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Actuation enhances patterning in human neural tube organoids.
Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.
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Engineered Mesenchymal Stem Cells Expressing CD::UPRT and TRAIL Exhibit Potent Anti-Tumor Effects in Glioblastoma Patient-Derived Organoids.
Glioblastoma (GBM) is a highly aggressive brain tumor with limited therapeutic options due to its invasive nature, therapeutic resistance, and the challenge of drug delivery across the blood-brain barrier (BBB). Mesenchymal stem cells (MSCs), owing to their tumor tropic properties and ability to cross the BBB, offer a promising platform for targeted anti-cancer delivery. We previously engineered MSCs to express CD::UPRT and TRAIL, along with chemokine receptors to enhance tumor homing (MSC-CD-TRAIL; BM03). This study evaluated the anti-tumor efficacy of BM03 using GBM patient-derived organoids (GBOs), clinically relevant in vitro models. Using a GBO-MSC co-culture system, BM03 significantly increased cell death and reduced viability in GBOs from four GBM patients compared with controls and MSC-WT groups. In 3D invasion assays, BM03-treated GBOs showed markedly reduced invasive outgrowth, accompanied by downregulation of EMT markers (Zeb1 and Snail) and stem-like markers (Olig2 and Sox2), particularly in invasive regions. GFAP expression remained unchanged, suggesting selective targeting of tumor stem-like cells. Live-cell imaging further demonstrated BM03 infiltration into GBOs, which was associated with increased apoptosis, as evidenced by elevated cleaved caspase-3 levels. These findings provide organoid-based preclinical evidence supporting further evaluation of BM03 as an MSC-based therapeutic strategy for GBM.
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Reverse engineering the beat: Assembloid modeling of sympathetic control of the heart.
Liu et al.1 generate human sympathetic ganglion organoids and assemble them with heart-forming organoids to build functionally connected sympathetic-cardiac organoids in vitro. Their platform captures autonomic development, neuromodulation of heart function, and metabolic injury, opening new opportunities to study human autonomic disorders and cardiometabolic disease.2.
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Human Cerebrospinal Fluid Promotes Neuronal Circuit Maturation of Human Induced Pluripotent Stem Cell-Derived 3D Neural Aggregates.
Human induced pluripotent stem cell (hiPSC)-derived in vitro neural and organoid models resemble fetal, rather than adult brain properties, indicating that currently applied cultivation media and supplements are insufficient to achieve neural maturation beyond the fetal stage. In vivo, cerebrospinal fluid molecules are regulating the transition of the immature fetal human brain into a mature adult brain. By culturing hiPSC-3D neural aggregates in human cerebrospinal fluid (hCSF) obtained from healthy adult individuals, we demonstrate that hCSF rapidly triggers neurogenesis, gliogenesis, synapse formation, neurite outgrowth, suppresses proliferation of residing neural stem cells, and results in the formation of synchronously active neuronal circuits in vitro within 3 days. Thus, a physiologically relevant and adult brain-like milieu triggers maturation of hiPSC-3D neural aggregates into highly functional neuronal circuits in vitro. The approach presented here opens a new avenue to identify novel physiological factors for the improvement of hiPSC neural in vitro models.
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Human Retinal Organoid Modeling Defines Developmental Window and Therapeutic Vulnerabilities in MYCN-Amplified Retinoblastoma.
