Cerebral Organoid Model Research Articles

A microglia-containing cerebral organoid model to study early life immune challenges.

Prenatal infections and activation of the maternal immune system have been proposed to contribute to causing neurodevelopmental disorders (NDDs), chronic conditions often linked to brain abnormalities. Microglia are the resident immune cells of the brain and play a key role in neurodevelopment. Disruption of microglial functions can lead to brain abnormalities and increase the risk of developing NDDs. How the maternal as well as the fetal immune system affect human neurodevelopment and contribute to NDDs remains unclear. An important reason for this knowledge gap is the fact that the impact of exposure to prenatal risk factors has been challenging to study in the human context. Here, we characterized a model of cerebral organoids (CO) with integrated microglia (COiMg). These organoids express typical microglial markers and respond to inflammatory stimuli. The presence of microglia influences cerebral organoid development, including cell density and neural differentiation, and regulates the expression of several ciliated and mesenchymal cell markers. Moreover, COiMg and organoids without microglia show similar but also distinct responses to inflammatory stimuli. Additionally, IFN-γ induced significant transcriptional and structural changes in the cerebral organoids, that appear to be regulated by the presence of microglia. Specifically, interferon-gamma (IFN-γ) was found to alter the expression of genes linked to autism. This model provides a valuable tool to study how inflammatory perturbations and microglial presence affect neurodevelopmental processes.

A tumorigenicity evaluation platform for cell therapies based on brain organoids

BackgroundTumorigenicity represents a critical challenge in stem cell-based therapies requiring rigorous monitoring. Conventional approaches for tumorigenicity evaluation are based on animal models and have numerous limitations. Brain organoids, which recapitulate the structural and functional complexity of the human brain, have been widely used in neuroscience research. However, the capacity of brain organoids for tumorigenicity evaluation needs to be further elucidated.MethodsA cerebral organoid model produced from human pluripotent stem cells (hPSCs) was employed. Meanwhile, to enhance the detection sensitivity for potential tumorigenic cells, we created a glioblastoma-like organoid (GBM organoid) model from TP53−/−/PTEN−/− hPSCs to provide a tumor microenvironment for injected cells. Midbrain dopamine (mDA) cells from human embryonic stem cells were utilized as a cell therapy product. mDA cells, hPSCs, mDA cells spiked with hPSCs, and immature mDA cells were then injected into the brain organoids and NOD SCID mice. The injected cells within the brain organoids were characterized, and compared with those injected in vivo to evaluate the capability of the brain organoids for tumorigenicity evaluation. Single-cell RNA sequencing was performed to identify the differential gene expression between the cerebral organoids and the GBM organoids.ResultsBoth cerebral organoids and GBM organoids supported maturation of the injected mDA cells. The hPSCs and immature mDA cells injected in the GBM organoids showed a significantly higher proliferative capacity than those injected in the cerebral organoids and in NOD SCID mice. Furthermore, the spiked hPSCs were detectable in both the cerebral organoids and the GBM organoids. Notably, the GBM organoids demonstrated a superior capacity to enhance proliferation and pluripotency of spiked hPSCs compared to the cerebral organoids and the mouse model. Kyoto Encyclopedia of Genes and Genomes analysis revealed upregulation of tumor-related metabolic pathways and cytokines in the GBM organoids, suggesting that these factors underlie the high detection sensitivity for tumorigenicity evaluation.ConclusionsOur findings suggest that brain organoids could represent a novel and effective platform for evaluating the tumorigenic risk in stem cell-based therapies. Notably, the GBM organoids offer a superior platform that could complement or potentially replace traditional animal-based models for tumorigenicity evaluation.

Dietary Flavonoid Chrysin Functions as a Dual Modulator to Attenuate Amyloid-β and Tau Pathology in the Models of Alzheimer's Disease.

