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Cross-site reproducibility of human cortical organoids reveals consistent cell type composition and architecture.
While guided human cortical organoid (hCO) protocols reproducibly generate cortical cell types at one site, variability in hCO phenotypes across sites using a harmonized protocol has not yet been evaluated. To determine the cross-site reproducibility of hCO differentiation, three independent research groups assayed hCOs in multiple differentiation replicates from one induced pluripotent stem cell (iPSC) line using a harmonized miniaturized spinning bioreactor protocol across 3 months. hCOs were mostly cortical progenitor and neuronal cell types in reproducible proportions that were consistently organized in cortical wall-like buds. Cross-site differences were detected in hCO size and expression of metabolism and cellular stress genes. Variability in hCO phenotypes correlated with stem cell gene expression prior to differentiation and technical factors associated with seeding, suggesting iPSC quality and treatment are important for differentiation outcomes. Cross-site reproducibility of hCO cell type proportions and organization encourages future prospective meta-analytic studies modeling neurodevelopmental disorders in hCOs.
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Time-Lapse Acquisition of Both Freely Secreted Proteome and Exosome Encapsulated Proteome in Live Organoids' Microenvironment.
Proteomic communications in neighboring microenvironments during early organ development is a dynamic process that continuously reshapes human embryonic stem cells (hESCs) developmental fate. Such dynamic proteomic alteration in the microenvironment consists of both freely secreted proteome and exosome-encapsulated proteome. Simultaneous monitoring of the time-lapse shift of both proteomes with live organoids remains technically challenging. Here, a continuous organoid secretion/encapsulation proteome tandem LC-MS/MS (COSEP-LCM) is introduced, which permits time-lapse monitoring of proteomic alterations both in free secretion form and in exosome encapsulated form at live organoids' microenvironment. Continuous growth of human cerebral organoids (COs) and free-secretion/exosome-encapsulation proteomics acquisition with COSEP-LCM for 60 days is demonstrated. SERPINF1, F5, and EFNB1 are initially enriched inside exosomes as encapsulated excretion and then gradually enriched outside exosomes as freely secreted excretion, while C3 is initially enriched outside exosomes as freely secreted excretion and gradually enriched inside exosomes as encapsulated excretion. Such dynamic excretion pattern paradigm shift may imply critical developmental strategy evolution during early human cerebral development. COSEP-LCM offers a platform technique for continuous inside/outside exosome proteomics co-analysis in live organoids' microenvironment.
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Interleukin-6 is an activator of pituitary stem cells upon local damage, a competence quenched in the aging gland.
Stem cells in the adult pituitary are quiescent yet show acute activation upon tissue injury. The molecular mechanisms underlying this reaction are completely unknown. We applied single-cell transcriptomics to start unraveling the acute pituitary stem cell activation process as occurring upon targeted endocrine cell-ablation damage. This stem cell reaction was contrasted with the aging (middle-aged) pituitary, known to have lost damage-repair capacity. Stem cells in the aging pituitary show regressed proliferative activation upon injury and diminished in vitro organoid formation. Single-cell RNA sequencing uncovered interleukin-6 (IL-6) as being up-regulated upon damage, however only in young but not aging pituitary. Administering IL-6 to young mice promptly triggered pituitary stem cell proliferation, while blocking IL-6 or associated signaling pathways inhibited such reaction to damage. By contrast, IL-6 did not generate a pituitary stem cell activation response in aging mice, coinciding with elevated basal IL-6 levels and raised inflammatory state in the aging gland (inflammaging). Intriguingly, in vitro stem cell activation by IL-6 was discerned in organoid culture not only from young but also from aging pituitary, indicating that the aging gland's stem cells retain intrinsic activatability in vivo, likely impeded by the prevailing inflammatory tissue milieu. Importantly, IL-6 supplementation strongly enhanced the growth capability of pituitary stem cell organoids, thereby expanding their potential as an experimental model. Our study identifies IL-6 as a pituitary stem cell activator upon local damage, a competence quenched at aging, concomitant with raised IL-6/inflammatory levels in the older gland. These insights may open the way to interfering with pituitary aging.
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In vitro derivation of midbrain dopaminergic neurons from porcine embryonic stem cells in multi-dimensional conditions.