MYCN amplification without concurrent RB1 mutations characterizes a rare yet highly aggressive subtype of retinoblastoma; however, its precise developmental origins and therapeutic vulnerabilities remain incompletely understood. Here, we modeled this subtype by lentiviral-mediated MYCN overexpression in human pluripotent stem cell-derived retinal organoids, revealing a discrete developmental window (days 70-120) during which retinal progenitors showed heightened susceptibility to transformation. Tumors arising in this period exhibited robust proliferation, expressed SOX2, and lacked CRX, consistent with origin from primitive retinal progenitors. MYCN-overexpressing organoids generated stable cell lines that reproducibly gave rise to MYCN-driven tumors when xenografted into immunodeficient mice. Transcriptomic profiling demonstrated that MYCN-overexpressing organoids closely recapitulated molecular features of patient-derived MYCN-amplified retinoblastomas, particularly through activation of MYC/E2F and mTORC1 signaling pathways. Pharmacological screening further identified distinct therapeutic vulnerabilities, demonstrating distinct subtype-specific sensitivity of MYCN-driven cells to transcriptional inhibitors (THZ1, Flavopiridol) and the cell-cycle inhibitor Volasertib, indicative of a unique oncogene-addicted state compared to RB1-deficient retinoblastoma cells. Collectively, our study elucidates the developmental and molecular mechanisms underpinning MYCN-driven retinoblastoma, establishes a robust and clinically relevant human retinal organoid platform, and highlights targeted transcriptional inhibition as a promising therapeutic approach for this aggressive pediatric cancer subtype.
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Tumor Treating Fields Enhance Temozolomide-Associated Growth Suppression in Glioblastoma Cells and Patient-Derived Organoids Through Cell-Cycle Blockade, Apoptosis, and SMAD-Associated Signaling.
AIMS: Glioblastoma (GBM) remains difficult to treat despite multimodal therapy. Tumor Treating Fields (TTF) disrupts mitosis and provides clinical benefit in GBM, but the molecular responses associated with TTF and the effects of combining TTF with temozolomide (TMZ) remain incompletely defined. METHODS: TMZ, TTF, and TMZ + TTF were evaluated in U251 and SHG140 GBM cells and patient-derived organoids. TTF was applied at 200 kHz and 1.75 V/cm RMS. Viability, colony formation, migration-related phenotypes, apoptosis, cell-cycle distribution, Western blotting, qRT-PCR, RNA-seq, and organoid morphology/immunofluorescence analyses were performed. SB525334 was used to assess SMAD-associated signaling. RESULTS: Combined TMZ + TTF treatment more strongly suppressed cell viability, clonogenic growth, and migration-related phenotypes than either monotherapy, produced greater disruption of organoid structural integrity, and was accompanied by increased cleaved caspase-3 activation and apoptotic populations. RNA-seq identified TTF-associated transcriptomic changes involving chromatin-associated processes, DNA replication, and cell-cycle pathways. TTF was associated with selected SMAD-related signaling changes, G2/M accumulation, altered checkpoint signaling, and enhanced proapoptotic signaling. SB525334 partially attenuated TTF-associated SMAD phosphorylation, growth suppression, and cleaved caspase-3 induction. CONCLUSION: TTF enhances TMZ-associated antitumor responses in GBM cell lines and organoids, accompanied by cell-cycle blockade, apoptosis-associated changes, and selected SMAD-related signaling responses.
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Modeling human neuronal migration deficits in 3D.
During the past few decades, we have witnessed an impressive gain in the knowledge regarding the basic mechanisms underlying human neuronal migration disorders by the usage of mouse models. Nevertheless, despite the remarkable conservation both in the genetic encoded information and the developmental processes, there are still numerous important differences between human and mouse. This may explain the vast excitement following the realization that technological breakthroughs enabled generating tissue-like human-based organoids for modeling human neuronal migration diseases. This review will provide a short introduction on human and mouse neuronal migration processes, and highlight human brain organoid models of neuronal migration diseases.
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Evaluation of Neurotoxicity With Human Pluripotent Stem Cell-Derived Cerebral Organoids.