Growing evidence indicates that healthy diets are associated with a slower progression of Alzheimer's disease (AD). Flavonoids are among the most abundant natural products in diets beneficial to AD, such as the Mediterranean diet. However, the effect and mechanism of these dietary flavonoids on AD remains incompletely understood. Here, we found that a representative dietary natural flavonoid, chrysin (Chr), significantly ameliorated cognitive impairment and AD pathology in APP/PS1 mice. Furthermore, mechanistic studies showed that Chr significantly reduced the levels of amyloid-β (Aβ) and phosphorylated tau (p-tau), along with dual inhibitory activity against β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and glycogen synthase kinase 3β (GSK3β). Moreover, the effect of Chr was further confirmed by EW233, a structural analog of Chr that exhibited an improved pharmacokinetic profile. To further verify the role of Chr and EW233, we utilized our previously established chimeric human cerebral organoid (chCO) model for AD, in which astrogenesis was promoted to mimic the neuron-astrocyte ratio in human brain tissue, and similar dual inhibition of Aβ and p-tau was also observed. Altogether, our study not only reveals the molecular mechanisms through which dietary flavonoids, such as Chr, mitigate AD pathology, but also suggests that identifying a specific constituent that mimics some of the benefits of these healthy diets could serve as a promising approach to discover new treatments for AD.

Molecular pathology, developmental changes and synaptic dysfunction in (pre-) symptomatic human C9ORF72-ALS/FTD cerebral organoids

A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human brain imaging and experimental studies indicate early changes in brain structure and connectivity in C9-ALS/FTD, even before symptom onset. Because these early disease phenotypes remain incompletely understood, we generated iPSC-derived cerebral organoid models from C9-ALS/FTD patients, presymptomatic C9ORF72-HRE (C9-HRE) carriers, and controls. Our work revealed the presence of all three C9-HRE-related molecular pathologies and developmental stage-dependent size phenotypes in cerebral organoids from C9-ALS/FTD patients. In addition, single-cell RNA sequencing identified changes in cell type abundance and distribution in C9-ALS/FTD organoids, including a reduction in the number of deep layer cortical neurons and the distribution of neural progenitors. Further, molecular and cellular analyses and patch-clamp electrophysiology detected various changes in synapse structure and function. Intriguingly, organoids from all presymptomatic C9-HRE carriers displayed C9-HRE molecular pathology, whereas the extent to which more downstream cellular defects, as found in C9-ALS/FTD models, were detected varied for the different presymptomatic C9-HRE cases. Together, these results unveil early changes in 3D human brain tissue organization and synaptic connectivity in C9-ALS/FTD that likely constitute initial pathologies crucial for understanding disease onset and the design of therapeutic strategies.

Effects of the oxoaporphine alkaloid hernandonine on dengue virus. Evidence for its mechanisms of action

BackgroundDengue, caused by the dengue virus (Orthoflavivirus dengue, encompassing DENV types 1-4), is a member of the Flaviviridae family. The symptoms of dengue range from subclinical or mild manifestations to potentially fatal complications. The management of severe dengue is exceptionally challenging due to the absence of effective antiviral medications. In this context, natural products, whether in the form of pure compounds or standardized plant extracts, have emerged as a promising source for the development of novel antiviral therapeutics. Hernandonine, an oxoaporphine alkaloid found in Hernandia nymphaeifolia (C. Presl) Kubitzki. serves both as a metabolite and an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase. PurposeThis study investigated the ability of hernandonine to inhibit DENV infection and explored its potential mechanisms. Study designTo assess the in vitro anti-DENV activity, cells or induced pluripotent stem cell (iPSC)-derived cerebral organoids were exposed to hernandonine before or after infection with DENV. Along with hernandonine, the endocytosis modulators, genistein, wortmannin, Methyl-β-cyclodextrin (MβCD) and lovastatin, were used in the assays. MethodsThe DENV infectivity and virion production in cells or cerebral organoids treated with compounds were determined. Various methods, including cell and cerebral organoids imaging, protein and gene detection were conducted to explore their antiviral mechanisms. ResultsThe results revealed notable antiviral properties of hernandonine, particularly in inhibiting DENV during the early stages of infection. Mechanistic analysis demonstrated that, akin to genistein, wortmannin, methyl-β-cyclodextrin (MβCD), and lovastatin, hernandonine exerted an influence on cholesterol-rich lipid rafts. It also restrained the pseudopodial movement ability of cells, potentially through the downregulation of cytoskeleton and endocytosis regulatory genes or protein expression. Moreover, hernandonine's virucidal activity was demonstrated. Hernandonine's inhibition of DENV infection was further validated in a disease-relevant iPSC-derived cerebral organoids model, a novel DENV-2 infection system worthy of further application. ConclusionThis study evidenced the potential of hernandonine as a novel candidate in the fight against DENV infection.

