Background: Human Mesenchymal Stromal Cells (hMSCs) are multi-potent cells that have are known to secrete therapeutic factors that can provide trophic support, immunomodulation, and vascular repair. However, like most cell therapies, using whole hMSCs comes with risks, such immune rejection and tumor formation. We wanted to address this challenge and find a way to improve hMSC therapies to make them safer and more effective. The therapeutic secreted products of hMSCs can be packaged into tiny particles called extracellular vesicles (EVs). We investigated the method of isolating EVs from hMSCs after transfecting them with mRNA, encoding for an anti-inflammatory compound called interleukin 10 (IL-10). Our goals were to determine if mRNA can be used for transient production of IL-10 by hMSCs, and to assess the IL-10 levels expressed freely and the amount being packaged into EVs from the genetically modified cells.
Hypothesis: We hypothesized that hMSCs transfected with IL-10 mRNA will produce more IL-10 free floating in the media and contained within the EVs than the GFP transfected cells.
Methods: To test this, hMSCs were cultured to 80-90% confluency and transfected with IL-10 mRNA (IL10-MSCs) or GFP mRNA (GFP-MSCs) using Lipofectamine for four hours. Cells were then incubated in complete media for one day, followed by serum-free conditioned media for two days. The conditioned media was collected and EVs were isolated using size exclusion chromatography. Nanoparticle tracking analysis was used to confirm successful EV isolation and the amount of IL-10 in whole media or EVs was quantified using ELISA.
Results: Our results confirmed we successfully isolated EVs from conditioned media with ~65% of EVs being in the exosome size range (50nm-150nm) and ~33% in the microvesicle range (100nm-1000nm). The average concentration of EVs was 5.8 x 109 particles/mL. We also demonstrated that IL-10 over secreting hMSCs produced significantly more IL-10 than GFP controls in whole media and showed a trend in increased levels in EVs. IL-10 was detected freely in whole media and in EVs; however, the majority was being secreted freely into the media.
Conclusion: In conclusion, we demonstrated that hMSCs can be transfected with IL-10 mRNA and produce IL-10 freely and also package it into EVs at greater amounts than the control. Future studies can focus on improving the loading efficiency of IL-10 into EVs, through more genetic modifications. The currently mRNA technology tested here is effect for enhancing hMSC cell therapies, with great potential to also be used for cell-free therapy that is safer, more targeted, and potentially more effective for treating many diseases and injuries.