BACKGROUND: . Porcine pluripotent stem cells hold significant promise as a large-animal model for human neurodevelopment and disease modeling. However, efficient protocols for their directed differentiation into midbrain dopaminergic (mDA) neurons and the generation of 3D midbrain-like organoids remain limited. This study aimed to establish species-optimized conditions for the derivation of functional mDA neurons from porcine embryonic stem cells (pESCs) under both two- and three-dimensional environments. METHODS: . In vitro fertilization (IVF)- and parthenogenetic activation (PA)-derived pESCs were subjected to neural induction using stepwise exposure to dual SMAD inhibition, SHH, CHIR99021, and FGF8. For monolayer culture, adherent monolayers were differentiated on Matrigel- or poly-L-ornithine/laminin I/fibronectin-coated surfaces. For 3D culture, porcine midbrain-like organoids (pMLOs) were formed under low-adhesion conditions. Functional and molecular characterization was performed via immunofluorescence, patch-clamp electrophysiology, microelectrode array (MEA) recordings, dopamine ELISA, and RNA sequencing. RESULTS: . Porcine embryonic stem cells required higher SHH and GSK3 inhibition thresholds to efficiently induce FOXA2⁺ ventral midbrain progenitors, revealing a species-specific divergence from human protocols. IVF-derived pESCs showed markedly enhanced dopaminergic differentiation and functional maturation compared to PA-derived pESCs. Notably, in 3D culture, neuroepithelial structures emerged as early as day 5, and functionally mature mDA neurons-confirmed by TH expression, dopamine release, and spontaneous synaptic activity-were detected by day 28. This timeline represents an accelerated maturation relative to comparable human mDA differentiation systems, where functional properties typically arise after day 35. Single-cell RNA sequencing further delineated dynamic dopaminergic lineage trajectories and revealed porcine-specific gene expression patterns associated with early mDA identity acquisition. CONCLUSIONS: . This study presents the first robust platform for generating functionally validated mDA neurons and midbrain-like organoids from porcine stem cells. The findings highlight species-specific signaling dynamics and establish porcine in vitro models as scalable, translational systems for investigating neurodevelopment and disease mechanisms.
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Shared effects of DISC1 disruption and elevated WNT signaling in human cerebral organoids.
The development of three-dimensional culture methods has allowed for the study of developing cortical morphology in human cells. This provides a new tool to study the neurodevelopmental consequences of disease-associated mutations. Here, we study the effects of isogenic DISC1 mutation in cerebral organoids. DISC1 has been implicated in psychiatric disease based on genetic studies, including its interruption by a balanced translocation that increases the risk of major mental illness. Isogenic wild-type and DISC1-disrupted human-induced pluripotent stem cells were used to generate cerebral organoids, which were then examined for morphology and gene expression. We show that DISC1-mutant cerebral organoids display disorganized structural morphology and impaired proliferation, which is phenocopied by WNT agonism and rescued by WNT antagonism. Furthermore, there are many shared changes in gene expression with DISC1 disruption and WNT agonism, including in neural progenitor and cell fate markers, regulators of neuronal migration, and interneuron markers. These shared gene expression changes suggest mechanisms for the observed morphologic dysregulation with DISC1 disruption and points to new avenues for future studies. The shared changes in three-dimensional cerebral organoid morphology and gene expression with DISC1 interruption and WNT agonism further strengthens the link between DISC1 mutation, abnormalities in WNT signaling, and neuropsychiatric disease.
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Human cerebellar organoids with functional Purkinje cells.
Research on human cerebellar development and disease has been hampered by the need for a human cell-based system that recapitulates the human cerebellum's cellular diversity and functional features. Here, we report a human organoid model (human cerebellar organoids [hCerOs]) capable of developing the complex cellular diversity of the fetal cerebellum, including a human-specific rhombic lip progenitor population that have never been generated in vitro prior to this study. 2-month-old hCerOs form distinct cytoarchitectural features, including laminar organized layering, and create functional connections between inhibitory and excitatory neurons that display coordinated network activity. Long-term culture of hCerOs allows healthy survival and maturation of Purkinje cells that display molecular and electrophysiological hallmarks of their in vivo counterparts, addressing a long-standing challenge in the field. This study therefore provides a physiologically relevant, all-human model system to elucidate the cell-type-specific mechanisms governing cerebellar development and disease.
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[Technical characteristics of Alzheimer model based on organ technology (organoid)].
Alzheimer's disease (AD) is a serious neurodegenerative disorder that manifests itself as progressive damage to memory and knowledge and is the main cause of dementia in the elderly. AD is characterized by extracellular deposition of amyloid-β plate (Aβ) and by the formation of neurofibrillary tangles, composed of hyperphosphorylated Tau protein. These modifications lead to neuronal cell death, vascular dysfunction and inflammatory disorders. Described as "elderly disease", AD is an escalating threat to developed countries as life expectancy is increasing. Because of its severity, AD has been the subject of extensive studies that address the pathogenesis of the disease. However, its main cause remains unknown. Most research on neurological conditions has been applied to animal models. However, due to their high cost and the uncertain translation of their results to humans along with moral concerns, in recent years, there has been a growing need for in vitro modeling to mimic the brain. The creation of the aforementioned models aims at a better understanding of the factors contributing to the onset of the disease and the faster development of the treatment of diseases affecting the nervous system. Given this need, in this review, new approaches to study neurodegenerative disease were recorded. A three-dimensional (3D) neurosphere-based microfluid chip has been reported and this model imitates the in vivo microenvironment of the brain and provides a steady flow of fluid that is observed in the brain's space. Uniform neurospheres, with cell interactions and contacts in all directions, were formed in a hollow microfuge and a steady interstitial flow rate was maintained using a small pump osmotic system. In this model it was possible to control the toxic effects of amyloid-β. At the end, it was observed that the deposition of amyloid-β through an osmotic micro-pump significantly reduced the viability of the neurospheres and caused destruction of the neuronal networks. Therefore, this model was proposed as an in vitro brain model for neurodegenerative disease and high-throughput drugs.
