# Molecular and functional characterization of GMP-manufactured neural stem cells and their extracellular vesicles for innovative therapeutic applications

## TL;DR

This study characterizes human neural stem cells and their extracellular vesicles under GMP conditions, showing their potential for regenerative medicine and anti-inflammatory therapies.

## Contribution

The study provides a detailed molecular and functional characterization of GMP-manufactured hNSCs and their EVs for therapeutic applications.

## Key findings

- hNSCs showed stable growth and expressed neural stem cell markers.
- hNSC-EVs reduced inflammation in microglial and monocytic cells.
- hNSC-EVs have potential as alternatives or enhancers to cell-based therapies.

## Abstract

Human neural stem cells (hNSCs) are promising candidates for regenerative medicine due to their self-renewal capacity, differentiation potential, and ability to modulate inflammation. However, several reports showed that the regenerative properties of stem cells are tied to the extracellular vesicles (EVs) they secrete. This study aimed at characterizing hNSCs produced under Good Manufacturing Practice (GMP) conditions and at elucidating the molecular and functional properties of their secreted extracellular vesicles (hNSC-EVs). hNSCs were first assessed for proliferation, and differentiation potential, showing a stable growth profile and expression of neural stem cell markers. High-resolution proteomic analysis identified over 5000 proteins, with about 40% overlap with previous NSCs studies. hNSCs expressed mostly markers for different cell lineage precursors. The molecular characterization of hNSC-derived EVs (hNSC-EVs) showed a size distribution, as measured by nanoparticle tracking analysis, ranging from 140 to 200 nm and an enrichment in EV markers, detected by western blotting. Functional analyses showed that hNSC-EVs, reduce nitric oxide generation and inducible nitric oxide expression in LPS-treated microglial cells and inhibit caspase-1 activation in monocytic cell models through uptake-dependent and independent mechanism, respectively. Our findings show that hNSC possess a strong stemness signature and secrete EVs with immunomodulatory properties, suggesting the worth of hNSC-EVs as either alternative to cell-based therapies or primer to boost anti-inflammatory properties of hNSCs in the treatment of neurological disorders.

The online version contains supplementary material available at 10.1186/s13287-026-04904-x.