# Coincident Fluorescence‐Burst Analysis of Actin Cargo Molecules in Secreted Single Diffusing Extracellular Vesicles From Human Induced Pluripotent Stem Cells

## TL;DR

A new method tracks actin molecules in extracellular vesicles to study how cells communicate and change during neurodegeneration.

## Contribution

A novel two-color fluorescence platform quantifies actin cargo in extracellular vesicles from human stem cells under different conditions.

## Key findings

- Actin green fluorescent protein burst frequency increases in EVs from partially differentiated cells compared to undifferentiated ones.
- FCCS analysis confirms enhanced actin packaging into EVs under partially differentiated conditions.
- The platform quantifies EV size and actin cargo number, linking to neurodegenerative biogenesis patterns.

## Abstract

Extracellular vesicles (EVs) mediate cellular communication via cargoes of nucleic acids, proteins, and miRNAs, and play key roles in neurodegeneration. However, quantification of specific cargo changes in EVs is challenging due to their small size and heterogeneity. Here, we develop a two‐color fluorescence cross‐correlation spectroscopy (FCCS) and coincident‐burst analysis platform to quantify actin‐enhanced green fluorescent protein and micro red fluorescent protein dual‐labeled EVs secreted from human induced pluripotent stem cells in undifferentiated, partially differentiated, and amyloid beta‐treated conditions. First, a two‐color time trace with pulsed interleaved excitation reveals a markedly increased actin green fluorescent protein burst frequency in EVs derived from partially differentiated cells compared with undifferentiated ones, suggesting altered secretion dynamics under stress. Second, FCCS analysis directly confirms the loading yield elevation based on coincident‐burst analysis of single EVs derived under a partially differentiated condition, indicating enhanced packaging of actin cargo into EVs. Third, the proposed methodology directly quantifies the EV size and the number of actin molecules carried to serve as biogenesis patterns linked to neurodegenerative pathology. Altogether, our platform offers a quantitative, highly specific tool to monitor cytoskeletal disruption via EVs and uncover molecular changes in neuronal differentiation, which is critical for developing therapies for neurological disorders.

This study presents a two‐color FCCS and coincident‐burst analysis platform to quantify actin cargo in dual‐labeled EVs secreted from hiPSCs. The approach tracks secretion dynamics, confirms actin loading, and measures EV size and cargo number, offering insights into cytoskeletal disruption and neuronal differentiation relevant to neurodegenerative disease mechanisms.