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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This content will become publicly available on December 1, 2024
Space microgravity increases expression of genes associated with proliferation and differentiation in human cardiac spheres
Efficient generation of cardiomyocytes from human-induced pluripotent stem cells (hiPSCs) is important for their application in basic and translational studies. Space microgravity can significantly change cell activities and function. Previously, we reported upregulation of genes associated with cardiac proliferation in cardiac progenitors derived from hiPSCs that were exposed to space microgravity for 3 days. Here we investigated the effect of long-term exposure of hiPSC-cardiac progenitors to space microgravity on global gene expression. Cryopreserved 3D hiPSC-cardiac progenitors were sent to the International Space Station (ISS) and cultured for 3 weeks under ISS microgravity and ISS 1 G conditions. RNA-sequencing analyses revealed upregulation of genes associated with cardiac differentiation, proliferation, and cardiac structure/function and downregulation of genes associated with extracellular matrix regulation in the ISS microgravity cultures compared with the ISS 1 G cultures. Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes mapping identified the upregulation of biological processes, molecular function, cellular components, and pathways associated with cell cycle, cardiac differentiation, and cardiac function. Taking together, these results suggest that space microgravity has a beneficial effect on the differentiation and growth of cardiac progenitors.
more » « less- Award ID(s):
- 1926387
- NSF-PAR ID:
- 10478844
- Publisher / Repository:
- Springer Nature
- Date Published:
- Journal Name:
- npj Microgravity
- Volume:
- 9
- Issue:
- 1
- ISSN:
- 2373-8065
- 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. -
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Abstract A systematic study on the biological effects of simulated microgravity (sµg) on human pluripotent stem cells (hPSC) is still lacking. Here, we used a fast-rotating 2-D clinostat to investigate the sµg effect on proliferation, self-renewal, and cell cycle regulation of hPSCs. We observed significant upregulation of protein translation of pluripotent transcription factors in hPSC cultured in sµg compared to cells cultured in 1g conditions. In addition to a significant increase in expression of telomere elongation genes. Differentiation experiments showed that hPSC cultured in sµg condition were less susceptible to differentiation compared to cells in 1g conditions. These results suggest that sµg enhances hPSC self-renewal. Our study revealed that sµg enhanced the cell proliferation of hPSCs by regulating the expression of cell cycle-associated kinases. RNA-seq analysis indicated that in sµg condition the expression of differentiation and development pathways are downregulated, while multiple components of the ubiquitin proteasome system are upregulated, contributing to an enhanced self-renewal of hPSCs. These effects of sµg were not replicated in human fibroblasts. Taken together, our results highlight pathways and mechanisms in hPSCs vulnerable to microgravity that imposes significant impacts on human health and performance, physiology, and cellular and molecular processes.
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During mammalian development, the left and right ventricles arise from early populations of cardiac progenitors known as the first and second heart fields, respectively. While these populations have been extensively studied in non-human model systems, their identification and study in vivo human tissues have been limited due to the ethical and technical limitations of accessing gastrulation-stage human embryos. Human-induced pluripotent stem cells (hiPSCs) present an exciting alternative for modeling early human embryogenesis due to their well-established ability to differentiate into all embryonic germ layers. Here, we describe the development of a TBX5/MYL2 lineage tracing reporter system that allows for the identification of FHF- progenitors and their descendants including left ventricular cardiomyocytes. Furthermore, using single-cell RNA sequencing (scRNA-seq) with oligonucleotide-based sample multiplexing, we extensively profiled differentiating hiPSCs across 12 timepoints in two independent iPSC lines. Surprisingly, our reporter system and scRNA-seq analysis revealed a predominance of FHF differentiation using the small molecule Wnt-based 2D differentiation protocol. We compared this data with existing murine and 3D cardiac organoid scRNA-seq data and confirmed the dominance of left ventricular cardiomyocytes (>90%) in our hiPSC-derived progeny. Together, our work provides the scientific community with a powerful new genetic lineage tracing approach as well as a single-cell transcriptomic atlas of hiPSCs undergoing cardiac differentiation.more » « less
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Abstract Background Fetal alcohol syndrome (FAS) due to gestational alcohol exposure represents one of the most common causes of nonheritable lifelong disability worldwide. In vitro and in vivo models have successfully recapitulated multiple facets of the disorder, including morphological and behavioral deficits, but far less is understood regarding the molecular and genetic mechanisms underlying FAS.
Methods In this study, we utilized an in vitro human pluripotent stem cell‐based (hPSC) model of corticogenesis to probe the effects of early, chronic intermittent alcohol exposure on the transcriptome of first trimester‐equivalent cortical neurons.
Results We used RNA sequencing of developing hPSC‐derived neurons treated for 50 days with 50 mM ethanol and identified a relatively small number of biological pathways significantly altered by alcohol exposure. These included cell‐type specification, axon guidance, synaptic function, and regional patterning, with a notable upregulation of WNT signaling‐associated transcripts observed in alcohol‐exposed cultures relative to alcohol‐naïve controls. Importantly, this effect paralleled a shift in gene expression of transcripts associated with regional patterning, such that caudal forebrain‐related transcripts were upregulated at the expense of more anterior ones. Results from H9 embryonic stem cells were largely replicated in an induced pluripotent stem cell line (IMR90‐4), indicating that these patterning alterations are not cell line‐specific.
Conclusions We found that a major effect of chronic intermittent alcohol on the developing cerebral cortex is an overall imbalance in regionalization, with enrichment of gene expression related to the production of posterodorsal progenitors and a diminution of anteroventral progenitors. This finding parallels behavioral and morphological phenotypes observed in animal models of high‐dose prenatal alcohol exposure, as well as patients with FAS.
