Human Forebrain Organoid-Derived Extracellular Vesicle Labeling with Iron Oxides for In Vitro Magnetic Resonance Imaging

Abstract

The significant roles of extracellular vesicles (EVs) as intracellular mediators, disease biomarkers, and therapeutic agents, make them a scientific hotspot. In particular, EVs secreted by human stem cells show significance in treating neurological disorders, such as Alzheimer’s disease and ischemic stroke. However, the clinical applications of EVs are limited due to their poor targeting capabilities and low therapeutic efficacies after intravenous administration. Superparamagnetic iron oxide (SPIO) nanoparticles are biocompatible and have been shown to improve the targeting ability of EVs. In particular, ultrasmall SPIO (USPIO, <50 nm) are more suitable for labeling nanoscale EVs due to their small size. In this study, induced forebrain neural progenitor cortical organoids (iNPCo) were differentiated from human induced pluripotent stem cells (iPSCs), and the iNPCo expressed FOXG1, Nkx2.1, α-catenin, as well as β-tubulin III. EVs were isolated from iNPCo media, then loaded with USPIOs by sonication. Size and concentration of EV particles were measured by nanoparticle tracking analysis, and no significant changes were observed in size distribution before and after sonication, but the concentration decreased after labeling. miR-21 and miR-133b decreased after sonication. Magnetic resonance imaging (MRI) demonstrated contrast visualized for the USPIO labeled EVs embedded in agarose gel phantoms. Upon calculation, USPIO labeled EVs exhibited considerably shorter relaxation times, quantified as T2 and T2* values, reducing the signal intensity and generating higher MRI contrast compared to unlabeled EVs and gel only. Our study demonstrated that USPIO labeling was a feasible approach for in vitro tracking of brain organoid-derived EVs, which paves the way for further in vivo examination.

Research progress in in vivo tracing technology for extracellular vesicles

“…The donor cancer cells were subjected to treatment with tetraacetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz), and the resulting azide groups were labeled using near-infrared fluorescent dye-conjugated dibenzylcyclooctyne (DBCO-Cy5). Subsequently, non-invasive tracking and imaging of Cy5-Exo were conducted through nearinfrared fluorescence (NIRF) imaging in mice with tumors [41] .…”

Extracellular vesicle biogenesis of three‐dimensional human pluripotent stem cells in a novel Vertical‐Wheel bioreactor

“…It was also observed that some therapeutically relevant protein and microRNA cargo were promoted in the EVs produced in the VWBRs (Jeske et al., 2023). Our previous studies also have isolated and characterized hPSC‐derived EVs, including undifferentiated hPSCs, forebrain organoids, cerebellar organoids, choroid plexus organoids and retinal organoids, mostly in static culture (Arthur et al., 2023; Hua et al., 2022; Liu et al., 2022; Marzano et al., 2019, 2021; Muok et al., 2023). Given the potential needs for animal and clinical studies, the biomanufacturing of hPSC‐EVs in a scalable bioreactor such as VWBRs has become important.…”

Inflammatory Response and Exosome Biogenesis of Choroid Plexus Organoids Derived from Human Pluripotent Stem Cells

“…Our previous studies evaluated the effects of Wnt signaling in modulating YAP and Notch activities, as well as the microRNAs that modulate Wnt signaling in cortical and cerebellar organoids patterning from hiPSCs [19][20][21][22][23]. In this study, the inflammatory response to Aβ42 oligomer stimulation was evaluated for the ChP organoids derived from hiPSCs in the presence or absence of CHIR.…”