Suppression of HSV-1 infection and viral reactivation by CRISPR-Cas9 gene editing in 2D and 3D culture models

Although our understanding of HSV-1 biology has been considerably enhanced, developing therapeutic strategies to eliminate HSV-1 in latently infected individuals remains a public health concern. Current antiviral drugs used for the treatment of HSV-1 complications are not specific and do not address latent infection. We recently developed a CRISPR/Cas9-based gene editing platform to specifically target the HSV-1 genome. In this study, we further used 2D Vero cell culture and 3D hiPSC-derived cerebral organoid (CO) models to assess the effectiveness of our editing constructs targeting viral ICP0 or ICP27 gene. We found that targeting the ICP0 or ICP27 gene with AAV2-CRISPR/Cas9 vectors in Vero cells drastically suppressed HSV-1 replication. In addition, we productively infected COs with HSV-1, characterized the viral replication kinetics, and established a viral latency model. Finally, we discovered that ICP0- or ICP27-targeting AAV2-CRISPR/Cas9 vector significantly reduced viral rebound in the COs that were latently infected with HSV-1. In summary, our results suggest that CRISPR/Cas9 gene editing of HSV-1 is an efficient therapeutic approach to eliminate the latent viral reservoir and treat HSV-1 associated complications.

NFS-22. INVESTIGATING MALIGNANT TRANSFORMATION IN NF1 PEDIATRIC GLIOMAS USING AN IPSCS-DERIVED CEREBRAL ORGANOID MODEL

Abstract BACKGROUND Neurofibromatosis Type 1 (NF1) is an autosomal dominant tumor predisposition syndrome affecting 1 in 3000 children around the world. Although NF1 inactivation is recognized as the cause of low-grade glioma (LGG) development, little is known about subsequent genetic alterations that lead to malignant transformation. One of the limitations in understanding the molecular events underlying the malignant transformation of NF1 gliomas is the lack of appropriate NF1 preclinical models for its comprehensive profiling. OBJECTIVE Brain organoids have emerged as powerful in vitro models for studying the interaction between tumor cells and the tumor microenvironment. In this study, we present a novel cerebral organoid model, developed to investigate the molecular events leading to malignant transformation in NF1 pediatric gliomas. METHODS Induced pluripotent stem cells (iPSCs)-derived cerebral organoids were generated using NF1wt and NF1mut iPSCs, the latter reprogrammed from patients with plexiform neurofibromas (Carlos III Health Institute, Spain). Immunofluorescence analysis was performed at four different developmental stages to confirm their correct cytoarchitectural development and differentiation into heterogeneous cell populations. Furthermore, four mCherry-labelled isogenic glioma cells lines, RES 186 wild type, NF1-/-, CDKN2a-/-,NF1-/-/CDKN2a-/- were co-cultured with the developed cerebral organoids and their migration potential and invasiveness were compared using two-photon microscopy. RESULTS/DISCUSSION NF1mut cerebral organoids display ventricular-like zone at early stages of development (d15-d30), characterized by luminal Sox2+ neural stem cells surrounded by Tuj1+ immature neuron. These organized structures disappear at later stages, while progenitor cells start to differentiate into fully mature neurons and glial cells (d45-d60). Additionally, after 10 days of co-culture, all four RES186 isogenic cell lines infiltrated the organoid structure with different invasion patterns. The established organoid model will provide a platform for evaluating the impact of sequential mutational events on LGG transformation, taking into consideration the role exerted by the tumor microenvironment in supporting tumor cell invasion and migration.

