Exosomes From Induced Pluripotent Stem Cell-Derived Cardiomyocytes Promote Autophagy for Myocardial Repair.
Journal of the American Heart Association
2020; 9 (6): e014345
CXCR2 Ligands and mTOR Activation Enhance Reprogramming of Human Somatic Cells to Pluripotent Stem Cells
STEM CELLS AND DEVELOPMENT
Background Induced pluripotent stem cells and their differentiated cardiomyocytes (iCMs) have tremendous potential as patient-specific therapy for ischemic cardiomyopathy following myocardial infarctions, but difficulties in viable transplantation limit clinical translation. Exosomes secreted from iCMs (iCM-Ex) can be robustly collected in vitro and injected in lieu of live iCMs as a cell-free therapy for myocardial infarction. Methods and Results iCM-Ex were precipitated from iCM supernatant and characterized by protein marker expression, nanoparticle tracking analysis, and functionalized nanogold transmission electron microscopy. iCM-Ex were then used in in vitro and in vivo models of ischemic injuries. Cardiac function in vivo was evaluated by left ventricular ejection fraction and myocardial viability measurements by magnetic resonance imaging. Cardioprotective mechanisms were studied by JC-1 (tetraethylbenzimidazolylcarbocyanine iodide) assay, immunohistochemistry, quantitative real-time polymerase chain reaction, transmission electron microscopy, and immunoblotting. iCM-Ex measured 140nm and expressed CD63 and CD9. iCM and iCM-Ex microRNA profiles had significant overlap, indicating that exosomal content was reflective of the parent cell. Mice treated with iCM-Ex demonstrated significant cardiac improvement post-myocardial infarction, with significantly reduced apoptosis and fibrosis. In vitro iCM apoptosis was significantly reduced by hypoxia and exosome biogenesis inhibition and restored by treatment with iCM-Ex or rapamycin. Autophagosome production and autophagy flux was upregulated in iCM-Ex groups in vivo and in vitro. Conclusions iCM-Ex improve post-myocardial infarction cardiac function by regulating autophagy in hypoxic cardiomyoytes, enabling a cell-free, patient-specific therapy for ischemic cardiomyopathy.
View details for DOI 10.1161/JAHA.119.014345
View details for PubMedID 32131688
Stem Cell-Derived Exosomes Protect Astrocyte Cultures From in vitro Ischemia and Decrease Injury as Post-stroke Intravenous Therapy
FRONTIERS IN CELLULAR NEUROSCIENCE
Exosomes Generated From iPSC-Derivatives New Direction for Stem Cell Therapy in Human Heart Diseases
2017; 120 (2): 407-417
Induced pluripotent stem cell (iPSC) technology has great promise in regenerative medicine and disease modeling. In this study, we show that human placenta-derived cell conditioned medium stimulates chemokine (C-X-C motif) receptor 2 (CXCR2) in human somatic cells ectopically expressing the pluripotency-associated transcription factors Oct4, Sox2, Klf4, and cMyc (OSKM), leading to mechanistic target of rapamycin (mTOR) activation. This causes an increase in endogenous cMYC levels and a decrease in autophagy, thereby enhancing the reprogramming efficiency of human somatic cells into iPSCs. These findings were reproduced when human somatic cells after OSKM transduction were cultured in a widely used reprogramming medium (mTeSR) supplemented with CXCR2 ligands interleukin-8 and growth-related oncogene α or an mTOR activator (MHY1485). To our knowledge, this is the first report demonstrating that mTOR activation in human somatic cells with ectopic OSKM expression significantly enhances the production of iPSCs. Our results support the development of convenient protocols for iPSC generation and further our understanding of somatic cell reprogramming.
View details for DOI 10.1089/scd.2019.0188
View details for Web of Science ID 000505721100001
View details for PubMedID 31808362
CXCR2 Inhibition in Human Pluripotent Stem Cells Induces Predominant Differentiation to Mesoderm and Endoderm Through Repression of mTOR, beta-Catenin, and hTERT Activities
STEM CELLS AND DEVELOPMENT
2016; 25 (13): 1006-1019
Cardiovascular disease (CVD) is the leading cause of death in modern society. The adult heart innately lacks the capacity to repair and regenerate the damaged myocardium from ischemic injury. Limited understanding of cardiac tissue repair process hampers the development of effective therapeutic solutions to treat CVD such as ischemic cardiomyopathy. In recent years, rapid emergence of induced pluripotent stem cells (iPSC) and iPSC-derived cardiomyocytes presents a valuable opportunity to replenish the functional cells to the heart. The therapeutic effects of iPSC-derived cells have been investigated in many preclinical studies. However, the underlying mechanisms of iPSC-derived cell therapy are still unclear, and limited engraftment of iPSC-derived cardiomyocytes is well known. One facet of their mechanism is the paracrine effect of the transplanted cells. Microvesicles such as exosomes secreted from the iPSC-derived cardiomyocytes exert protective effects by transferring the endogenous molecules to salvage the injured neighboring cells by regulating apoptosis, inflammation, fibrosis, and angiogenesis. In this review, we will focus on the current advances in the exosomes from iPSC derivatives and discuss their therapeutic potential in the treatment of CVD.
