Biogenesis of Extracellular Vesicles Produced from Human-Stem-Cell-Derived Cortical Spheroids Exposed to Iron Oxides

Abstract

Stem-cell-derived extracellular vesicles (EVs) are promising tools for therapeutic delivery and imaging in the medical research fields. EVs that arise from endosomal compartments or plasma membrane budding consist of exosomes and microvesicles, which range between 30 and 200 nm and 100–1000 nm, respectively. Iron oxide nanoparticles can be used to label stem cells or possibly EVs for magnetic resonance imaging. This could be a novel way to visualize areas in the body that are affected by neurological disorders such as stroke. Human induced pluripotent stem cells (iPSK3 cells) were plated on low-attachment plates and treated with SB431542 and LDN193189 during the first week for the induction of cortical spheroid formation and grown with fibroblast growth factor 2 and cyclopamine during the second week for the neural progenitor cell (iNPC) differentiation. iNPCs were then grown on attachment plates and treated with iron oxide (Fe3O4) nanoparticles at different sizes (8, 15, and 30 nm in diameter) and concentrations (0.1, 10, and 100 μM). The spheroids and media collected from these cultures were used for iron oxide detection as well as EV isolation and characterizations, respectively. MTT assay demonstrated that the increased size and concentration of the iron oxide nanoparticles had little effect on the metabolic activity of iNPCs. In addition, the Live/Dead assay showed high viability in all the nanoparticle treated groups and the untreated control. The EVs isolated from these culture groups were analyzed and displayed similar or higher EV counts compared with control. The observed EV size averaged 200–250 nm, and electron microscopy revealed the expected exosome morphology for EVs from all groups. RT-PCR analysis of EV biogenesis markers (CD63, CD81, Alix, TSG101, Syntenin1, ADAM10, RAB27b, and Syndecan) showed differential expression between the iron-oxide-treated cultures and nontreated cultures, as well as between adherent and nonadherent 3D cultures. Iron oxide nanoparticles were detected inside the cortical spheroid cells but not EVs by MRI. The addition of iron oxide nanoparticles does not induce significant cytotoxic effects to cortical spheroids. In addition,, nanoparticles may stimulate the biogenesis of EVs when added to cortical spheroids in vitro.

Nanomaterial Exposure, Extracellular Vesicle Biogenesis and Adverse Cellular Outcomes: A Scoping Review

“…In this ScR, we found that the variety of NMs exposed to the different biological models increased exosome secretion (see Figure 3 B), whereas no significant differences were observed regarding their size or morphology [ 26 , 39 , 40 , 41 , 44 , 45 , 46 , 49 , 51 ]. Many studies reported vesicles with a cup-shaped appearance in the negative-stain TEM with HSP 70, CD63, CD81, CD9, LAMP1, and TSG101 as characteristic markers, suggesting that the vesicular secretome that was isolated also comprised exosomes.…”

“…Regarding the physicochemical characteristics of NMs, 78% of studies used nanoparticles of different chemical compositions as test substances (e.g., metallic, polymeric, metallic oxides, inorganic, etc.) [ 24 , 40 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 ]. The remaining studies used nanotubes, nanoclusters, nanosheets, as well as dendrimers [ 26 , 49 , 50 , 51 ].…”

“…No article dealt with isolated apoptotic bodies, and in one article, isolated microvesicles were investigated. It is important to note that the term EV was also part of the nomenclature of isolated vesicles in different studies [ 20 , 43 , 46 , 50 ].…”

“…Only eight studies reported analysis regarding EV size, with studies referring to ‘exosome’ isolation reporting mean diameters ranging from 30–200 nm while EVs generally range from 60–1000 nm. Compared with the control, six studies reported no significant differences in EV size in response to NM exposure [ 26 , 39 , 41 , 45 , 49 , 51 ], while one study observed a decrease in EV size [ 46 ], and one study reported an increase in EV size [ 25 ]. Interestingly, the impact of NMs on EV biogenesis is evident.…”

“…Interestingly, the impact of NMs on EV biogenesis is evident. Among the eighteen articles, fourteen studies showed that NMs increased EV secretion, especially for studies specifically referring to exosomes [ 20 , 24 , 26 , 40 , 41 , 42 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 ]. Twelve studies explored EV-enriched biomarkers (with CD81, CD63, CD9, and TSG101 being the predominantly analyzed markers), while three studies analyzed additional non-EV markers (GAPDH, Calnexin, and Calreticulin) [ 40 , 43 , 48 ].…”