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Differential Effects of Extracellular Vesicles of Lineage-Specific Human Pluripotent Stem Cells on the Cellular Behaviors of Isogenic Cortical Spheroids.

ABSTRACT: Extracellular vesicles (EVs) contribute to a variety of signaling processes and the overall physiological and pathological states of stem cells and tissues. Human induced pluripotent stem cells (hiPSCs) have unique characteristics that can mimic embryonic tissue development. There is growing interest in the use of EVs derived from hiPSCs as therapeutics, biomarkers, and drug delivery vehicles. However, little is known about the characteristics of EVs secreted by hiPSCs and paracrine signaling during tissue morphogenesis and lineage specification. Methods: In this study, the physical and biological properties of EVs isolated from hiPSC-derived neural progenitors (ectoderm), hiPSC-derived cardiac cells (mesoderm), and the undifferentiated hiPSCs (healthy iPSK3 and Alzheimer's-associated SY-UBH lines) were analyzed. Results: Nanoparticle tracking analysis and electron microscopy results indicate that hiPSC-derived EVs have an average size of 100-250 nm. Immunoblot analyses confirmed the enrichment of exosomal markers Alix, CD63, TSG101, and Hsc70 in the purified EV preparations. MicroRNAs including miR-133, miR-155, miR-221, and miR-34a were differently expressed in the EVs isolated from distinct hiPSC lineages. Treatment of cortical spheroids with hiPSC-EVs in vitro resulted in enhanced cell proliferation (indicated by BrdU+ cells) and axonal growth (indicated by β-tubulin III staining). Furthermore, hiPSC-derived EVs exhibited neural protective abilities in Aβ42 oligomer-treated cultures, enhancing cell viability and reducing oxidative stress. Our results demonstrate that the paracrine signaling provided by tissue context-dependent EVs derived from hiPSCs elicit distinct responses to impact the physiological state of cortical spheroids. Overall, this study advances our understanding of cell‒cell communication in the stem cell microenvironment and provides possible therapeutic options for treating neural degeneration.

SUBMITTER: Marzano M

PROVIDER: S-EPMC6770916 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:Extracellular vesicles (EVs) secreted by human brain cells have great potential as cell-free therapies in various diseases, including stroke. However, because of the significant amount of EVs needed in preclinical and clinical trials, EV application is still challenging. Vertical-Wheel Bioreactors (VWBRs) have designed features that allow for scaling up the generation of human forebrain spheroid EVs under low shear stress. In this study, EV secretion by human forebrain spheroids derived from induced pluripotent stem cells as 3D aggregates and on Synthemax II microcarriers in VWBRs were investigated with static aggregate culture as a control. The spheroids were characterized by metabolite and transcriptome analysis. The isolated EVs were characterized by nanoparticle tracking analysis, electron microscopy, and Western blot. The EV cargo was analyzed using proteomics and miRNA sequencing. The in vitro functional assays of an oxygen and glucose-deprived stroke model were conducted. Proof of concept in vivo study was performed, too. Human forebrain spheroid differentiated on microcarriers showed a higher growth rate than 3D aggregates. Microcarrier culture had lower glucose consumption per million cells and lower glycolysis gene expression but higher EV biogenesis genes. EVs from the three culture conditions showed no differences in size, but the yields from high to low were microcarrier cultures, dynamic aggregates, and static aggregates. The cargo is enriched with proteins (proteomics) and miRNAs (miRNA-seq), promoting axon guidance, reducing apoptosis, scavenging reactive oxygen species, and regulating immune responses. Human forebrain spheroid EVs demonstrated the ability to improve recovery in an in vitro stroke model and in vivo. Human forebrain spheroid differentiation in VWBR significantly increased the EV yields (up to 240-750 fold) and EV biogenesis compared to static differentiation due to the dynamic microenvironment and metabolism change. The biomanufactured EVs from VWBRs have exosomal characteristics and more therapeutic cargo and are functional in in vitro assays, which paves the way for future in vivo stroke studies.

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Differential Effects of Extracellular Vesicles of Lineage-Specific Human Pluripotent Stem Cells on the Cellular Behaviors of Isogenic Cortical Spheroids.

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