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

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 993 ◽  
Author(s):  
Mark Marzano ◽  
Julie Bejoy ◽  
Mujeeb R. Cheerathodi ◽  
Li Sun ◽  
Sara B. York ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Vesicles ◽  
Pluripotent Stem Cells ◽  
Physiological State ◽  
Cell Communication ◽  
Biological Properties ◽  
Cardiac Cells ◽  
Paracrine Signaling ◽  
Average Size

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.

scholarly journals Very Long-Chain C24:1 Ceramide Is Increased in Serum Extracellular Vesicles with Aging and Can Induce Senescence in Bone-Derived Mesenchymal Stem Cells

Cells ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 37 ◽  
Author(s):  
Andrew Khayrullin ◽  
Priyanka Krishnan ◽  
Luis Martinez-Nater ◽  
Bharati Mendhe ◽  
Sadanand Fulzele ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Extracellular Vesicles ◽  
Cardiovascular Health ◽  
Rhesus Macaques ◽  
Cell Communication ◽  
Size Exclusion ◽  
Target Cells ◽  
Serum Samples

Extracellular vesicles (EVs), including exosomes and microvesicles, function in cell-to-cell communication through delivery of proteins, lipids and microRNAs to target cells via endocytosis and membrane fusion. These vesicles are enriched in ceramide, a sphingolipid associated with the promotion of cell senescence and apoptosis. We investigated the ceramide profile of serum exosomes from young (24–40 yrs.) and older (75–90 yrs.) women and young (6–10 yrs.) and older (25–30 yrs.) rhesus macaques to define the role of circulating ceramides in the aging process. EVs were isolated using size-exclusion chromatography. Proteomic analysis was used to validate known exosome markers from Exocarta and nanoparticle tracking analysis used to characterize particle size and concentration. Specific ceramide species were identified with lipidomic analysis. Results show a significant increase in the average amount of C24:1 ceramide in EVs from older women (15.4 pmol/sample) compared to those from younger women (3.8 pmol/sample). Results were similar in non-human primate serum samples with increased amounts of C24:1 ceramide (9.3 pmol/sample) in older monkeys compared to the younger monkeys (1.8 pmol/sample). In vitro studies showed that primary bone-derived mesenchymal stem cells (BMSCs) readily endocytose serum EVs, and serum EVs loaded with C24:1 ceramide can induce BMSC senescence. Elevated ceramide levels have been associated with poor cardiovascular health and memory impairment in older adults. Our data suggest that circulating EVs carrying C24:1 ceramide may contribute directly to cell non-autonomous aging.

scholarly journals From Human Pluripotent Stem Cells to 3D Cardiac Microtissues: Progress, Applications and Challenges

2020 ◽  
Vol 7 (3) ◽  
pp. 92
Author(s):  
Mariana A. Branco ◽  
Joaquim M.S. Cabral ◽  
Maria Margarida Diogo
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
High Throughput Screening ◽  
Cardiac Development ◽  
Human Pluripotent Stem Cells ◽  
Cardiac Cells ◽  
Cardiac Disorders ◽  
The Impact

The knowledge acquired throughout the years concerning the in vivo regulation of cardiac development has promoted the establishment of directed differentiation protocols to obtain cardiomyocytes (CMs) and other cardiac cells from human pluripotent stem cells (hPSCs), which play a crucial role in the function and homeostasis of the heart. Among other developments in the field, the transition from homogeneous cultures of CMs to more complex multicellular cardiac microtissues (MTs) has increased the potential of these models for studying cardiac disorders in vitro and for clinically relevant applications such as drug screening and cardiotoxicity tests. This review addresses the state of the art of the generation of different cardiac cells from hPSCs and the impact of transitioning CM differentiation from 2D culture to a 3D environment. Additionally, current methods that may be employed to generate 3D cardiac MTs are reviewed and, finally, the adoption of these models for in vitro applications and their adaptation to medium- to high-throughput screening settings are also highlighted.

Abstract 010: A Separable Upstream Promoter of the Slc8a1 (NCX1) Gene Efficiently Marks Cardiac Cell Populations Derived from Human Pluripotent Stem Cells

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Alejandro Hidalgo-Gonzalez ◽  
Dmitry A Ovchinnikov ◽  
James Hudson ◽  
Justin Cooper-White ◽  
Wolvetang Ernst
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Human Pluripotent Stem Cells ◽  
Cardiac Cells ◽  
Protein Quantification ◽  
Egfp Expression ◽  
Cardiac Cell