The recent development of human cerebral organoids provides an invaluable in vitro model of human brain development to assess the toxicity of natural or man-made toxic substances. By recapitulating key aspects of early human neurodevelopment, investigators can evaluate with this three-dimensional (3D) model the effect of certain compounds on the formation of neuronal networks and their electrophysiological properties with more physiological relevance than neurons grown in monolayers and in cultures composed of a unique cell type. This promising potential has contributed to the development of a large number of diverse protocols to generate human cerebral organoids, making interlaboratory comparisons of results difficult. Based on a previously published protocol to generate human cortical organoids (herein called cerebral organoids), we detail several approaches to evaluate the effect of chemicals on neurogenesis, apoptosis, and neuronal function when exogenously applied to cultured specimens. Here, we take as an example 4-aminopyridine, a potassium channel blocker that modulates the activity of neurons and neurogenesis, and describe a simple and cost-effective way to test the impact of this agent on cerebral organoids derived from human induced pluripotent stem cells. We also provide tested protocols to evaluate neurogenesis in cerebral organoids with ethynyl deoxyuridine labeling and neuronal activity with live calcium imaging and microelectrode arrays. Together, these protocols should facilitate the implementation of cerebral organoid technologies in laboratories wishing to evaluate the effects of specific compounds or conditions on the development and function of human neurons with only basic cell culture equipment. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of human cerebral organoids from pluripotent stem cells Support Protocol 1: Human pluripotent stem cell culture Basic Protocol 2: Evaluation of neurogenesis in cerebral organoids with ethynyl deoxyuridine labeling Basic Protocol 3: Calcium imaging in cerebral organoids Basic Protocol 4: Electrophysiological evaluation of cerebral organoids with microelectrode arrays Support Protocol 2: Immunostaining of cerebral organoids.
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Aluminum hydroxide exposure induces neurodevelopmental impairment in hESC-derived cerebral organoids.
Aluminum (Al) has been classified as a cumulative environmental pollutant that endangers human health. There is increasing evidence to suggest the toxic effects of Al, but the specific action on human brain development remains unclear. Al hydroxide (Al(OH)3), the most common vaccine adjuvant, is the major source of Al and poses risks to the environment and early childhood neurodevelopment. In this study, we explored the neurotoxic effect of 5 μg/ml or 25 μg/ml Al(OH)3 for six days on neurogenesis by utilizing human cerebral organoids from human embryonic stem cells (hESCs). We found that early Al(OH)3 exposure in organoids caused a reduction in the size, deficits in basal neural progenitor cell (NPC) proliferation, and premature neuron differentiation in a time and dose-dependent manner. Transcriptomes analysis revealed a markedly altered Hippo-YAP1 signaling pathway in Al(OH)3 exposed cerebral organoid, uncovering a novel mechanism for Al(OH)3-induced detrimental to neurogenesis during human cortical development. We further identified that Al(OH)3 exposure at day 90 mainly decreased the production of outer radial glia-like cells(oRGs) but promoted NPC toward astrocyte differentiation. Taken together, we established a tractable experimental model to facilitate a better understanding of the impact and mechanism of Al(OH)3 exposure on human brain development.
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Transcriptomic and morphological maturation of human astrocytes in cerebral organoids.
Cerebral organoids (CerOrgs) derived from human induced pluripotent stem cells (iPSCs) are a valuable tool to study human astrocytes and their interaction with neurons and microglia. The timeline of astrocyte development and maturation in this model is currently unknown and this limits the value and applicability of the model. Therefore, we generated CerOrgs from three healthy individuals and assessed astrocyte maturation after 5, 11, 19, and 37 weeks in culture. At these four time points, the astrocyte lineage was isolated based on the expression of integrin subunit alpha 6 (ITGA6). Based on the transcriptome of the isolated ITGA6-positive cells, astrocyte development started between 5 and 11 weeks in culture and astrocyte maturation commenced after 11 weeks in culture. After 19 weeks in culture, the ITGA6-positive astrocytes had the highest expression of human mature astrocyte genes, and the predicted functional properties were related to brain homeostasis. After 37 weeks in culture, a subpopulation of ITGA6-negative astrocytes appeared, highlighting the heterogeneity within the astrocytes. The morphology shifted from an elongated progenitor-like morphology to the typical bushy astrocyte morphology. Based on the morphological properties, predicted functional properties, and the similarities with the human mature astrocyte transcriptome, we concluded that ITGA6-positive astrocytes have developed optimally in 19-week-old CerOrgs.