(592a) Exosome Biogenesis of Three-Dimensional Human Pluripotent Stem Cells in a Novel Vertical Wheel Bioreactor

Background: Extracellular vesicles (EVs) secreted by human induced pluripotent stem cells (hiPSCs) have great potential in cell-free based therapies in various diseases, such as preventing blood brain barrier senescence. EVs are membrane-covered nanoscale particles that carry therapeutic protein and miRNA cargo. However, there are still challenges in preclinical and clinical use of hiPSC-EVs due to the need for a large amount of EVs. The novel Vertical-Wheel bioreactors (VWBRs) have design features of low shear stress for hiPSC-EV production.

Methods: In this study, the EV secretion by undifferentiated hiPSCs expanded as 3-D aggregates and on Synthemax II microcarriers in VWBRs were investigated. Additionally, two types of EV collection media, mTeSR and HBM, were also compared. The expanded hiPSCs were characterized by metabolite and transcriptome analysis as well as EV biogenesis markers. The isolated hiPSC-EVs were characterized by nanoparticle tracking analysis, electron microscopy, and Western blot. Protein cargo was analyzed by proteomics and miRNA-sequencing was performed to identify miRNA cargo. The in vitro functional assays of microglia stimulation and proliferation analysis were conducted.

Results: hiPSCs expanded as 3-D aggregates and on microcarriers had comparable cell number, while microcarrier culture had higher glucose consumption, higher glycolysis gene expression and lower autophagy genes. The microcarrier cultures had at least 17-23 fold higher EV secretion, and EV collection in mTesR had 2.7-3.7 fold higher yield than HBM medium. Microcarrier culture with mTeSR EV collection had smaller EV size than other groups, and the cargo enriched with proteins and miRNAs reducing apoptosis and promoting cell proliferation (Wnt-related pathway). hiPSC-EVs demonstrated the ability of immuno-modulation and stimulating proliferation with in vitro microglia cultures.

Conclusions: hiPSC expansion on microcarriers produces much higher yields of EVs than hiPSC aggregates in VWBRs. EV collection in mTeSR has higher yield than HBR medium. The generated EVs have exosome characteristics and are functional in in vitro assays, which paves the ways for future in vivo study.

References:

[1] X. Yuan, L. Sun, R. Jeske, D. Nkosi, S. York, Y. Liu, S.C. Grant, D.G.J. Meckes, Y. Li, Engineering Extracellular Vesicles by Three-dimensional Dynamic Culture of Human Mesenchymal Stem Cells, Journal of Extracellular Vesicles, 11 (2022) e12235.

[2] R. Jeske, C. Liu, L. Duke, M.L. Canonicco Castro, L. Muok, P. Arthur, M. Singh, L. Sung, L. Sun, Y. Li, Upscaling Human Mesenchymal Stem Cell Production in a Novel Vertical Wheel Bioreactor Enhances Extracellular Vesicle Secretion and Cargo Profile, Bioactive Materials, 25 (2023) 732-747.

This project is supported by NIH R01NS125016.