Brain spheroids or organoids derived from human pluripotent stem cells (hiPSCs) are still not capable of completely recapitulating in vivo human brain tissue, and one of the limitations is lack of microglia. To add built-in immune function, coculture of the dorsal forebrain spheroids with isogenic microglia-like cells (D-MG) was performed in our study. The three-dimensional D-MG spheroids were analyzed for their transcriptome and compared with isogenic microglia-like cells (MG). Cortical spheroids containing microglia-like cells displayed different metabolic programming, which may affect the associated phenotype. The expression of genes related to glycolysis and hypoxia signaling was increased in cocultured D-MG spheroids, indicating the metabolic shift to aerobic glycolysis, which is in favor of M1 polarization of microglia-like cells. In addition, the metabolic pathways and the signaling pathways involved in cell proliferation, cell death, PIK3/AKT/mTOR signaling, eukaryotic initiation factor 2 pathway, and Wnt and Notch pathways were analyzed. The results demonstrate the activation of mTOR and p53 signaling, increased expression of Notch ligands, and the repression of NF- κ B and canonical Wnt pathways, as well as the lower expression of cell cycle genes in the cocultured D-MG spheroids. This analysis indicates that physiological 3-D microenvironment may reshape the immunity of in vitro cortical spheroids and better recapitulate in vivo brain tissue function for disease modeling and drug screening.
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Differential Effects of Extracellular Vesicles of Lineage-Specific Human Pluripotent Stem Cells on the Cellular Behaviors of Isogenic Cortical Spheroids
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.
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- Award ID(s):
- 1652992
- NSF-PAR ID:
- 10113235
- Date Published:
- Journal Name:
- Cells
- Volume:
- 8
- Issue:
- 9
- ISSN:
- 2073-4409
- Page Range / eLocation ID:
- 993
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Extracellular vesicles (EVs) secreted by human‐induced pluripotent stem cells (hiPSCs) have great potential as cell‐free therapies in various diseases, including prevention of blood–brain barrier senescence and stroke. However, there are still challenges in pre‐clinical and clinical use of hiPSC‐EVs due to the need for large‐scale production of a large quantity. Vertical‐Wheel bioreactors (VWBRs) have design features that allow the biomanufacturing of hiPSC‐EVs using a scalable aggregate or microcarrier‐based culture system under low shear stress. 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 compared. The hiPSCs were characterized by metabolite and transcriptome analysis as well as EV biogenesis markers. Protein and microRNA cargo were analysed by proteomics and microRNA‐seq, respectively. The
in vitro functional assays of microglia stimulation and proliferation were conducted. HiPSCs expanded as 3‐D aggregates and on microcarriers had comparable cell number, while microcarrier culture had higher glucose consumption, higher glycolysis and lower autophagy gene expression based on mRNA‐seq. 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 a smaller EV size than other groups, and the cargo was enriched with proteins (proteomics) and miRNAs (microRNA‐seq) reducing apoptosis and promoting cell proliferation (e.g. Wnt‐related pathways). hiPSC‐EVs demonstrated the ability of stimulating proliferation and M2 polarization of microgliain vitro . HiPSC expansion on microcarriers produces much higher yields of EVs than hiPSC aggregates in VWBRs. EV collection in mTeSR increases yield compared to HBM. The biomanufactured EVs from microcarrier culture in mTeSR have exosomal characteristics and are functional in microglia stimulation, which paves the ways for future in vivo anti‐aging study. -
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.more » « less
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Cardiomyocytes (CMs) are heart cells responsible for heart contraction and relaxation. CMs can be derived from human induced pluripotent stem cells (hiPSCs) with high yield and purity. Mature CMs can potentially replace dead and dysfunctional cardiac tissue and be used for screening cardiac drugs and toxins. However, hiPSCs-derived CMs (hiPSC-CMs) are immature, which limits their utilization. Therefore, it is crucial to understand how experimental variables, especially tunable ones, of hiPSC expansion and differentiation phases affect the hiPSC-CM maturity stage. This study applied clustering algorithms to day 30 cardiac differentiation data to investigate if any maturity-related cell features could be related to the experimental variables. The best models were obtained using k-means and Gaussian mixture model clustering algorithms based on the evaluation metrics. They grouped the cells based on eccentricity and elongation. The cosine similarity between the clustering results and the experimental parameters revealed that the Gaussian mixture model results have strong similarities of 0.88, 0.94, and 0.93 with axial ratio, diameter, and cell concentration.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/cells8090993