A microglia-containing cerebral organoid model to study early life immune challenges.

Prenatal infections and activation of the maternal immune system have been proposed to contribute to causing neurodevelopmental disorders (NDDs), chronic conditions often linked to brain abnormalities. Microglia are the resident immune cells of the brain and play a key role in neurodevelopment. Disruption of microglial functions can lead to brain abnormalities and increase the risk of developing NDDs. How the maternal as well as the fetal immune system affect human neurodevelopment and contribute to NDDs remains unclear. An important reason for this knowledge gap is the fact that the impact of exposure to prenatal risk factors has been challenging to study in the human context. Here, we characterized a model of cerebral organoids (CO) with integrated microglia (COiMg). These organoids express typical microglial markers and respond to inflammatory stimuli. The presence of microglia influences cerebral organoid development, including cell density and neural differentiation, and regulates the expression of several ciliated mesenchymal cell markers. Moreover, COiMg and organoids without microglia show similar but also distinct responses to inflammatory stimuli. Additionally, IFN-γ induced significant transcriptional and structural changes in the cerebral organoids, that appear to be regulated by the presence of microglia. Specifically, interferon-gamma (IFN-γ) was found to alter the expression of genes linked to autism. This model provides a valuable tool to study how inflammatory perturbations and microglial presence affect neurodevelopmental processes.

KCNJ2 inhibition mitigates mechanical injury in a human brain organoid model of traumatic brain injury

Traumatic brain injury (TBI) strongly correlates with neurodegenerative disease. However, it remains unclear which neurodegenerative mechanisms are intrinsic to the brain and which strategies most potently mitigate these processes. We developed a high-intensity ultrasound platform to inflict mechanical injury to induced pluripotent stem cell (iPSC)-derived cortical organoids. Mechanically injured organoids elicit classic hallmarks of TBI, including neuronal death, tau phosphorylation, and TDP-43 nuclear egress. We found that deep-layer neurons were particularly vulnerable to injury and that TDP-43 proteinopathy promotes cell death. Injured organoids derived from C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) patients displayed exacerbated TDP-43 dysfunction. Using genome-wide CRISPR interference screening, we identified a mechanosensory channel, KCNJ2, whose inhibition potently mitigated neurodegenerative processes invitro and invivo, including in C9ORF72 ALS/FTD organoids. Thus, targeting KCNJ2 may reduce acute neuronal death after brain injury, and we present a scalable, genetically flexible cerebral organoid model that may enable the identification of additional modifiers of mechanical stress.

Microglial over-pruning of synapses during development in autism-associated SCN2A-deficient mice and human cerebral organoids.

Autism spectrum disorder (ASD) is a major neurodevelopmental disorder affecting 1 in 36 children in the United States. While neurons have been the focus of understanding ASD, an altered neuro-immune response in the brain may be closely associated with ASD, and a neuro-immune interaction could play a role in the disease progression. As the resident immune cells of the brain, microglia regulate brain development and homeostasis via core functions including phagocytosis of synapses. While ASD has been traditionally considered a polygenic disorder, recent large-scale human genetic studies have identified SCN2A deficiency as a leading monogenic cause of ASD and intellectual disability. We generated a Scn2a-deficient mouse model, which displays major behavioral and neuronal phenotypes. However, the role of microglia in this disease model is unknown. Here, we reported that Scn2a-deficient mice have impaired learning and memory, accompanied by reduced synaptic transmission and lower spine density in neurons of the hippocampus. Microglia in Scn2a-deficient mice are partially activated, exerting excessive phagocytic pruning of post-synapses related to the complement C3 cascades during selective developmental stages. The ablation of microglia using PLX3397 partially restores synaptic transmission and spine density. To extend our findings from rodents to human cells, we established a microglia-incorporated human cerebral organoid model carrying an SCN2A protein-truncating mutation identified in children with ASD. We found that human microglia display increased elimination of post-synapse in cerebral organoids carrying the SCN2A mutation. Our study establishes a key role of microglia in multi-species autism-associated models of SCN2A deficiency from mouse to human cells.

