amount of these proteins per mitochondrion. Since the quantity of TFAM corresponds to the amount of mtDNA, it provides an indirect estimation of the number of mtDNA copies per mitochondrial content. This entire procedure can be completed within a span of 2-3 h. Crucially, it allows for the concurrent quantification of multiple mitochondrial parameters, including both total and specific levels relative to the mitochondrial mass.

Stress induces behavioral abnormalities by increasing expression of phagocytic receptor MERTK in astrocytes to promote synapse phagocytosis

Childhood neglect and/or abuse can induce mental health conditions with unknown mechanisms. Here, we identified stress hormones as strong inducers of astrocyte-mediated synapse phagocytosis. Using invitro, invivo, and human brain organoid experiments, we showed that stress hormones increased the expression of the Mertk phagocytic receptor in astrocytes through glucocorticoid receptor (GR). In post-natal mice, exposure to early social deprivation (ESD) specifically activated the GR-MERTK pathway in astrocytes, but not in microglia. The excitatory post-synaptic density in cortical regions was reduced in ESD mice, and there was an increase in the astrocytic engulfment of these synapses. The loss of excitatory synapses, abnormal neuronal network activities, and behavioral abnormalities in ESD mice were largely prevented by ablating GR or MERTK in astrocytes. Our work reveals the critical roles of astrocytic GR-MERTK activation in evoking stress-induced abnormal behaviors in mice, suggesting GR-MERTK signaling as a therapeutic target for stress-induced mental health conditions.

Genetics of human brain development.

Brain development in humans is achieved through precise spatiotemporal genetic control, the mechanisms of which remain largely elusive. Recently, integration of technological advances in human stem cell-based modelling with genome editing has emerged as a powerful platform to establish causative links between genotypes and phenotypes directly in the human system. Here, we review our current knowledge of complex genetic regulation of each key step of human brain development through the lens of evolutionary specialization and neurodevelopmental disorders and highlight the use of human stem cell-derived 2D cultures and 3D brain organoids to investigate human-enriched features and disease mechanisms. We also discuss opportunities and challenges of integrating new technologies to reveal the genetic architecture of human brain development and disorders.

Modelling host- Trypanosoma brucei gambiense interactions in vitro using human induced pluripotent stem cell-derived cortical brain organoids.

Background: Sleeping sickness is caused by the extracellular parasite Trypanosoma brucei and is associated with neuroinflammation and neuropsychiatric disorders, including disruption of sleep/wake patterns, and is now recognised as a circadian disorder. Sleeping sickness is traditionally studied using murine models of infection due to the lack of alternative in vitro systems that fully recapitulate the cellular diversity and functionality of the human brain. The aim of this study is to develop a much-needed in vitro system that reduces and replaces live animals for the study of infections in the central nervous system, using sleeping sickness as a model infection. Methods: We developed a co-culture system using induced pluripotent stem cell (iPSC)-derived cortical human brain organoids and the human pathogen T. b. gambiense to model host-pathogen interactions in vitro. Upon co-culture, we analysed the transcriptional responses of the brain organoids to T. b. gambiense over two time points. Results: We detected broad transcriptional changes in brain organoids exposed to T. b. gambiense, mainly associated with innate immune responses, chemotaxis, and blood vessel differentiation compared to untreated organoids. Conclusions: Our co-culture system provides novel, more ethical avenues to study host-pathogen interactions in the brain as alternative models to experimental infections in mice. Although our data support the use of brain organoids to model host-pathogen interactions during T. brucei infection as an alternative to in vivo models, future work is required to increase the complexity of the organoids ( e.g., addition of microglia and vasculature). We envision that the adoption of organoid systems is beneficial to researchers studying mechanisms of brain infection by protozoan parasites. Furthermore, organoid systems have the potential to be used to study other parasites that affect the brain significantly reducing the number of animals undergoing moderate and/or severe protocols associated with the study of neuroinflammation and brain infections.

