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Reelin Regulates Developmental Desynchronization Transition of Neocortical Network Activity.
During the first and second stages of postnatal development, neocortical neurons exhibit a wide range of spontaneous synchronous activity (SSA). Towards the end of the second postnatal week, the SSA is replaced by a more sparse and desynchronized firing pattern. The developmental desynchronization of neocortical spontaneous neuronal activity is thought to be intrinsically generated, since sensory deprivation from the periphery does not affect the time course of this transition. The extracellular protein reelin controls various aspects of neuronal development through multimodular signaling. However, so far it is unclear whether reelin contributes to the developmental desynchronization transition of neocortical neurons. The present study aims to investigate the role of reelin in postnatal cortical developmental desynchronization using a conditional reelin knockout (RelncKO) mouse model. Conditional reelin deficiency was induced during early postnatal development, and Ca2+ recordings were conducted from organotypic cultures (OTCs) of the somatosensory cortex. Our results show that both wild type (wt) and RelncKO exhibited an SSA pattern during the early postnatal week. However, at the end of the second postnatal week, wt OTCs underwent a transition to a desynchronized network activity pattern, while RelncKO activity remained synchronous. This changing activity pattern suggests that reelin is involved in regulating the developmental desynchronization of cortical neuronal network activity. Moreover, the developmental desynchronization impairment observed in RelncKO was rescued when RelncKO OTCs were co-cultured with wt OTCs. Finally, we show that the developmental transition to a desynchronized state at the end of the second postnatal week is not dependent on glutamatergic signaling. Instead, the transition is dependent on GABAAR and GABABR signaling. The results suggest that reelin controls developmental desynchronization through GABAAR and GABABR signaling.
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Organ-on-chip models: Implications in drug discovery and clinical applications.
Before a lead compound goes through a clinical trial, preclinical studies utilize two-dimensional (2D) in vitro models and animal models to study the pharmacodynamics and pharmacokinetics of that lead compound. However, these current preclinical studies may not accurately represent the efficacy and safety of a lead compound in humans, as there has been a high failure rate of drugs that enter clinical trials. All of these failures and the associated costs demonstrate a need for more representative models of human organ systems for screening in the preclinical phase of drug development. In this study, we review the recent advances in in vitro modeling including three-dimensional (3D) organoids, 3D microfabrication, and 3D bioprinting for various organs including the heart, kidney, lung, gastrointestinal tract (intestine-gut-stomach), liver, placenta, adipose, retina, bone, and brain as well as multiorgan models. The availability of organ-on-chip models provides a wealth of opportunities to understand the pathogenesis of human diseases and provide a potentially better model to screen a drug, as these models utilize a dynamic 3D environment similar to the human body. Although there are limitations of organ-on-chip models, the emergence of new technologies have refined their capability for translational research as well as precision medicine.
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A cost- and time-efficient method for high-throughput cryoprocessing and tissue analysis using multiplexed tissue molds.
Cryosectioning remains the gold standard for antibody and transcriptomic/in situ hybridization tissue analysis. However, tissue processing is time-consuming and costly, limiting routine and diagnostic use. Currently, no commercially available protocols or products exist for multiplexing this process. Here, we introduce multiplexed tissue molds (MTMs) that enable high-throughput cryoprocessing-cutting costs and workload by up to 96% while permitting the processing of tissues of various sizes and origins. We demonstrate compatibility with heterogeneous tissues by processing 19 different adult mouse tissues in parallel. Furthermore, we process up to ∼110 neural organoids of different ages and sizes simultaneously and assess their neural differentiation marker expression. MTMs allow sectioning-based tissue analysis when labor, time, and cost are limiting factors. MTMs could be used to compare high specimen numbers in histopathological settings, organism-wide antigen and antibody targeting studies, high-throughput tissue screens, and defined tissue section positioning for, e.g., spatial transcriptomics experiments.
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Automated and scalable expansion of human liver organoids for translational applications.