Recapitulating and reversing human brain ribosomopathy defects via the maladaptive integrated stress response.

Animal or human models recapitulating brain ribosomopathies are incomplete, hampering development of urgently needed therapies. Here, we generated genetic mouse and human cerebral organoid models of brain ribosomopathies, caused by mutations in small nucleolar RNA (snoRNA) SNORD118. Both models exhibited protein synthesis loss, proteotoxic stress, and p53 activation and led to decreased proliferation and increased death of neural progenitor cells (NPCs), resulting in brain growth retardation, recapitulating features in human patients. Loss of SNORD118 function resulted in an aberrant upregulation of p-eIF2α, the mediator of integrated stress response (ISR). Using human iPSC cell-based screen, we identified small-molecule 2BAct, an ISR inhibitor, which potently reverses mutant NPC defects. Targeting ISR by 2BAct mitigated ribosomopathy defects in both cerebral organoid and mouse models. Thus, our SNORD118 mutant organoid and mice recapitulate human brain ribosomopathies and cross-validate maladaptive ISR as a key disease-driving mechanism, pointing to a therapeutic intervention strategy.

Apically localized PANX1 impacts neuroepithelial expansion in human cerebral organoids

Dysfunctional paracrine signaling through Pannexin 1 (PANX1) channels is linked to several adult neurological pathologies and emerging evidence suggests that PANX1 plays an important role in human brain development. It remains unclear how early PANX1 influences brain development, or how loss of PANX1 alters the developing human brain. Using a cerebral organoid model of early human brain development, we find that PANX1 is expressed at all stages of organoid development from neural induction through to neuroepithelial expansion and maturation. Interestingly, PANX1 cellular distribution and subcellular localization changes dramatically throughout cerebral organoid development. During neural induction, PANX1 becomes concentrated at the apical membrane domain of neural rosettes where it co-localizes with several apical membrane adhesion molecules. During neuroepithelial expansion, PANX1−/− organoids are significantly smaller than control and exhibit significant gene expression changes related to cell adhesion, WNT signaling and non-coding RNAs. As cerebral organoids mature, PANX1 expression is significantly upregulated and is primarily localized to neuronal populations outside of the ventricular-like zones. Ultimately, PANX1 protein can be detected in all layers of a 21–22 post conception week human fetal cerebral cortex. Together, these results show that PANX1 is dynamically expressed by numerous cell types throughout embryonic and early fetal stages of human corticogenesis and loss of PANX1 compromises neuroepithelial expansion due to dysregulation of cell-cell and cell-matrix adhesion, perturbed intracellular signaling, and changes to gene regulation.

Neural cell engraftment therapy for sporadic Creutzfeldt-Jakob disease restores neuroelectrophysiological parameters in a cerebral organoid model

BackgroundSporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is a fatal neurodegenerative disease with currently no treatment options. Stem cell therapy for neurodegenerative diseases is emerging as a possible treatment option. However, while there are a few clinical trials for other neurodegenerative disorders such as Parkinson’s disease, prion disease cell therapy research has so far been confined to animal models.MethodsHere, we use a novel approach to study cell therapies in sCJD using a human cerebral organoid model. Cerebral organoids can be infected with sCJD prions allowing us to assess how neural precursor cell (NPC) therapy impacts the progression of sCJD. After 90 days of sCJD or mock infection, organoids were either seeded with NPCs or left unseeded and monitored for cellular composition changes, prion infection parameters and neuroelectrophysiological function at 180 days post-infection.ResultsOur results showed NPCs integrated into organoids leading to an increase in neuronal markers and changes in cell signaling irrespective of sCJD infection. Although a small, but significant, decrease in protease-resistant PrP deposition was observed in the CJD-infected organoids that received the NPCs, other disease-associated parameters showed minimal changes. However, the NPCs had a beneficial impact on organoid function following infection. sCJD infection caused reduction in neuronal spike rate and mean burst spike rate, indicative of reduced action potentials. NPC seeding restored these electrophysiological parameters to the uninfected control level.ConclusionsTogether with the previous animal studies, our results support that cell therapy may have some functional benefit for the treatment of human prion diseases.