Stem cell-derived brain organoids for controlled studies of transcranial neuromodulation

Transcranial neuromodulation methods have the potential to diagnose and treat brain disorders at their neural source in a personalized manner. However, it has been difficult to investigate the direct effects of transcranial neuromodulation on neurons in human brain tissue. Here, we show that human brain organoids provide a detailed and artifact-free window into neuromodulation-evoked electrophysiological effects. We derived human cortical organoids from induced pluripotent stem cells and implanted 32-channel electrode arrays. Each organoid was positioned in the center of the human skull and subjected to low-intensity transcranial focused ultrasound. We found that ultrasonic stimuli modulated network activity in the gamma and delta ranges of the frequency spectrum. The effects on the neural networks were a function of the ultrasound stimulation frequency. High gamma activity remained elevated for at least 20 minutes following stimulation offset. This approach is expected to provide controlled studies of the effects of ultrasound and other transcranial neuromodulation modalities on human brain tissue.

Are human brain organoids cloned human individuals? An ethical analysis

In recent years, there has been much ethical debate about human brain organoid research. However, the possibility that human brain organoids might be human individuals has not been examined. This is an important issue because if human brain organoids are human individuals, then the creation of human brain organoids could be a form of human reproductive cloning. We examine the implications of two mainstream bioethical views on the nature of human individuals for human brain organoids: (1) Functional Integration Theory, the view that the capacity to integrate vital functions is the criterion for human individuality, and (2) Mental State Theory, the view that some mental capacity is the criterion for human individuality. We find that under either theory of human individuality, human brain organoids could be human individuals in the future. According to a version of Functional Integration Theory that locates the integrative function in the human brainstem (Brainstem Theory), a well-developed human brainstem organoid can be a human individual. Further, on Mental State Theory, a conscious human brain organoid can be a human individual. Our results, combined with the ban on human reproductive cloning, may provide some reasons to be more cautious in human brain organoid research. Alternatively, in light of the progress in human brain organoid research, it may be necessary to reconsider the ethics and laws surrounding human reproductive cloning. This research paves the way for the first time to examine the question of great ethical importance whether human brain organoids can be human individuals, and encourages further consideration.

Scientific and ethical challenges of brain chimeras converge on an ‘enriched’ approach

Transplantation of human brain organoids into nonhuman animals has the potential to advance our molecular understanding of human cognition. Yet hybridized nervous systems raise novel scientific and ethical questions that appear to be oriented toward opposing ends – as models become more sophisticated, ethical concerns become more salient. I posit that scientific and ethical challenges arising from interspecies human brain engraftment may be akin to ‘birds of a feather’ that are more entangled, rather than in tension. The scientific value of engrafted organoids depends on the extent to which they provide insights into human neurobiology, while ethical concerns tend to focus on the wellbeing of engrafted animals. Based on a synthesis of empirical and theoretical evidence from neuroscience and philosophical bioethics, respectively, I argue that scientific and ethical challenges converge on a shared interest in achieving the fullest possible maturation of the host animal. The conditions which are favorable to the wellbeing of animals are also consistent with factors that contribute to the elaboration of neuronal features during development and later behavioral traits. Treating engrafted animals as if they already possess higher moral status may, in fact, be necessary for them to develop the neuronal, cognitive, and behavioral traits that are most relevant to our understanding of the human brain. Studies have shown that enriched environments provide the conditions for neurons to mature, more fully, into neural circuits that contribute to complex behaviors, while supporting the wellbeing and individuation of animals. I conceptualize an ‘enriched’ approach toward research using interspecies brain engraftment and offer a case study examining how enriched environments can be used to study the neurobiology of human speech circuits in grafted animals. The uncertainties posed by interspecies brain engraftment illustrates how joint consideration of scientific and ethical perspectives can reveal convergent pathways for advancing human brain research.

Advances in the knowledge and therapeutics of schizophrenia, major depression disorder, and bipolar disorder from human brain organoid research.