View details for DOI 10.1161/CIRCRESAHA.116.309307
View details for PubMedID 28104773
A strategy for culturing human pluripotent stem cells for translational research
Stem Cell & Translational Investigation
2016; 3: e1134
A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
On the basis of our previous report verifying that chemokine (C-X-C motif) receptor 2 (CXCR2) ligands in human placenta-derived cell conditioned medium (hPCCM) support human pluripotent stem cell (hPSC) propagation without exogenous basic fibroblast growth factor (bFGF), this study was designed to identify the effect of CXCR2 manipulation on the fate of hPSCs and the underlying mechanism, which had not been previously determined. We observed that CXCR2 inhibition in hPSCs induces predominant differentiation to mesoderm and endoderm with concomitant loss of hPSC characteristics and accompanying decreased expression of mammalian target of rapamycin (mTOR), β-catenin, and human telomerase reverse transcriptase (hTERT). These phenomena are recapitulated in hPSCs propagated in conventional culture conditions, including bFGF as well as those in hPCCM without exogenous bFGF, suggesting that the action of CXCR2 on hPSCs might not be associated with a bFGF-related mechanism. In addition, the specific CXCR2 ligand growth-related oncogene α (GROα) markedly increased the expression of ectodermal markers in differentiation-committed embryoid bodies derived from hPSCs. This finding suggests that CXCR2 inhibition in hPSCs prohibits the propagation of hPSCs and leads to predominant differentiation to mesoderm and endoderm owing to the blockage of ectodermal differentiation. Taken together, our results indicate that CXCR2 preferentially supports the maintenance of hPSC characteristics as well as facilitates ectodermal differentiation after the commitment to differentiation, and the mechanism might be associated with mTOR, β-catenin, and hTERT activities.
View details for DOI 10.1089/scd.2015.0395
View details for Web of Science ID 000378654300005
View details for PubMedID 27188501
CXCR2 and Its Related Ligands Play a Novel Role in Supporting the Pluripotency and Proliferation of Human Pluripotent Stem Cells
STEM CELLS AND DEVELOPMENT
2015; 24 (8): 948-961
The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.
View details for DOI 10.3791/53204
View details for Web of Science ID 000361537100051
View details for PubMedID 26275004
Mel-18, a mammalian Polycomb gene, regulates angiogenic gene expression of endothelial cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2010; 400 (4): 523-530
Basic fibroblast growth factor (bFGF) is a crucial factor sustaining human pluripotent stem cells (hPSCs). We designed this study to search the substitutive factors other than bFGF for the maintenance of hPSCs by using human placenta-derived conditioned medium without exogenous bFGF (hPCCM-), containing chemokine (C-X-C motif) receptor 2 (CXCR2) ligands, including interleukin (IL)-8 and growth-related oncogene α (GROα), which were developed on the basis of our previous studies. First, we confirmed that IL-8 and/or GROα play independent roles to preserve the phenotype of hPSCs. Then, we tried CXCR2 blockage of hPSCs in hPCCM- and verified the significant decrease of pluripotency-associated genes expression and the proliferation of hPSCs. Interestingly, CXCR2 suppression of hPSCs in mTeSR™1 containing exogenous bFGF decreased the proliferation of hPSCs while maintaining pluripotency characteristics. Lastly, we found that hPSCs proliferated robustly for more than 35 passages in hPCCM- on a gelatin substratum. Higher CXCR2 expression of hPSCs cultured in hPCCM- than those in mTeSR™1 was observable. Our findings suggest that CXCR2 and its related ligands might be novel factors comparable to bFGF supporting the characteristics of hPSCs and hPCCM- might be useful for the maintenance of hPSCs as well as for the accurate evaluation of CXCR2 role in hPSCs without the confounding influence of exogenous bFGF.
View details for DOI 10.1089/scd.2014.0381
View details for Web of Science ID 000352322200004
View details for PubMedID 25390768
Decreased Level and Defective Function of Circulating Endothelial Progenitor Cells in Children With Moyamoya Disease
JOURNAL OF NEUROSCIENCE RESEARCH
2010; 88 (3): 510-518
Mel-18 is a mammalian homolog of Polycomb group (PcG) genes. Microarray analysis revealed that Mel-18 expression was induced during endothelial progenitor cell (EPC) differentiation and correlates with the expression of EC-specific protein markers. Overexpression of Mel-18 promoted EPC differentiation and angiogenic activity of ECs. Accordingly, silencing Mel-18 inhibited EC migration and tube formation in vitro. Gene expression profiling showed that Mel-18 regulates angiogenic genes including kinase insert domain receptor (KDR), claudin 5, and angiopoietin-like 2. Our findings demonstrate, for the first time, that Mel-18 plays a significant role in the angiogenic function of ECs by regulating endothelial gene expression.
