Complementary 2D and 3D cultures deriving from a single human cortical differentiation process recapitulate in vitro neurodevelopmental and neurogenerative features of juvenile Huntington’s Disease

<title>Abstract</title> <p>The development of reliable human-based models that accurately recapitulate the pathophysiology of neurological disorders remains a major challenge in translational research. Human induced pluripotent stem cells (iPSCs) have emerged as a powerful tool to overcome the limitations of animal models, enabling the generation of disease-relevant cell types. While both two-dimensional (2D) neuronal cultures and three-dimensional (3D) brain organoids provide valuable insights, they rely on distinct differentiation protocols and present complementary advantages and limitations in terms of complexity, reproducibility, and experimental accessibility. Here, we describe an integrated differentiation strategy that combines sequential 2D and 3D culture steps to generate, from a single iPSC batch, both cortical neuronal monolayers and cortical organoids with comparable maturation and regional specification in terms of both neuronal and cortical markers expression. This approach recapitulates key neurodevelopmental stages, including embryoid bodies, neural epithelium, and neural spheres, and does not require extracellular matrix scaffolding or specialized equipment such as bioreactors. By unifying the differentiation process, our method reduces variability, experimental time, and resource consumption, while enabling the simultaneous production of two complementary in vitro platforms. To validate this system, we employed iPSC lines derived from healthy donors and a juvenile Huntington’s disease (jHD) patient carrying 85 CAG repeats. Both 2D and 3D cultures recapitulated disease-associated phenotypes, revealing alterations in the neural epithelium structure, cortical markers expression and layering, electrophysiological maturation and mutant Huntingtin aggregates, consistent with known neurodevelopmental defects and unfolding novel relevant pathological features. Comprehensive molecular, cellular, and functional characterization confirmed the robustness and translational relevance of this platform. Overall, our study provides a versatile and scalable cortical modeling framework that bridges the gap between monolayer cultures and organoids, offering a powerful tool for investigating neurodevelopmental and neurodegenerative disorders and for advancing drug discovery applications.</p>