DEVELOPMENT OF CELLULAR ENERGY METABOLISM DURING <EM >IN VITRO</EM> DIFFERENTIATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS INTO CORTICAL NEURONS

Neuronal differentiation requires extensive metabolic remodeling to support increasing energy and biosynthetic demands. Here, we present an integrated multi-omics and functional characterization of metabolic remodeling during early differentiation of human induced pluripotent stem cells (iPSCs) into excitatory cortical neurons using doxycycline-inducible overexpression of neurogenin-2 (NGN2). Parental iPSCs (day 0) and induced neurons at days 7 and 14 of differentiation were analyzed using a comprehensive methodological approach combining gene expression profiling, label-free quantitative proteomics, high-resolution respirometry, fluorescence lifetime imaging microscopy (FLIM), and ¹³C₆-glucose metabolic flux analysis. Neuronal differentiation was associated with enhanced oxidative phosphorylation, increased mitochondrial content, and respiratory capacity, with proteomics identifying the first week of differentiation as a critical window of coordinated metabolic specialization. FLIM revealed a gradual increase in enzyme-bound NAD(P)H, consistent with a shift toward oxidative metabolism. ¹³C₆-glucose tracing showed delayed labeling of the intracellular pool of fully labeled glucose and tricarboxylic acid cycle intermediates, accompanied by enhanced labeling of pentose phosphate pathway metabolites and glutathione, indicating redirection of glucose toward biosynthetic and antioxidant pathways. Nonetheless, differentiated neurons retained substantial glycolytic flux, highlighting metabolic flexibility during maturation. Together, these findings establish NGN2-iPSC-derived cortical neurons as a versatile and well-characterized platform for studying human early developmental bioenergetic remodeling. This metabolic baseline provides a foundation for future studies linking patient-specific genetic variants to metabolic alterations in disease-relevant contexts, with a focus on epilepsy and oncogenic processes. Supported by the Ministry of Health of the Czech Republic in cooperation with the Czech Health Research Council under project NU23-08-00460.