Communications Between Adipose-Derived Mesenchymal Stem Cells (AdMSCs) and Retinal Pigment Epithelial Cell Line (RPE-1) in Different Stress Environments via Tunneling Nanotubes

Abstract

Purpose : Paracrine supportive effects of MSCs are thought to be at least partially accomplished by TNTs which allow direct intercellular communication and cargo transfer. This study aims to demonstrates formation of TNTs between AdMSCs and RPE-1 and their alterations in response to experimental stress conditions.

Methods : Serum starvation and oxidative stress were used as stress conditions to induce TNTs. Texas Red™-X Phalloidin for binding F-actin of tunneling tubes, anti β-tubulin for cell structure, DAPI for nuclei were used for immunofluorosence staining. Scanning electron microscopy (SEM) imaging determined the average thickness of TNTs. CTG (CellTiter-Glo®) assay determined cell survival due to TNTs. Reactive oxygen species (ROS) quantification within the cells group was performed. Fluo-4AM staining was used to determine Ca²⁺ concentrations.

Results : Fluorescence microscopy revealed that TNTs, which are not anchored to the substratum, regularly connect between cultured RPE-1 and AdMSCs in stress environments and contain F-actin but no microtubules. SEM showed the ultrastructure of TNTs were straight connections between cells with a diameter ranging from 35 to 200 nm and a length of up to 120 mm. We observed that RPE-1 have lower cell viability when seeded under stress without AdMSCs than in the group co-cultured (p=0.0015). Decreased total ROS production in RPE-1 under stress conditions was observed with coculture (p=0.017) and it suggested that TNT-mediated mitochondrial transport may have occurred. In hypoxic condition, TNTs was determined more widely than in serum starvation (p=0.0001). Ca²⁺ imaging has been shown that calcium signals are transmitted by intercellular communication via TNTs and increase in stress environments (p=0.0001).

Conclusions : Our observations demostrated the signal trafficking via TNTs between RPE-1 and AdMSCs. This communication may support the ability to survive in response to stress conditions. These results can provide a new perspective on stem cell-based therapy in ophthalmologic diseases that may help develop a new strategy to manipulate direct signal, organelle, or molecule transfer for retinal pigment epithelial damage.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Fluorescence images represent the TNTs structures between the AdMSCs and RPE-1