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Enhanced cortical neural stem cell identity through short SMAD and WNT inhibition in human cerebral organoids facilitates emergence of outer radial glial cells.
Cerebral organoids exhibit broad regional heterogeneity accompanied by limited cortical cellular diversity despite the tremendous upsurge in derivation methods, suggesting inadequate patterning of early neural stem cells (NSCs). Here we show that a short and early Dual SMAD and WNT inhibition course is necessary and sufficient to establish robust and lasting cortical organoid NSC identity, efficiently suppressing non-cortical NSC fates, while other widely used methods are inconsistent in their cortical NSC-specification capacity. Accordingly, this method selectively enriches for outer radial glia NSCs, which cyto-architecturally demarcate well-defined outer sub-ventricular-like regions propagating from superiorly radially organized, apical cortical rosette NSCs. Finally, this method culminates in the emergence of molecularly distinct deep and upper cortical layer neurons, and reliably uncovers cortex-specific microcephaly defects. Thus, a short SMAD and WNT inhibition is critical for establishing a rich cortical cell repertoire that enables mirroring of fundamental molecular and cyto-architectural features of cortical development and meaningful disease modelling.
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iPEX enables micrometre-resolution deep spatial proteomics via tissue expansion.
The number of spatial omics technologies being developed is increasing1. However, a missing tool is one that can locate proteins in tissues in an untargeted manner at high spatial resolution and coverage. Here we present in situ imaging proteomics via expansion (iPEX), which integrates isotropic tissue magnification2 with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging. iPEX provides scalable spatial resolution down to the micrometre scale and substantially increases the sensitivity of protein identification by 10-100-fold. Using the retina as a model, iPEX enabled the construction of spatial proteomic maps with high precision, the visualization of single-cell layers and extrasomatic structures and the identification of colocalized proteins. iPEX was readily applied to diverse tissues, including brain, intestine, liver and organoids, detecting 600-1,500 proteins at 1-5-µm effective pixel size. The application of iPEX to depict spatial proteomic maps in brains of mice with 5xFAD Alzheimer's disease revealed an early-onset mitochondrial aberrancy. Notably, in young mice, the peroxisomal acetyl-CoA acyltransferase ACAA1A-of which the N392S mutant is a monogenic risk factor in Alzheimer's disease3-was downregulated. ACAA1 depletion blocked the biosynthesis of long-chain polyunsaturated fatty acids, including docosahexaenoic acid, in multiple cellular contexts. These lipidome alterations were restored in cells overexpressing wild-type ACAA1 but not ACAA1(N392S), which suggests that the dysregulation of long-chain polyunsaturated fatty acids has an early role in neurodegeneration. Together, these results demonstrate that iPEX facilitates untargeted spatial proteomics at micrometre resolution for diverse applications.
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PTEN variant and genetic backgrounds combine to modify cerebellar neuronal differentiation in autism spectrum disorder.
Mutations in the PTEN gene have been implicated in autism spectrum disorders (ASD), particularly among individuals with comorbid macrocephaly. In our previous study, we demonstrated that the PTEN p.Ile135Leu variant, in an ASD-related genetic background dependent fashion, disrupts both cortical neurogenesis and gliogenesis. While abnormal cerebellar development is a recognized feature of ASD, the specific cellular targets and timing of disruptions during cerebellar differentiation and development remain poorly understood. To investigate these aspects, we applied our previously established cerebellar organoid protocol and used isogenic human iPSC lines harboring this PTEN-variant. We examined the expression of Purkinje cells, granule cells, interneurons, and glial cells prior to 22 weeks of differentiation, assessed genes expression at 8 weeks, and evaluated spontaneous spikes activity in Purkinje cells after 11 weeks. We observed that cell-type-specific expression patterns differed between the PTEN p.Ile135Leu variant in control versus ASD-genetic backgrounds. However, these background differences were diminished in PTEN knockout lines across both backgrounds. Our single-cell RNA sequencing (scRNA-seq) dataset revealed that the PTEN p.Ile135Leu variant increased the number of interneuron progenitor cells, whereas PTEN knockout led to an expansion of meningeal-like cells in both genetic contexts. Moreover, both the PTEN p.Ile135Leu variant and PTEN knockout abolished spontaneous simple spikes activity in Purkinje cells across both backgrounds, including PTEN-corrected patient-derived lines. Together, these findings provide direct evidence linking PTEN dysfunction and genetic background to altered cerebellar differentiation and neuronal network activity in human cerebellar organoids.