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Optimized Pipeline for Personalized Neurobiological Insights from Single Patient-Derived Neurospheres

ABSTRACT: Quantitative proteomic approaches such as TMT labeling enable the amplification of biological material by combining multiple samples into a single analysis. This technique maximizes the data extracted from each sample and allows for precise comparative quantitative analysis between different experimental groups. In this study, we aimed to optimize the workflow for proteomic investigations using neurospheres derived from induced pluripotent stem cells (iPSCs). To improve both the efficiency and accuracy of our proteomic analysis, we compared different protocols and employed Tandem Mass Tag (TMT) labeling. Human neurospheres are three-dimensional, free-floating clusters primarily composed of neural progenitor cells, early-stage neurons, and radial glia. This diverse cell population more closely replicates the complexity of neural development compared to traditional 2D cultures, providing a more relevant model for studying neurogenesis, cell differentiation, and the underlying mechanisms of neurological diseases. Enhancing in vitro models through advanced cell culture techniques is crucial for reducing reliance on animal studies. In neural research, where access to human-relevant data is increasingly feasible, such improvements foster a deeper understanding of neural mechanisms and potential therapeutic targets. The use of 3D cultures represents a significant step forward in accurately modeling neural behavior and disease progression. By optimizing proteomic workflows and employing advanced labeling techniques, this study contributes to the development of more reliable and comprehensive in vitro neural models. This, in turn, supports the broader goal of advancing neural research while reducing reliance on animal models, leading to more ethical and potentially more accurate scientific outcomes that are clinically relevant to humans.

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ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture, Fibroblast

SUBMITTER: Guillaume Nugue

LAB HEAD: Magno Rodrigues Junqueira

PROVIDER: PXD057496 | Pride | 2026-05-19

REPOSITORIES: Pride

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Project description:Neural organoids are invaluable model systems for studying neurodevelopment and neurological diseases. For this purpose, reproducible differentiation protocols are needed that minimize inter-organoid variability whilst generating neural organoids that physiologically resemble the brain area of interest. Currently, two main approaches are used: guided, where the differentiation towards neuroectoderm and subsequently specific CNS regions is driven by applying extrinsic signalling molecules, and unguided, where the intrinsic capability of pluripotent stem cells to generate neuroectoderm without external signalling is promoted. Despite the importance for the field, the resulting differences between these models have not been directly investigated. To obtain an unbiased comparison, we performed a multi-omic analysis of forebrain organoids generated using a guided and unguided approach focusing on proteomic, lipidomic and metabolomic differences. Furthermore, we characterised differences in phosphorylation and sialylation states of proteins, two key post-translational modifications (PTMs) in neurodevelopment, and performed single cell transcriptomics (scRNAseq). The multi-omic analysis revealed considerable differences in neuronal-, synaptic and glial content, indicating that guided forebrain organoids contain a larger proportion of neurons, including GABAergic interneurons, and synapses whereas unguided organoids contain significantly more GFAP+ cells and choroid plexus. Furthermore, substantial differences in mitochondrial- and metabolic profiles were identified, pointing to increased levels of oxidative phosphorylation and fatty acid β-oxidation in unguided forebrain organoids and a higher reliance on glycolysis in guided forebrain organoids. Overall, our study comprises a thorough description of the multi-omic differences arising when generating guided and unguided forebrain organoids and provide an important resource for the organoid field studying neurodevelopment and -disease.

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Optimized Pipeline for Personalized Neurobiological Insights from Single Patient-Derived Neurospheres

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