Tridimensional cultures of human induced pluripotent cells (iPSCs) experimentally directed to neural differentiation, termed "brain organoids" are now employed as an in vitro assay that recapitulates early developmental stages of nervous tissue differentiation. Technical progress in culture methodology enabled the generation of regionally specialized organoids with structural and neurochemical characters of distinct encephalic regions. The technical process of organoid elaboration is undergoing progressively implementation, but current robustness of the assay has attracted the attention of psychiatric research to substitute/complement animal experimentation for analyzing the pathophysiology of psychiatric disorders. Numerous morphological, structural, molecular and functional insights of psychiatric disorders have been uncovered by comparing brain organoids made with iPSCs obtained from control healthy subjects and psychiatric patients. Brain organoids were also employed for analyzing the response to conventional treatments, to search for new drugs, and to anticipate the therapeutic response of individual patients in a personalized manner. In this review, we gather data obtained by studying cerebral organoids made from iPSCs of patients of the three most frequent serious psychiatric disorders: schizophrenia, major depression disorder, and bipolar disorder. Among the data obtained in these studies, we emphasize: (i) that the origin of these pathologies takes place in the stages of embryonic development; (ii) the existence of shared molecular pathogenic aspects among patients of the three distinct disorders; (iii) the occurrence of molecular differences between patients bearing the same disorder, and (iv) that functional alterations can be activated or aggravated by environmental signals in patients bearing genetic risk for these disorders.

Donor perspectives on informed consent and use of biospecimens for brain organoid research.

Debates about the ethics of human brain organoids have proceeded without the input of individuals whose brains are being modeled. Interviews with donors of biospecimens for brain organoid research revealed overall enthusiasm for brain organoids as a tool for biomedical discovery, alongside a desire for ongoing engagement with research teams to learn the results of the research, to allow transfer of decision-making authority over time, and to ensure ethical boundaries are not crossed. Future work is needed to determine the most feasible and resource-efficient way to longitudinally engage donors participating in brain organoid research.

Study of Human Microglia Phenotypes via an In Vivo Neuroimmune Organoid Model

Microglia are specialized brain-resident macrophages that play crucial roles in brain development, homeostasis, and disease. However, until now, the ability to model interactions between the human brain environment and microglia has been severely limited. To overcome these limitations, Schafer and colleagues developed an in vivo xenotransplantation approach that allows us to study functionally mature human microglia (hMGs) that operate within a physiologically relevant, vascularized immunocompetent human brain organoid (iHBO) model. Developing an in vivo neuroimmune organoid model to study human microglia phenotypes would involve creating a three-dimensional structure that mimics the complexity of the human brain and immune system. Such a model could provide a valuable tool to investigate the behavior and function of microglia, the resident immune cells of the central nervous system, in a more physiologically relevant context. Data from Schafer and colleagues show that organoid-resident hMGs gain human-specific transcriptomic signatures that closely resemble their in vivo counterparts. In vivo two-photon imaging reveals that hMGs actively engage in surveilling the human brain environment, react to local injuries, and respond to systemic inflammatory cues. Finally, they demonstrate that the transplanted iHBOs developed here offer the unprecedented opportunity to study functional human microglia phenotypes in health and disease and provide experimental evidence for a brain-environment-induced immune response in a patient-specific model of autism with macrocephaly. Cell. 2023 May 11;186(10):2111-2126.e20. doi: 10.1016/j.cell.2023.04.022.

Increased G3BP2-Tau interaction in tauopathies is a natural defense against Tau aggregation

Many RNA-binding proteins (RBPs), particularly those associated with RNA granules, promote pathological protein aggregation in neurodegenerative diseases. Here, we demonstrate that G3BP2, a core component of stress granules, directly interacts with Tau and inhibits Tau aggregation. In the human brain, the interaction of G3BP2 and Tau is dramatically increased in multiple tauopathies, and it is independent of neurofibrillary tangle (NFT) formation in Alzheimer's disease (AD). Surprisingly, Tau pathology is significantly elevated upon loss of G3BP2 in human neurons and brain organoids. Moreover, we found that G3BP2 masks the microtubule-binding region (MTBR) of Tau, thereby inhibiting Tau aggregation. Our study defines a novel role for RBPs as a line of defense against Tau aggregation in tauopathies.

Schizophrenia-associated NRXN1 deletions induce developmental-timing- and cell-type-specific vulnerabilities in human brain organoids

De novo mutations and copy number deletions in NRXN1 (2p16.3) pose a significant risk for schizophrenia (SCZ). It is unclear how NRXN1 deletions impact cortical development in a cell type-specific manner and disease background modulates these phenotypes. Here, we leveraged human pluripotent stem cell-derived forebrain organoid models carrying NRXN1 heterozygous deletions in isogenic and SCZ patient genetic backgrounds and conducted single-cell transcriptomic analysis over the course of brain organoid development from 3 weeks to 3.5 months. Intriguingly, while both deletions similarly impacted molecular pathways associated with ubiquitin-proteasome system, alternative splicing, and synaptic signaling in maturing glutamatergic and GABAergic neurons, SCZ-NRXN1 deletions specifically perturbed developmental trajectories of early neural progenitors and accumulated disease-specific transcriptomic signatures. Using calcium imaging, we found that both deletions led to long-lasting changes in spontaneous and synchronous neuronal networks, implicating synaptic dysfunction. Our study reveals developmental-timing- and cell-type-dependent actions of NRXN1 deletions in unique genetic contexts.

Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids

Herpes simplex encephalitis is a life-threatening disease of the central nervous system caused by herpes simplex viruses (HSVs). Following standard of care with antiviral acyclovir treatment, most patients still experience various neurological sequelae. Here we characterize HSV-1 infection of human brain organoids by combining single-cell RNA sequencing, electrophysiology and immunostaining. We observed strong perturbations of tissue integrity, neuronal function and cellular transcriptomes. Under acyclovir treatment viral replication was stopped, but did not prevent HSV-1-driven defects such as damage of neuronal processes and neuroepithelium. Unbiased analysis of pathways deregulated upon infection revealed tumour necrosis factor activation as a potential causal factor. Combination of anti-inflammatory drugs such as necrostatin-1 or bardoxolone methyl with antiviral treatment prevented the damages caused by infection, indicating that tuning the inflammatory response in acute infection may improve current therapeutic strategies.

SARS-CoV-2 infection and viral fusogens cause neuronal and glial fusion that compromises neuronal activity.

Numerous viruses use specialized surface molecules called fusogens to enter host cells. Many of these viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can infect the brain and are associated with severe neurological symptoms through poorly understood mechanisms. We show that SARS-CoV-2 infection induces fusion between neurons and between neurons and glia in mouse and human brain organoids. We reveal that this is caused by the viral fusogen, as it is fully mimicked by the expression of the SARS-CoV-2 spike (S) protein or the unrelated fusogen p15 from the baboon orthoreovirus. We demonstrate that neuronal fusion is a progressive event, leads to the formation of multicellular syncytia, and causes the spread of large molecules and organelles. Last, using Ca2+ imaging, we show that fusion severely compromises neuronal activity. These results provide mechanistic insights into how SARS-CoV-2 and other viruses affect the nervous system, alter its function, and cause neuropathology.

Human Brain Organoids: Why There Can Be Moral Concerns If They Grow Up in the Lab and Are Transplanted or Destroyed.

Human brain organoids (HBOs) are three-dimensional biological entities grown in the laboratory in order to recapitulate the structure and functions of the adult human brain. They can be taken to be novel living entities for their specific features and uses. As a contribution to the ongoing discussion on the use of HBOs, the authors identify three sets of reasons for moral concern. The first set of reasons regards the potential emergence of sentience/consciousness in HBOs that would endow them with a moral status whose perimeter should be established. The second set of moral concerns has to do with an analogy with artificial womb technology. The technical realization of processes that are typically connected to the physiology of the human body can create a manipulatory and instrumental attitude that can undermine the protection of what is human. The third set concerns the new frontiers of biocomputing and the creation of chimeras. As far as the new frontier of organoid intelligence is concerned, it is the close relationship of humans with new interfaces having biological components capable of mimicking memory and cognition that raises ethical issues. As far as chimeras are concerned, it is the humanization of nonhuman animals that is worthy of close moral scrutiny. A detailed description of these ethical issues is provided to contribute to the construction of a regulative framework that can guide decisions when considering research in the field of HBOs.

Human brain organoid transplantation: ethical implications of enhancing specific cerebral functions in small-animal models

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.

Functional reconstruction of the impaired cortex and motor function by hMGEOs transplantation in stroke

Stroke is the leading cause of death and long-term disability worldwide. But treatments are not available to promote functional recovery, and efficient therapies need to be investigated. Stem cell-based therapies hold great promise as potential technologies to restore function in brain disorders. Loss of GABAergic interneurons after stroke may result in sensorimotor defects. Here, by transplanting human brain organoids resembling the MGE domain (human MGE organoids, hMGEOs) derived from human induced pluripotent stem cells (hiPSCs) into the infarcted cortex of stroke mice, we found that grafted hMGEOs survived well and primarily differentiated into GABAergic interneurons and significantly restored the sensorimotor deficits of stroke mice for a long time. Our study offers the feasibility of stem cell replacement therapeutics strategy for stroke.

