# Development of Cellular Energy Metabolism During Differentiation of Human iPSCs into Cortical Neurons

## TL;DR

This study tracks how human stem cells change their energy use as they develop into brain neurons, showing increased reliance on mitochondria and antioxidant pathways.

## Contribution

The study provides the first detailed multi-omics characterization of metabolic changes during early human iPSC differentiation into cortical neurons.

## Key findings

- Differentiating neurons show increased mitochondrial function and oxidative phosphorylation.
- Metabolic flux analysis reveals a shift toward biosynthetic and antioxidant glucose utilization in neurons.
- Differentiated neurons maintain glycolytic activity, indicating metabolic flexibility.

## Abstract

Neuronal differentiation requires extensive metabolic remodeling to support increased energetic and biosynthetic demands. Here, we present an integrated multi-omics and functional characterization of metabolic transitions during early differentiation of human induced pluripotent stem cells (iPSCs) into excitatory cortical neurons using doxycycline-inducible overexpression of neurogenin-2 (NGN2). We analyzed parental iPSCs and induced neurons (iNs) at days 7 and 14 of differentiation, integrating gene expression profiling, label-free quantitative proteomics, high-resolution respirometry, fluorescence lifetime imaging microscopy (FLIM), and 13C₆-glucose metabolic flux analysis. Our data reveal progressive metabolic remodeling associated with neuronal maturation, including enhanced oxidative phosphorylation, increased mitochondrial content, and respiratory capacity. Proteomic analyses showed upregulation of mitochondrial and antioxidant pathways, while FLIM indicated a progressive increase in enzyme-bound NAD(P)H, consistent with a shift toward oxidative metabolism. Notably, 13C₆-glucose tracing revealed delayed labeling of the intracellular pool of fully labeled glucose and tricarboxylic acid cycle metabolites, together with enhanced labeling of pentose phosphate pathway intermediates and glutathione in iNs, indicating a shift toward biosynthetic and antioxidant glucose utilization during differentiation. Despite this enhancement in mitochondrial function, differentiated neurons maintained glycolytic activity, suggesting metabolic flexibility. Our results define the first week of differentiation as a critical window of metabolic specialization and establish NGN2-iPSC-derived cortical neurons as a versatile and well-characterized model system for investigating bioenergetic remodeling during early human neurodevelopment. It provides a robust foundation for mechanistic insights and high-throughput evaluation of metabolic pathways relevant to human disease.

The online version contains supplementary material available at 10.1007/s12035-025-05284-8.