The sarcolemmal Na+/Ca2+ exchanger SLC8A1(NCX) regulates intracellular Ca+ in cardiomyocytes from early developmental stages. The upstream-most SLC8A1(NCX1) promoter is well conserved amongst the homoeothermic animals and contains putative binding sites for transcription factors of the NKX, GATA, STAT and CDX families. We hypothesized that functional cardiac cells with mature cardiac structural markers will express the sarcolemmal Na+/Ca2+ calcium antiporting channel, important for proper functional contractivity of in vitro differentiated human pluripotent stem cells. Pseudotyped lentiviral particles delivering NCX1cp-EGFP reporter cassette were used to confirm the efficiency and specificity of the reporter in rodent foetal cardiac cell isolates, and to establish stable human pluripotent stem cell lines. Cells were differentiated using a 2D induction protocol, and gene expression analysis and protein quantification carried at day 16. Initial NCX1cp-EGFP expression was observed from day 10-11 of cardiac differentiation. Beating foci were visualized 1-2 day after initial NCXCP-EGFP expression, reporter expression was confined to the grouped and individual beating cells, and highly correlated with the efficiency of spontaneously contractile cell production. At later stages, NCX1cp-EGFP expression correlated with clusters of formed spontaneously contractile units harbouring essentially all cardiomyocytes present in cultures, as evidenced by colocalization of high levels of cardiac troponin T (cTnT) and α-actinin proteins. The EGFP+ sorted fraction of differentiated cultures was found to be highly enriched in both early (ISL1, TBX5) and late (cTnT, MYH6) cardiomyocyte markers when compared to the EGFP- fraction. We conclude that a ~3 kb genomic fragment of the distal cardiac-specific promoter of the SLC8A1(NCX1) containing the upstream-most exon of the gene is sufficient to drive the expression of a lentiviral reporter in both rodent heart-derived primary and human (embryonic and induced) pluripotent stem cell-derived cardiac cells. Isolation of a homogenous and functional cardiomyogenic population represents one of the key objectives for cardiac tissue engineering, and in particular in vitro drug screening applications.

scholarly journals miR-212/132-Enriched Extracellular Vesicles Promote Differentiation of Induced Pluripotent Stem Cells Into Pancreatic Beta Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Chunyu Bai ◽  
Qiwei Ren ◽  
Haifeng Liu ◽  
Xiangchen Li ◽  
Weijun Guan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Beta Cell ◽  
Induced Pluripotent Stem Cells ◽  
Beta Cells ◽  
Extracellular Vesicles ◽  
Pluripotent Stem Cells ◽  
Pancreatic Beta Cells ◽  
Pancreatic Beta Cell ◽  
Induced Pluripotent

Pancreatic beta cell transplantation is the ideal method for treatment of type 1 diabetes mellitus (T1DM), and the generation of beta cells from induced pluripotent stem cells (iPSCs) of patients is a promising strategy. In this study, we improved a previous strategy to produce beta cells using extracellular vesicles (EVs) derived from mature beta cells and differentiated beta cells from iPSCs (i-Beta cells), which secreted insulin under glucose stimulation in vitro and ameliorated hyperglycemia in vivo. Mechanistic analyses revealed that EV-carried microRNA (miR)-212/132 (EV-miR-212/132) directly bound to the 3′ UTR of FBW7 to prevent its translation and FBW7 combined with NGN3 to accelerate its proteasomal degradation. EV-miR-212/132 stabilized NGN3 expression to promote differentiation of endocrine cells from induced iPSCs. Moreover, NGN3 bound to PDX1 to enhance transcription of endogenous miR-212/132 and formed a positive regulatory circuit that maintained the functions of mature pancreatic beta cells.ConclusionThis study describes a novel approach for beta cell production and supports the use of iPSCs for cell replacement therapy of T1DM.

scholarly journals Extracellular Vesicles from Skeletal Muscle Cells Efficiently Promote Myogenesis in Induced Pluripotent Stem Cells

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1527 ◽  
Author(s):  
Denisa Baci ◽  
Maila Chirivì ◽  
Valentina Pace ◽  
Fabio Maiullari ◽  
Marika Milan ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Extracellular Vesicles ◽  
Pluripotent Stem Cells ◽  
Myogenic Differentiation ◽  
Bioactive Molecules ◽  
Patient Specific ◽  
Induced Pluripotent