BACKGROUND: Human tissue-derived organoids hold strong potential for personalized medicine and cell therapy, but this requires large cell quantities. Conventional organoid culture systems remain labor-intensive, are difficult to scale, and lack process control. Here, we present a novel strategy using an automated bioreactor platform that enables large-scale expansion of human liver organoids. METHODS: Human liver organoids were expanded for 14 days in a single bioreactor suspension culture bag and compared with spinner flasks and static dome cultures. Cell yield, viability, fold expansion, morphology, and phenotypic markers (LGR5, E-cadherin, Vimentin, Ki67) were assessed. The system’s uninterrupted workflow enabled seamless transition to differentiation: using integrated perfusion, we performed a direct medium switch from expansion to hepatic differentiation without harvesting or disrupting the culture. Commitment to the hepatic lineage was evaluated by expression of ALB, CYP3A4, MRP2, and HNF4A. RESULTS: By day 14, the bioreactor generated an average of 5.63 × 108 (± 1.1 × 108) viable cells, while spinner flasks reached 1.22 × 108 (± 4.26 × 107) cells, while static cultures yielded only 4.02 × 105 (± 2.81 × 105), making the bioreactor’s output ~ 1400 times greater than static cultures (p = 0.022) and nearly five times higher than spinner flasks. This substantial gain in absolute cell yield is a promising indicator for downstream translation. Organoids preserved phenotypic integrity and proliferative capacity as shown by sustained expression of LGR5, E-cadherin, and Ki67. Bioreactor-cultured organoids exhibited robust growth and intact cyst-like morphology with a large size, due to the absence of mechanical fragmentation and related cellular stress. As a proof-of-principle, bioreactor-grown organoids differentiated efficiently toward the hepatic lineage, as evidenced by a downregulated gene expression of LGR5 and Ki67, with elevated gene expression of ALB, CYP3A4, MRP2, and HNF4A, along with an upregulated secretion of Albumin. CONCLUSION: The system establishes a closed, monitored, and scalable upstream workflow for liver organoid expansion. This work represents a significant step toward organoid production for future cell therapy and regenerative medicine applications, while maintaining phenotypic stability and differentiation capacity.
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Advances in modelling the human microbiome-gut-brain axis in vitro.
The human gut microbiome has emerged as a key player in the bidirectional communication of the gut-brain axis, affecting various aspects of homeostasis and pathophysiology. Until recently, the majority of studies that seek to explore the mechanisms underlying the microbiome-gut-brain axis cross-talk, relied almost exclusively on animal models, and particularly gnotobiotic mice. Despite the great progress made with these models, various limitations, including ethical considerations and interspecies differences that limit the translatability of data to human systems, pushed researchers to seek for alternatives. Over the past decades, the field of in vitro modelling of tissues has experienced tremendous growth, thanks to advances in 3D cell biology, materials, science and bioengineering, pushing further the borders of our ability to more faithfully emulate the in vivo situation. The discovery of stem cells has offered a new source of cells, while their use in generating gastrointestinal and brain organoids, among other tissues, has enabled the development of novel 3D tissues that better mimic the native tissue structure and function, compared with traditional assays. In parallel, organs-on-chips technology and bioengineered tissues have emerged as highly promising alternatives to animal models for a wide range of applications. Here, we discuss how recent advances and trends in this area can be applied in host-microbe and host-pathogen interaction studies. In addition, we highlight paradigm shifts in engineering more robust human microbiome-gut-brain axis models and their potential to expand our understanding of this complex system and hence explore novel, microbiome-based therapeutic approaches.
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Neural organoids for disease phenotyping, drug screening and developmental biology studies.
Human induced pluripotent stem cells (hiPSCs) can theoretically yield limitless supplies of cells fated to any cell type that comprise the human organism, making them a new tool by which to potentially overcome caveats in current biomedical research. In vitro derivation of central nervous system (CNS) cell types has the potential to provide material for drug discovery and validation, safety and toxicity assays, cell replacement therapy and the elucidation of previously unknown disease mechanisms. However, current two-dimensional (2D) CNS differentiation protocols do not faithfully recapitulate the spatial organization of heterogeneous tissue, nor the cell-cell interactions, cell-extracellular matrix interactions, or specific physiological functions generated within complex tissue such as the brain. In an effort to overcome 2D protocol limitations, there have been advancements in deriving highly complicated 3D neural organoid structures. Herein we provide a synopsis of the derivation and application of neural organoids and discuss recent advancements and remaining challenges on the full potential of this novel technological platform.