View details for DOI 10.1016/j.bbrc.2010.08.086
View details for Web of Science ID 000282850500013
View details for PubMedID 20801102
Multifarious proteomic signatures and regional heterogeneity in glioblastomas
JOURNAL OF NEURO-ONCOLOGY
2009; 94 (1): 31-39
Circulating endothelial progenitor cells (EPCs) play an important role in physiological and pathological neovascularization and may be involved in attenuating ischemic diseases. This study aimed to characterize circulating EPCs in moyamoya disease (MMD), one of the most common pediatric cerebrovascular diseases. Twenty-eight children with MMD prior to any surgical treatment and 12 healthy volunteers were recruited. Peripheral blood mononuclear cells (PBMNCs) were isolated and cultured in endothelial cell growth medium. Temporal change of phenotype of cells was analyzed on days 0 and 7. The formation of EPC clusters was evaluated on day 7. The CD34(+), CD133(+), and KDR(+) cells, and the number of EPC clusters was significantly reduced in children with MMD. In controls, CD34(+) cells were significantly decreased on day 7 compared with day 0, but in MMD they were only slightly decreased. The change in KDR(+) cells on day 7 compared with day 0 was the reverse of that for CD34(+) cells. Functional assay of EPC demonstrated less tube formation and increased senescent-like phenotype in children with MMD. Analysis of the circulating EPCs of MMD children reveals decreased level and defective function. This study suggests that circulating EPCs may be associated with MMD pathogenesis.
View details for DOI 10.1002/jnr.22228
View details for Web of Science ID 000274274700006
View details for PubMedID 19774676
Tissue Expression of Manganese Superoxide Dismutase Is a Candidate Prognostic Marker for Glioblastoma
2009; 77 (3-4): 178-181
To investigate the underlying intratumoral diversity of molecular profiles in glioblastomas, a proteomic approach was introduced to compare samples from regions of different histological grade. Using two-dimensional gel electrophoresis (2DE) with matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), we compared prospectively collected tissue samples of different histological grade areas of three glioblastoma patients. Consistent results showing relatively high expression of ubiquitin carboxyl-terminal esterase L1 in low-histological-grade areas (Grade 2 > Grades 3 and 4) and high expression of transthyretin in high-histological-grade areas (Grade 2 < Grades 3 and 4) were demonstrated. These results were confirmed by western blot (WB) analysis and immunohistochemical staining. This study provided the evidence of multifarious proteomic signatures according to regional and histological heterogeneity in glioblastomas.
View details for DOI 10.1007/s11060-009-9805-8
View details for Web of Science ID 000267683600003
View details for PubMedID 19219580
Investigation of molecular factors associated with malignant transformation of oligodendroglioma by proteomic study of a single case of rapid tumor progression
JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY
2008; 134 (2): 255-262
Characterization of a rare subgroup of glioblastoma patients who survive for more than 3 years (long-term survival glioblastoma, LTSGBL, patients) may be helpful to identify prognostic factors.A molecular-profiling proteomic approach using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to identify prognostic factors associated with glioblastoma by comparing frozen tumor tissue from LTSGBL patients with matched samples from short-term survival glioblastoma (STSGBL) patients. Western blot (WB) analysis, reverse-transcriptase polymerase chain reaction (RT-PCR) and immmunohistochemical (IHC) staining were used for confirmation.Among most candidate spots identified by 2-DE, lack of overexpression of manganese superoxide dismutase (MnSOD) in LTSGBL samples was consistently observed using WB and RT-PCR.These results suggest that MnSOD expression level in tumor tissue is a candidate marker for the prognosis of glioblastoma patients.
View details for DOI 10.1159/000231888
View details for Web of Science ID 000269573200005
View details for PubMedID 19641337
Frozen tumor tissues from a patient who showed rapid progression to anaplastic oligodendroglioma after near total resection of oligodendroglioma were used to examine differential expression of proteins to gain better understanding of the pathogenesis of malignant transformation.We have determined their protein profiles using a 2D gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry approach.Among 23 differentially expressed spots, overexpression of peroxiredoxin 6 and underexpression of rho GDP dissociation inhibitor alpha were confirmed to be valid after western blot and immunocytochemical analysis of oligodendroglioma tissue.Abnormal expression of peroxiredoxin 6 and rho GDP dissociation inhibitor alpha may be associated with malignant transformation in oligodendroglioma and these proteins might be candidates of molecular predictive factors.
View details for DOI 10.1007/s00432-007-0282-1
View details for Web of Science ID 000251487000017
View details for PubMedID 17653765