Alzheimer’s disease phenotypes in human induced pluripotent stem cell‐based models of Down syndrome

AbstractBackgroundBy age 40, all people with Down syndrome (DS) develop Alzheimer’s disease (AD) brain neuropathology, including Aβ plaques and neurofibrillary tangles of phosphorylated microtubule associated protein tau (MAPT), and most go on to develop early‐onset dementia. People with Down syndrome (DS) have triplication of chromosome 21 (trisomy 21) and they develop AD neuropathology due to triplication of the amyloid precursor protein gene (APP) which resides on chromosome 21. Here, we developed and characterized human induced pluripotent stem cell (hiPSC)‐derived neurons, astrocytes, microglia, and cerebral organoid (CO) models of DS and familial AD and used them to evaluate the AD‐related pathological mechanisms caused by trisomy 21.MethodWe obtained hiPSC lines derived from individuals with DS, healthy control individuals (HC), and individuals with familial AD, including duplication of APP on one chromosome (APPdup) or carrying the London mutation in APP (APPV717I). All donors had the apolipoprotein E (APOE) ε3/ε3 genotype. We generated hiPSC‐derived neurons, astrocytes, microglia, and COs using validated techniques. We evaluated the hiPSC‐derived neural cells and COs at different ages for AD‐related neuropathology, neuroinflammation, and neurodegeneration by performing RNA sequencing, immunohistochemistry, immunoblotting, and enzyme‐linked immunosorbent assay.ResultAD‐related size phenotypes characteristic of neurodegeneration were observed in DS hiPSC‐based models relative to HC models. Dysregulated gene and protein expression of APP, MAPT, and APOE, in addition to chromosome 21 genes, were also observed in DS hiPSC‐based models. In particular, neuroinflammatory markers, especially those previously shown in APOE‐related pathways, were also increased in DS models.ConclusionWe characterized new hiPSC‐based models of DS and familial AD which will be useful for identifying commonalities in disease mechanisms as well as for dissecting the roles of chromosome 21 genes other than APP. We identified several AD‐related phenotypes in hiPSC‐based DS related to neuropathology, neuroinflammation, and neurodegeneration. We expect that these hiPSC‐based models will be useful for testing therapeutics in the future to address AD in people with DS, for which there is no cure.

CNSC-33. MOLECULAR AND FUNCTIONAL DRIVERS OF ACTIVITY DEPENDENT GLIOBLASTOMA PROLIFERATION

Abstract Glioblastoma, the most common primary brain tumor in adults, is a major cause of neurological morbidity and mortality with no effective therapies. Its proliferation and invasion are regulated by direct and paracrine-mediated neuronal activity. In this study, we uncover molecular targets and protein-protein signaling driving activity dependent proliferation. To identify candidate molecular pathways, we used high-density electrode arrays in vivo to record human local field potentials. Single-cell RNA sequencing identified Netrin-G1 (NTNG1) and thrombospondin-1 (TSP1) elevation in glioblastoma clonal population that proliferates in response to the presence of neurons. The amino acid sequence of NTNG1 and the TSR1 domain of TSP1 were utilized to predict the 3D interaction surface of NTNG1 and TSP1. Compared to structure predictions of NTNG1 and its known binding partner NGL1, NTNG1 and TSP1 complex predictions showed agreement between machine learning predicted models and lower predicted aligned error in interdomain regions of the complex, suggesting higher confidence in the relative position of NTNG1-TSP1 as compared to NTNG1-NGL1. We performed 45 protein structure predictions and two molecular dynamics simulations of mutant and WT TSP1 in complex with NTNG1. Each simulation box consisted of >263,000 atoms, each run for 50 million 2 femtosecond timesteps for a total simulation time of 200 ns. These calculations confirmed a stable complex in the WT condition and significant conformational change in the R46A condition, emphasizing the importance of this residue in mediating NTNG1-TSP1 binding. Computational affinity models between NTNG1 and TSP1 were validated by pull-down assays and immunofluorescence labelling using cerebral organoid and human glioma-neuronal coculture models. Finally, we design small molecule and fusion-protein inhibitors disrupting this interaction. Here we advance the development of targeted precision-medicine therapies to treat glioblastoma proliferation through activity-dependent mechanisms.