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Neurotoxicity of phenylalanine on human iPSC-derived cerebral organoids.
Phenylketonuria (PKU) is a common genetic metabolic disorder that causes phenylalanine accumulation in the blood. The most serious symptoms are related to the brain, as intellectual disability, seizure, and microcephaly are commonly found in poorly treated PKU patients and the babies of maternal PKU. However, the mechanism of hyperphenylalaninemia on human neurodevelopment is still unclear. Here we utilized human induced pluripotent stem cell (iPSC)-derived cerebral organoids to investigate the neurotoxicity of hyperphenylalaninemia. Cerebral organoids at days 40 or 100 were treated with different concentrations of phenylalanine for 5 days. After phenylalanine treatments, the cerebral organoids displayed alterations in organoid size, induction of apoptosis, and depletion of neural progenitor cells. However, phenylalanine did not have an impact on neurons and glia, including astrocytes, immature oligodendrocytes, and mature oligodendrocytes. Remarkably, a reduction in the thickness of the cortical rosettes and a decrease in myelination at the intermediate zone were inspected with the elevated phenylalanine concentrations. RNA-seq of phenylalanine-treated organoids revealed that gene sets related to apoptosis, p53 signaling pathway, and TNF signaling pathway via NF-kB were enriched in upregulated genes, while those related to cell cycle and amino acid metabolism were enriched in downregulated genes. In addition, there were several microcephaly disease genes, such as ASPM, LMNB1, and CENPE, ranked at the top of the downregulated genes. These findings indicate that phenylalanine exposure may contribute to microcephaly, abnormal cortical expansion, and myelination lesions in the developing human brain.
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Characterization of Native Extracellular Matrix of Patient-Derived Glioblastoma Multiforme Organoids.
Model systems play a crucial role in biological and biomedical research, especially in the search for new treatments for challenging diseases such as glioblastoma multiforme (GBM). Organoids are 3D in vitro multicellular "middle-ground" model systems that recapitulate highly organized and heterogeneous in vivo organ-like systems, often through stem cell differentiation. Incorporating Matrigel™ or other exogenous extracellular matrices (ECMs) that do not naturally occur in the human body is common practice for organoid generation, ignoring the role of dynamic reciprocity between the cells and the ECM in tissue development. In this study, we describe a method to develop GBM organoids (GBOs) from cells without the need for exogenous ECM encapsulation and without cell culture media changes to produce stable tissue-like organoids that reach a 4 mm diameter in as little as 6 weeks. We observed a transition from homogenous cell populations to tissue-like structures when GBOs were larger than 1 mm in diameter. Transcriptomic analysis revealed that the greatest gene expression changes occurred when GBOs were 2 mm in diameter, with collagen VI as the most upregulated ECM-related gene. Quantitative and histochemical assessments further supported native ECM synthesis with significantly higher levels of glycosaminoglycans and collagen in GBOs compared with spheroids. To our knowledge, this study presents the first reproducibly large GBOs with natively produced ECMs. Organoids with natively synthesized ECMs promise to eliminate artifacts and variability from aged, homogeneic, or xenogeneic scaffolds and to provide insights for ECM-targeted drug development. Impact Statement Glioblastoma multiforme (GBM) is the most common and deadly brain tumor due to its complex tissue heterogeneity. Drug development for GBM is difficult because GBM models are not very translatable and are limited, leading to the need of GBM organoids (GBOs). Current GBO development is highly laborious and of questionable relevance because of the reliance on non-native, animal-derived extracellular matrix (ECM). This study describes a scalable and reproducible method of developing GBOs with natively generated ECMs. These GBOs allow for both the study of the early stages of GBM that are currently inaccessible and a quicker and more translatable tool for GBM drug screening and development.