Herpes Simplex Virus‐1 Proteins as Drivers for Alzheimer’s Disease Pathologies

AbstractBackgroundDNA and RNA of Herpes Simplex Virus 1 (HSV‐1) were found in the brains and serological samples of Alzheimer’s disease (AD) patients. Such molecular presence of HSV‐1 in AD patients is especially intriguing as HSV‐1 virions are rarely detected in AD brains.MethodTo follow the molecular footsteps detected, we imaged viral proteins in postmortem human AD brains at superior resolution using expansion microscopy, a tissue manipulation method that physically expands the samples by a factor of 4.5x, allowing a 40 nm imaging resolution, and immunolabeled herpetic proteins, AD pathologies and cell markers.ResultWe found an abundance of herpetic proteins, previously undetectable with standard methods, across large brain areas. Importantly, we found that HSV‐1 proteins strongly co‐localized with AD pathologies. Consequently, we hypothesized that expression of HSV‐1 proteins during latency may be linked to AD pathology. We are now in the process of characterizing the HSV‐1 proteome in AD brains by imaging key proteins in expanded AD brain slices and examining their colocalization with AD pathologies across brain areas and disease stages. As a complementary system to the fixed human brain slices, we are exposing live human brain organoids, to HSV‐1, and imaging the relationships between viral proteins and the formation of AD pathologies via expansion microscopy.ConclusionPathogens may be triggers of immune responses driving AD; this study would shed light on one common pathogen, HSV‐1, while serving as a framework to unveiling molecular causation between infectious agents and AD hallmarks.

Effects of acute low-moderate dose ionizing radiation to human brain organoids.

Human exposure to low-to-moderate dose ionizing radiation (LMD-IR) is increasing via environmental, medical, occupational sources. Acute exposure to LMD-IR can cause subclinical damage to cells, resulting in altered gene expression and cellular function within the human brain. It has been difficult to identify diagnostic and predictive biomarkers of exposure using traditional research models due to factors including lack of 3D structure in monolayer cell cultures, limited ability of animal models to accurately predict human responses, and technical limitations of studying functional human brain tissue. To address this gap, we generated brain/cerebral organoids from human induced pluripotent stem cells to study the radiosensitivity of human brain cells, including neurons, astrocytes, and oligodendrocytes. While organoids have become popular models for studying brain physiology and pathology, there is little evidence to confirm that exposing brain organoids to LMD-IR will recapitulate previous in vitro and in vivo observations. We hypothesized that exposing brain organoids to proton radiation would (1) cause a time- and dose-dependent increase in DNA damage, (2) induce cell type-specific differences in radiosensitivity, and (3) increase expression of oxidative stress and DNA damage response genes. Organoids were exposed to 0.5 or 2 Gy of 250 MeV protons and samples were collected at 30 minute, 24 hour, and 48 hour timepoints. Using immunofluorescence and RNA sequencing, we found time- and dose-dependent increases in DNA damage in irradiated organoids; no changes in cell populations for neurons, oligodendrocytes, and astrocytes by 24 hours; decreased expression of genes related to oligodendrocyte lineage, astrocyte lineage, mitochondrial function, and cell cycle progression by 48 hours; increased expression of genes related to neuron lineage, oxidative stress, and DNA damage checkpoint regulation by 48 hours. Our findings demonstrate the possibility of using organoids to characterize cell-specific radiosensitivity and early radiation-induced gene expression changes within the human brain, providing new avenues for further study of the mechanisms underlying acute neural cell responses to IR exposure at low-to-moderate doses.

Global governance of human brain organoid research and applications: A role for the World Health Organization?

Human brain organoid research and applications have generated considerable ethical discussions, yet as with other technologies, anticipating potential transnational issues associated with research and cross-border commercialization of organoids would allow a proactive approach as the field advances rapidly. This policy piece explores the potential role of the World Health Organization in the global governance of human brain organoid research applications by examining its legal authority and recent scientific activities in grounding foresight to harness the power of science to better global health.