CNSC-29. NKCC1 SIGNALING IN GLIOBLASTOMA REGULATES NEURONAL HYPEREXCITABILITY THROUGH GABAERGIC TONE

Abstract Over 90% of patients with glioma experience tumor-associated epilepsy, however first-line treatment fails to control seizures in most patients. Thus, there is a critical need to identify novel drivers of glioblastoma-induced neuronal hyperexcitability. We therefore used high-density electrode arrays to record local field potentials in vivo from gliomas. We then developed cerebral organoid and mouse models using primary glioblastoma cells from patients and neurons derived from induced pluripotent stem cells. Single-cell RNA sequencing (13,730 cells analyzed) in this system identified genes elevated in glioma-intrinsic neurons, including the sodium-potassium-chloride co-transporter 1 (NKCC1), which mediates chloride (Cl-) influx into cells. This elevated NKCC1 expression and subsequent increase of intracellular Cl- sets the reversal potential of GABAAR-mediated currents promoting depolarizing excitatory GABAergic responses. Electrophysiological analysis of glioma-neuron co-cultures using multi-electrode array and live-cell calcium imaging demonstrated increased network burst synchrony in the presence of NKCC1-high expressing tumor cells. Single-nucleus sequencing (54,000 cells analyzed) of embryonic neurons in co-culture with GBM cells further confirmed the elevated expression of circuit assembly genes including neuronal NKCC1. Live-cell imaging of iPSC-induced neuron organoids co-cultured with NKCC1-high-and low-GBM cells revealed increased migration and integration of NKCC1-elevated tumor cells. Mechanistic and functional studies validating therapeutic vulnerabilities to NKCC1 silencing by shRNA knockdown and by the FDA-approved diuretic drug, bumetanide was performed in vitro. The reduction in neuronal synchrony induced by NKCC1 downregulation correlated with a decrease in extracellular GABA and glutamate, suggesting a strong association between excitatory GABAergic and glutamate signaling with neuronal hypersynchrony in glioblastoma. Patient-derived xenograft in vivo experiments evaluated the effect of NKCC1 on tumor growth and progression and NKCC1-high GBM cells exhibited greater tumor burden compared to control mice. Collectively, these findings reveal a novel mechanism driving neuronal hyperactivity in glioblastoma and identifies potential therapeutic vulnerabilities for the treatment of glioma-associated epilepsy.

EXTH-27. ASSESING THE EFFECT OF ELECTRIC FIELD THERAPY ON PATIENT DERIVED GLIOMA STEM CELL LINES AND GLIOMA INFILTRATING CEREBRAL ORGANOIDS

Abstract Glioblastoma (GBM) is the most aggressive primary brain tumour in adults. Electric field therapy (EFT) is a new cancer treatment applied after safe maximal tumour resection that interferes with cancer cell division, slowing tumour recurrence and prolonging life. In GBM patients, this therapeutic modality has demonstrated an increase in median survival from ~16 to ~21 months. This study aimed to assess and decipher the effectiveness of EFT on patient-derived glioblastoma stem cell lines (GSC) as well as on advanced Glioma infiltrating cerebral organoid models (GLICOs). To achieve the project goals, we used a novel In-vitro EFT stimulation apparatus to test stimulation parameters on multiple patient-derived GSC lines as well as GLICO models derived from them. We used advanced microscopy and transcriptomic sequencing to visualize and decipher the causal effect of our stimulation regime on the in-vitro models. We observe a strong effect of our EFT regime on multiple GSC lines as well as on GLICOs when compared to their controls. Transcriptomic analysis showed multitude of differentially expressed mRNAs between the treated and control samples with multiple pathways being dysregulated from nuclear proteins, cell mitosis-related events and activation of non-apoptotic mediated programmed cell death pathways indicating a novel mechanism of action. Advanced causal inference studies using the data also provided novel insights on combinatorial therapies that could enhance the efficacy of the treatment. This study provides novel understanding of the mechanism of EFT with potential for optimised treatment regimens for GBM patients.