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A 3D pooled PBMC-organoid co-culture platform for profiling immune susceptibility and PD-1 blockade response in gastric cancer.
BACKGROUND: Peritoneal metastatic gastric cancer (PMGC) is associated with a dismal prognosis and limited benefit from immune checkpoint inhibitors (ICIs). Although PD-1/PD-L1 blockade has improved outcomes in selected patients, therapeutic responses remain highly heterogeneous, even among PD-L1-high tumors. The lack of preclinical models that functionally recapitulate tumor-immune interactions in a three-dimensional (3D) context has hindered the investigation of immune susceptibility and resistance mechanisms in PMGC. METHODS: We established a standardized allogeneic 3D co-culture platform integrating patient-derived PMGC organoids with pooled peripheral blood mononuclear cells (PBMCs) from healthy donors. An optimized Matrigel-embedded configuration was used to enable sustained immune cell infiltration and tumor-immune contact. Immune-mediated cytotoxicity, organoid susceptibility phenotypes, and responses to PD-1 blockade with pembrolizumab were functionally assessed. Comparative proteomic profiling was performed, and differential expression between groups was evaluated using appropriate statistical tests with false discovery rate correction. Group comparisons were conducted using Student's t-test. RESULTS: The optimized 3D embedded system enabled dynamic infiltration of activated T cells into organoid structures. PMGC organoids exhibited heterogeneous susceptibility to immune-mediated cytotoxicity, classifying them into cytolytic and noncytolytic phenotypes independent of HLA mismatching. Comparative proteomic profiling revealed that cytolytic lines were enriched in metabolic pathways, whereas noncytolytic lines showed enrichment of immune-related signaling. Notably, response to pembrolizumab varied even among PD-L1-high organoids. Nonresponsive PD-L1-high lines were characterized by elevated baseline expression of alternative immune checkpoint ligands, specifically CD112 (TIGIT ligand) and galectin-9 (TIM-3 ligand). CONCLUSIONS: We established a robust pooled PBMC-organoid co-culture platform that enables functional assessment of tumor-immune dynamics in PMGCs. This system serves as a functional ex vivo tool for evaluating immunotherapy responses and for examining the expression patterns of alternative immune checkpoint ligands associated with differential PD-1 blockade response. These findings support the utility of this ex vivo PMGC co-culture system for evaluating heterogeneous responses to PD-1 blockade and associated baseline differences in alternative immune checkpoint ligand expression among PD-L1-high organoids.
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Roles of Metastasis Associated Fibroblasts in Paclitaxel Resistance of Brain Metastatic Tumor Cells: Evaluation in Metastatic Organoids.
BACKGROUND: Some breast cancer patients experience the devastating effects of brain metastases. The metastasis-associated fibroblasts (MAFs), the most abundant cells in the metastatic microenvironment, have an important role in chemoresistance. The MAFs' roles in paclitaxel resistance of brain metastatic tumor cells were evaluated in metastatic organoids. METHODS: Brain metastatic cells, known as 4T1B, were isolated and expanded from the brains of cancerous mice. Alginate-based metastatic organoids were prepared from the culture of 4T1B and 3T3 fibroblasts in an alginate solution. Cytotoxic activity of paclitaxel against 4T1B in 3D (3-Dimensional) culture was evaluated by MTT. For molecular analysis, real-time PCR was done. RESULTS: Our results proved that in brain metastatic organoids, fibroblasts cause a significant increase in paclitaxel resistance. Molecular analysis showed that the expression level of α-smooth muscle actin (α-SMA) increased in MAF compared to normal fibroblasts. Surprisingly, the expression level of fibroblast-specific protein 1 (FSP-1) decreased compared to normal fibroblasts. CONCLUSION: Here, we reveal the pivotal roles of MAFs in chemoresistance and discuss their molecular properties. Our approach involves exploring various therapeutic techniques to comprehend MAFs and presenting therapeutic perspectives for metastatic cancer treatment.