(K-422) Cell-Dependent Response of Human Astrocytes and Neural Stem Cells to On-Demand Generated Reactive Oxygen Species

Friday, October 13, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row K - Poster # 422

Presenting Author(s)

KH

Katherine Hoang

Undergraduate Student Researcher
Trinity University
San Antonio, United States

Co-Author(s)

CL

Cora Lewis

Undergraduate Student Researcher
Trinity University, United States
AL

Austin Lee

Undergraduate Student Researcher
Trinity University, United States
MP

Mikayla Potts

Undergraduate Student Researcher
Trinity University, United States

Primary Investigator(s)

DM

Dany Munoz-Pinto

Primary Investigator
Trinity University, United States

Co-Author(s)

AD

Ana Maria Diaz Lasprilla

Researcher
Trinity University, United States
AJ

Andrea Jimenez-Vergar

Researcher
Trinity Univerisity, United States

Introduction:: Reactive oxygen species (ROS) are highly reactive molecules that play a critical role in inflammatory cell-signaling and metabolism. Oxidative stress due to ROS production is one of the key contributors to aging and neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Multiple in vitro studies have explored the use of hydrogen peroxide to generate ROS and to model their effect of cellular responses. However, controlling the ROS levels accurately and in temporal fashion in this type of model is challenging. In this work, we used a photoreactive molecule to generate free radicals and ROS species upon controlled exposure to low energy UV radiation. The use of a photoreactive system offers the opportunity to tightly control ROS levels on demand. The differential response of human astrocytes (hAs) and human neural stem cells (iHNSCs) to the ROS levels was evaluated at the gene and protein expression levels. This work will contribute to the basic understanding of the onset and propagation of inflammatory signaling and the potential role the selected Central Nervous System (CNS) cell types may play.

Materials and Methods:: In this research, we used two commercially available CNS cell types: hAs and iHNSCs provided by Celprogen and Cell Applications respectively. Each cell type was cultured following the instruction and culture protocols from each cell provider. Once expanded, cells were seeded onto cell culture well plates and exposed to 365 nm UV radiation at 6 mW/cm2 intensity in the presence of 1 mM photoreactive species for 0.0 min and 5 min. Following the UV radiation, cells were incubated for 24 hours, and protein and mRNA were extracted using the Dynabeads mRNA direct kit. We examined the changes in gene expression of both hAs and iHNSCs exposed to UV light by conducting qRT-PCR analyses targeting the senescence marker, P21, the inflammatory markers IL-1b, IL-6, MMP1, and TNFα and the reactive markers, GFAP, GLAS and Vimentin using β-Actin as housekeeping gene.

Results, Conclusions, and Discussions:: Preliminary qRT-PCR results revealed that the 5 min UV exposure time had limited effects on the response of hAs. Differences in mean values for the relative expression of the selected markers were not statistically different. In contrast, the relative expression of reactive and inflammatory markers in iHNSCs appears to be enhanced by the exposure of cells to free radicals and ROS (Figure 1) for up to 5 min exposure to longwave UV radiation. Results also indicate that the current platform may be a helpful tool to contribute to the understanding of ROS activity in CNS cells and that exposure to ROS molecules can be tightly regulated by the use of UV radiation. Future studies will be focused on complimenting the qRT-PCR result with protein analyses and the evaluation of paracrine signaling in a co-culture system.

Acknowledgements (Optional): :

This work was supported by a research grant from the National Science Foundation (NSF-2138684)

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