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Measuring Microglial Suppression with Multiple Assays

5 Introduction Many neurodegenerative diseases display chronic inflammation in the central nervous system (CNS). Microglia, the primary resident immune cell in the brain, activate rapidly when injured and release proinflammatory cytokines that contribute to CNS damage. Mesenchymal stromal cells (MSCs) have been widely tested as a cellular therapy for neuroinflammatory conditions due to their immunomodulatory properties. However, MSCs exhibit variable characteristics because of differences in donors, manufacturing methods, etc. It is important to establish a reliable way to determine MSC effectiveness in suppressing microglial activation. Objective We aimed to develop multiple assays to study how human umbilical cord tissue-derived MSCs inhibit the activation of microglia. We compared assays using organotypic cerebellar slice culture and primary microglial culture to look for an effective and high throughput way to screen for MSC lines that suppress microglia activation. Methods For slice cultures, 12 hours of lysophosphatidylcholine (LPC) treatment induced extensive microglia activation. After washing off of LPC, 25 000 MSCs were added. Five hours after the MSC treatment, the microglia morphology changes were measured using confocal microscopy. For the primary culture assay, cells were plated in 24-well microplate and cultured for 2 days before treating with lipopolysaccharide (LPS) for 4 hours. MSCs were added for 24 hours. The supernatant was then collected, and ELISA was performed to detect the release of mouse interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα). Results In slice culture, LPC treatment resulted in microglia activation, demonstrated by a shift from ramified to amoeboid microglia. MSC addition rescued microglia morphology back to a calm, ramified resting stage. Sholl analysis (Figure 1A) showed the number of microglia projections after MSC treatment increased compared with the LPC group. For primary microglia cultures, MSCs inhibited the production of mouse IL-6 by 63% and mTNFα by 77% (Figure 1B). Discussion Both cerebellar slice culture assay and primary microglia culture assay are effective in showing the inhibitory effects of MSCs on activated microglia. The primary microglia culture assay is less time-consuming, which makes it feasible for large scale screening. The primary microglia cells can be characterized and stored in freezers, which are ready to be used to screen for MSC lines at later time points. Figure 1 (A): Sholl analysis shows MSC treatment suppresses microglia activation. The number of projections in the MSC treatment group is comparable to the control group and significantly increased compared with the LPC group. (B): MSC suppress microglia activation in the primary microglia assay. MSCs reduce the release of mouse IL6 by 63% and mouse TNFα by 77% after LPS stimulation. STEM CELLS TRANSLATIONAL MEDICINE | StemCellsTM.com © AlphaMed Press 2020 PRECLINICAL STUDIES