TP53-PTEN-NF1 depletion in human brain organoids produces a glioma phenotype in vitro.

Glioblastoma (GBM) is fatal and the study of therapeutic resistance, disease progression, and drug discovery in GBM or glioma stem cells is often hindered by limited resources. This limitation slows down progress in both drug discovery and patient survival. Here we present a genetically engineered human cerebral organoid model with a cancer-like phenotype that could provide a basis for GBM-like models. Specifically, we engineered a doxycycline-inducible vector encoding shRNAs enabling depletion of the TP53, PTEN, and NF1 tumor suppressors in human cerebral organoids. Designated as inducible short hairpin-TP53-PTEN-NF1 (ish-TPN), doxycycline treatment resulted in human cancer-like cerebral organoids that effaced the entire organoid cytoarchitecture, while uninduced ish-TPN cerebral organoids recapitulated the normal cytoarchitecture of the brain. Transcriptomic analysis revealed a proneural GBM subtype. This proof-of-concept study offers a valuable resource for directly investigating the emergence and progression of gliomas within the context of specific genetic alterations in normal cerebral organoids.

Characterization of neural mechanotransduction response in human traumatic brain injury organoid model

The ability to model physiological systems through 3D neural in-vitro systems may enable new treatments for various diseases while lowering the need for challenging animal and human testing. Creating such an environment, and even more impactful, one that mimics human brain tissue under mechanical stimulation, would be extremely useful to study a range of human-specific biological processes and conditions related to brain trauma. One approach is to use human cerebral organoids (hCOs) in-vitro models. hCOs recreate key cytoarchitectural features of the human brain, distinguishing themselves from more traditional 2D cultures and organ-on-a-chip models, as well as in-vivo animal models. Here, we propose a novel approach to emulate mild and moderate traumatic brain injury (TBI) using hCOs that undergo strain rates indicative of TBI. We subjected the hCOs to mild (2 s−1) and moderate (14 s−1) loading conditions, examined the mechanotransduction response, and investigated downstream genomic effects and regulatory pathways. The revealed pathways of note were cell death and metabolic and biosynthetic pathways implicating genes such as CARD9, ENO1, and FOXP3, respectively. Additionally, we show a steeper ascent in calcium signaling as we imposed higher loading conditions on the organoids. The elucidation of neural response to mechanical stimulation in reliable human cerebral organoid models gives insights into a better understanding of TBI in humans.

A Human Cerebral Organoid Model of Neural Cell Transplantation.

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For the central nervous system, although xenotransplantation remains state-of-the-art, such models are technically challenging, limited in throughput, and expensive. Moreover, the environmental signals present do not perfectly cross-react with human cells. This paper presents an inexpensive, accessible, and high-throughput-compatible model for the transplantation and tracking of human neural cells into human cerebral organoids. These organoids can be easily generated from human induced pluripotent stem cells using commercial kits and contain the key cell types of the cerebrum. We first demonstrate this transplant protocol with the injection of EGFP-labeled human iPSC-derived neural progenitor cells (NPCs) into these organoids. We next discuss considerations for tracking the growth of these cells in the organoid by live-cell fluorescence microscopy and demonstrate the tracking of transplanted EGFP-labeled NPCs in an organoid over a 4 month period. Finally, we present a protocol for the sectioning, cyclic immunofluorescent staining, and imaging of the transplanted cells in their local context. The organoid transplantation model presented here allows the long-term (at least 4 months) tracking of transplanted human cells directly in a human microenvironment with an inexpensive and simple-to-perform protocol. It, thus, represents a useful model both for neural cell therapies (transplants) and, likely, for modeling central nervous system (CNS) tumors in a more microenvironmentally accurate manner.