A Robust Methodological Framework for Generating Whole‐Brain and Cortical Organoids From Diverse Healthy‐ and Patient‐Derived Induced Pluripotent Stem Cell Lines

BackgroundPatient‐derived neural organoids (NOs) have emerged as powerful tools for modeling human neurodevelopmental disorders, especially when animal models are unavailable or fail to recapitulate human‐specific cortical development. However, significant variability in differentiation potential, even among healthy donor lines, and especially in fragile patient‐derived induced pluripotent stem cells (iPSCs), poses major methodological challenges. Protocols that succeed in one line may fail in others, leading to poor organoid formation, reduced growth, impaired neuroepithelial patterning, or complete failure to generate neural tissues.MethodsBy systematically comparing multiple published protocols, we identified key sources of variability and ultimately developed optimized protocols for generating both whole‐brain (WB) and cortical organoids (CO) from human iPSCs (hiPSCs), including lines from progeroid Cockayne syndrome patients.ResultsThrough iterative refinement of critical parameters, including cell seeding density, Matrigel incorporation, and timing and type of pathway inhibition, we achieved consistent organoid growth and structural organization across all tested lines. The resulting pipeline is adaptable and can be further tailored for newly derived or particularly challenging hiPSC lines.ConclusionCollectively, this methodological framework enables robust and reproducible generation of NOs from genetically diverse hiPSC sources, providing a reliable platform for studying human neurodevelopment and disease mechanisms in progeroid and other patient‐specific contexts.