(247g) Engineering Extracellular Vesicles Secreted By Human Brain Organoids with Different Regional Identity

Extracellular vesicles (EVs) including the small size of subpopulation exosomes (30-150 nm) are membrane-bound vesicles that are secreted by all cell types. In particular, EVs secreted by human stem cells show significance in intercellular communications and high potential in therapeutic outcome (e.g., immunomodulation, angiogenesis, and neuroprotection) in treating neurological disorders. Most studies have been focusing on generating EVs from tissue or monolayer culture. In this study, EVs were isolated from the spent media of 3D brain organoids differentiated from human induced pluripotent stem cells (hiPSCs). In particular, a new type of EV subpopulation, matrix-bound nanovesicles (MBVs), were isolated from decellularized extracellular matrix (ECM) of brain organoids to compare with media EVs (MEVs). The hiPSC-derived forebrain cortical organoids (iFCo) were differentiated for 15 days, and then MEVs/MBVs were harvested on day 17. The hiPSC-derived hindbrain cerebellar organoids (iHCo) were differentiated for 35 days using two different protocols, and MEVs/MBVs were harvested on day 15 and during week 5-6. Nanoparticle tracking analysis showed that iFCo MBVs were larger than MEVs and their mode sizes were between 100-150 nm. iHCo MEVs and MBVs had size range of 130-140 nm. When normalizing to cell numbers, iFCo and iHCo generated one order magnitude more MBVs than MEVs. When normalizing to proteins, the MBVs showed much higher value than MEVs. Transmission electron microscopy images captured the typical cup shape morphology of exosomes for both MEVs and MBVs. Western blot revealed that MBVs lacked some of detectable EV markers such as syntenin-1, while MEVs expressed all the positive markers. In an immunomodulation assay, isogenic microglia were stimulated by Aβ42 oligomers with the addition of MEVs and MBVs derived from iFCo at different doses based on protein content. The EV-0.2 condition showed reduced mRNA expression of the TNFα and IL-6. For the MBV groups, the MBV-1 condition enhanced the expression of IL-10 and CD163. The ELISA analysis of IL-6 protein demonstrated that EV-0.2 condition had the lowest concentration, consistent with the mRNA result. The MBV group showed dose-depended effect on IL-6: the more MBVs added, the less IL-6 was observed. miRNA cargo analysis showed that 7 out of 8 miRNAs were more abundant in iFCo MBV than iHCo MBV. Most of ESCRT-dependent and independent EV biogenesis markers were comparable at day 35 for the two differentiation protocols. This study has the significance in advancing our understanding of the communication of EVs with the human brain tissue and in the design of cell-free based therapy for treating neurological disorders such as Alzheimer’s disease and ischemic stroke.