The recent advances, offered by cell therapy in the regenerative medicine field, offer a revolutionary potential for the development of innovative cures to restore compromised physiological functions or organs. Adult myogenic precursors, such as myoblasts or satellite cells, possess a marked regenerative capacity, but the exploitation of this potential still encounters significant challenges in clinical application, due to low rate of proliferation in vitro, as well as a reduced self-renewal capacity. In this scenario, induced pluripotent stem cells (iPSCs) can offer not only an inexhaustible source of cells for regenerative therapeutic approaches, but also a valuable alternative for in vitro modeling of patient-specific diseases. In this study we established a reliable protocol to induce the myogenic differentiation of iPSCs, generated from pericytes and fibroblasts, exploiting skeletal muscle-derived extracellular vesicles (EVs), in combination with chemically defined factors. This genetic integration-free approach generates functional skeletal myotubes maintaining the engraftment ability in vivo. Our results demonstrate evidence that EVs can act as biological “shuttles” to deliver specific bioactive molecules for a successful transgene-free differentiation offering new opportunities for disease modeling and regenerative approaches.

scholarly journals Toward an Understanding of Extracellular tRNA Biology

2021 ◽  
Vol 8 ◽  
Author(s):  
Adrian Gabriel Torres ◽  
Eulàlia Martí
Keyword(s):  
Extracellular Vesicles ◽  
Extracellular Space ◽  
Physiological State ◽  
Cell Communication ◽  
Signaling Molecules ◽  
Full Length ◽  
Target Cells ◽  
Paracrine Signaling ◽  
Trna Modifications ◽  
Promising Source

Extracellular RNAs (exRNAs) including abundant full length tRNAs and tRNA fragments (tRFs) have recently garnered attention as a promising source of biomarkers and a novel mediator in cell-to-cell communication in eukaryotes. Depending on the physiological state of cells, tRNAs/tRFs are released to the extracellular space either contained in extracellular vesicles (EVs) or free, through a mechanism that is largely unknown. In this perspective article, we propose that extracellular tRNAs (ex-tRNAs) and/or extracellular tRFs (ex-tRFs) are relevant paracrine signaling molecules whose activity depends on the mechanisms of release by source cells and capture by recipient cells. We speculate on how ex-tRNA/ex-tRFs orchestrate the effects in target cells, depending on the type of sequence and the mechanisms of uptake. We further propose that tRNA modifications may be playing important roles in ex-tRNA biology.

scholarly journals Human Induced Pluripotent Stem Cells Derived from a Cardiac Somatic Source: Insights for an In-Vitro Cardiomyocyte Platform

2020 ◽  
Vol 21 (2) ◽  
pp. 507
Author(s):  
Alessandra Maria Lodrini ◽  
Lucio Barile ◽  
Marcella Rocchetti ◽  
Claudia Altomare
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Somatic Cells ◽  
Cardiac Cells ◽  
Cardiac Differentiation ◽  
Human Ipscs ◽  
Wide Range ◽  
Induced Pluripotent

Reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) has revolutionized the complex scientific field of disease modelling and personalized therapy. Cardiac differentiation of human iPSCs into cardiomyocytes (hiPSC-CMs) has been used in a wide range of healthy and disease models by deriving CMs from different somatic cells. Unfortunately, hiPSC-CMs have to be improved because existing protocols are not completely able to obtain mature CMs recapitulating physiological properties of human adult cardiac cells. Therefore, improvements and advances able to standardize differentiation conditions are needed. Lately, evidences of an epigenetic memory retained by the somatic cells used for deriving hiPSC-CMs has led to evaluation of different somatic sources in order to obtain more mature hiPSC-derived CMs.

scholarly journals Mesenchymal and Induced Pluripotent Stem Cells-Derived Extracellular Vesicles: The New Frontier for Regenerative Medicine?

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1163 ◽  
Author(s):  
Maria Magdalena Barreca ◽  
Patrizia Cancemi ◽  
Fabiana Geraci
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Induced Pluripotent Stem Cells ◽  
Extracellular Vesicles ◽  
Pluripotent Stem Cells ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Cell Communication ◽  
Self Healing ◽  
Induced Pluripotent

Regenerative medicine aims to repair damaged, tissues or organs for the treatment of various diseases, which have been poorly managed with conventional drugs and medical procedures. To date, multimodal regenerative methods include transplant of healthy organs, tissues, or cells, body stimulation to activate a self-healing response in damaged tissues, as well as the combined use of cells and bio-degradable scaffold to obtain functional tissues. Certainly, stem cells are promising tools in regenerative medicine due to their ability to induce de novo tissue formation and/or promote organ repair and regeneration. Currently, several studies have shown that the beneficial stem cell effects, especially for mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) in damaged tissue restore are not dependent on their engraftment and differentiation on the injury site, but rather to their paracrine activity. It is now well known that paracrine action of stem cells is due to their ability to release extracellular vesicles (EVs). EVs play a fundamental role in cell-to-cell communication and are directly involved in tissue regeneration. In the present review, we tried to summarize the molecular mechanisms through which MSCs and iPSCs-derived EVs carry out their therapeutic action and their possible application for the treatment of several diseases.

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