FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3003060822> ?p ?o ?g. }

• W3003060822 endingPage "1197" @default.
• W3003060822 startingPage "1186" @default.
• W3003060822 abstract "Stem cell-based therapy is one of the most attractive approaches to ischemic heart diseases, such as myocardial infarction (MI). We evaluated the cardio-protective effects of the human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) stably expressing lymphoid enhancer-binding factor 1 (LEF1; LEF1/hUCB-MSCs) in a rat model of MI. LEF1 overexpression in hUCB-MSCs promoted cell-proliferation and anti-apoptotic effects in hypoxic conditions. For the application of its therapeutic effects in vivo, the LEF1 gene was introduced into an adeno-associated virus integration site 1 (AAVS1) locus, known as a safe harbor site on chromosome 19 by CRISPR/Cas9-mediated gene integration in hUCB-MSCs. Transplantation of LEF1/hUCB-MSCs onto the infarction region in the rat model significantly improved overall survival. The cardio-protective effect of LEF1/hUCB-MSCs was proven by echocardiogram parameters, including greatly improved left-ventricle ejection fraction (EF) and fractional shortening (FS). Moreover, histology and immunohistochemistry successfully presented reduced MI region and fibrosis by LEF1/hUCB-MSCs. We found that these overall positive effects of LEF1/hUCB-MSCs are attributed by increased proliferation and survival of stem cells in oxidative stress conditions and by the secretion of various growth factors by LEF1. In conclusion, this study suggests that the stem cell-based therapy, conjugated with genome editing of transcription factor LEF1, which promotes cell survival, could be an effective therapeutic strategy for cardiovascular disease. Stem cell-based therapy is one of the most attractive approaches to ischemic heart diseases, such as myocardial infarction (MI). We evaluated the cardio-protective effects of the human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) stably expressing lymphoid enhancer-binding factor 1 (LEF1; LEF1/hUCB-MSCs) in a rat model of MI. LEF1 overexpression in hUCB-MSCs promoted cell-proliferation and anti-apoptotic effects in hypoxic conditions. For the application of its therapeutic effects in vivo, the LEF1 gene was introduced into an adeno-associated virus integration site 1 (AAVS1) locus, known as a safe harbor site on chromosome 19 by CRISPR/Cas9-mediated gene integration in hUCB-MSCs. Transplantation of LEF1/hUCB-MSCs onto the infarction region in the rat model significantly improved overall survival. The cardio-protective effect of LEF1/hUCB-MSCs was proven by echocardiogram parameters, including greatly improved left-ventricle ejection fraction (EF) and fractional shortening (FS). Moreover, histology and immunohistochemistry successfully presented reduced MI region and fibrosis by LEF1/hUCB-MSCs. We found that these overall positive effects of LEF1/hUCB-MSCs are attributed by increased proliferation and survival of stem cells in oxidative stress conditions and by the secretion of various growth factors by LEF1. In conclusion, this study suggests that the stem cell-based therapy, conjugated with genome editing of transcription factor LEF1, which promotes cell survival, could be an effective therapeutic strategy for cardiovascular disease. Myocardial infarction (MI) is the most common coronary heart disease, which in turn, is the leading cause of morbidity and mortality worldwide.1GBD 2016 Causes of Death CollaboratorsGlobal, regional, and national age-sex specific mortality for 264 causes of death, 1980–016: a systematic analysis for the Global Burden of Disease Study 2016.Lancet. 2017; 390: 1151-1210Abstract Full Text Full Text PDF PubMed Scopus (2101) Google Scholar,2Jessup M. Brozena S. Heart failure.N. Engl. J. Med. 2003; 348: 2007-2018Crossref PubMed Scopus (1535) Google Scholar The main cause of the disease is an obstruction of the coronary artery that leads to a massive loss of cardiomyocytes, resulting in myocardial dysfunction and heart failure.3Neri M. Riezzo I. Pascale N. Pomara C. Turillazzi E. Ischemia/Reperfusion Injury following Acute Myocardial Infarction: A Critical Issue for Clinicians and Forensic Pathologists.Mediators Inflamm. 2017; 2017: 7018393Crossref PubMed Scopus (132) Google Scholar The primary therapy should be the restoration of lost cardiomyocytes, but there is a clear limitation to the regenerative capacity of the adult mammalian heart, meaning that additional therapeutic approaches are mandatory. Stem cell therapy has received constant attention as a potential strategy for the regeneration of infarcted hearts, and human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) are regarded as a promising candidate for this therapy due to its unique properties, such as multiple lineage potential, lack of teratoma formation, easy expansion, and low immunogenicity.4Nagamura-Inoue T. He H. Umbilical cord-derived mesenchymal stem cells: Their advantages and potential clinical utility.World J. Stem Cells. 2014; 6: 195-202Crossref PubMed Google Scholar Various trials have treated the injured myocardium using diverse MSC populations, including hUCB-MSCs. Some of these studies showed restoration of damaged cardiomyocytes, enhancement of cardiac function, and to certain degrees, reduced infarct size.5Amado L.C. Saliaris A.P. Schuleri K.H. St John M. Xie J.S. Cattaneo S. Durand D.J. Fitton T. Kuang J.Q. Stewart G. et al.Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction.Proc. Natl. Acad. Sci. USA. 2005; 102: 11474-11479Crossref PubMed Scopus (890) Google Scholar, 6Tang Y.L. Zhao Q. Qin X. Shen L. Cheng L. Ge J. Phillips M.I. Paracrine action enhances the effects of autologous mesenchymal stem cell transplantation on vascular regeneration in rat model of myocardial infarction.Ann. Thorac. Surg. 2005; 80 (discussion 236–237): 229-236Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 7Cai M. Shen R. Song L. Lu M. Wang J. Zhao S. Tang Y. Meng X. Li Z. He Z.X. Bone Marrow Mesenchymal Stem Cells (BM-MSCs) Improve Heart Function in Swine Myocardial Infarction Model through Paracrine Effects.Sci. Rep. 2016; 6: 28250Crossref PubMed Scopus (72) Google Scholar Although many studies have attempted to utilize hUCB-MSCs and provided positive outcomes in preclinical trials, there are still some hurdles to be surmounted, such as low graft and survival rates in the hostile microenvironment of the infarcted region with its insufficient supply of oxygen and nutrients. Therefore, to enhance hUCB-MSCs’ functionality, additional strategies, such as cell patch and genome editing of the hUCB-MSCs, are required to improve cell-survival rate and paracrine effects. There have been a few trials with different target genes that obtained the expected outcomes and addressed these issues. Our recent study reported that hUCB-MSCs overexpressing the vascular endothelial growth factor (VEGF) gene controlled by doxycycline (Dox) induction successfully improved cardiac function via the increase of angiogenesis in MI.8Cho H.M. Kim P.H. Chang H.K. Shen Y.M. Bonsra K. Kang B.J. Yum S.Y. Kim J.H. Lee S.Y. Choi M.C. et al.Targeted Genome Engineering to Control VEGF Expression in Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells: Potential Implications for the Treatment of Myocardial Infarction.Stem Cells Transl. Med. 2017; 6: 1040-1051Crossref PubMed Scopus (29) Google Scholar VEGF is an angiogenic factor that can promote endothelial cell survival and can be suggested as a therapeutic reagent.9Ferrara N. Gerber H.P. LeCouter J. The biology of VEGF and its receptors.Nat. Med. 2003; 9: 669-676Crossref PubMed Scopus (7294) Google Scholar Although methods were varied, introduction of islet (ISL) and hepatocyte growth factor (HGF) also enhanced therapeutic effects of stem cells.10Xiang Q. Liao Y. Chao H. Huang W. Liu J. Chen H. Hong D. Zou Z. Xiang A.P. Li W. ISL1 overexpression enhances the survival of transplanted human mesenchymal stem cells in a murine myocardial infarction model.Stem Cell Res. Ther. 2018; 9: 51Crossref PubMed Scopus (10) Google Scholar,11Zhao L. Liu X. Zhang Y. Liang X. Ding Y. Xu Y. Fang Z. Zhang F. Enhanced cell survival and paracrine effects of mesenchymal stem cells overexpressing hepatocyte growth factor promote cardioprotection in myocardial infarction.Exp. Cell Res. 2016; 344: 30-39Crossref PubMed Scopus (63) Google Scholar However, there is still room to improve the functionality of hUCB-MSCs via not only endocrine and paracrine growth factors but also autocrine effects. It is ultimately necessary to survey as many therapeutic target genes as possible. We indeed focused on the activation of canonical wingless/integrase-1 (Wnt) signaling pathways that have been known to contribute to mouse embryonic stem cell (ESC) self-renewal and help maintain the undifferentiated status of ESCs through modulation of Oct4 and Nanog.12Li J. Li J. Chen B. Oct4 was a novel target of Wnt signaling pathway.Mol. Cell. Biochem. 2012; 362: 233-240Crossref PubMed Scopus (43) Google Scholar, 13Huang C. Qin D. Role of Lef1 in sustaining self-renewal in mouse embryonic stem cells.J. Genet. Genomics. 2010; 37: 441-449Crossref PubMed Scopus (17) Google Scholar, 14Kim C.G. Chung I.Y. Lim Y. Lee Y.H. Shin S.Y. A Tcf/Lef element within the enhancer region of the human NANOG gene plays a role in promoter activation.Biochem. Biophys. Res. Commun. 2011; 410: 637-642Crossref PubMed Scopus (17) Google Scholar Lymphoid enhancer-binding factor-1 (LEF1), a 48-kD nuclear protein, is a crucial transcription factor for proliferation and survival of B and T cells.15Okamura R.M. Sigvardsson M. Galceran J. Verbeek S. Clevers H. Grosschedl R. Redundant regulation of T cell differentiation and TCRalpha gene expression by the transcription factors LEF-1 and TCF-1.Immunity. 1998; 8: 11-20Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar,16Staal F.J. Luis T.C. Tiemessen M.M. WNT signalling in the immune system: WNT is spreading its wings.Nat. Rev. Immunol. 2008; 8: 581-593Crossref PubMed Scopus (383) Google Scholar The involvement of LEF1 in canonical Wnt signaling pathways has been studied, and activation of the pathways via the overexpression of LEF1 has been shown to contribute to mouse ESC self-renewal.13Huang C. Qin D. Role of Lef1 in sustaining self-renewal in mouse embryonic stem cells.J. Genet. Genomics. 2010; 37: 441-449Crossref PubMed Scopus (17) Google Scholar Furthermore, it has also been reported that the gene regulates the follicle morphogenesis and proliferation of neural progenitor cells.17Armenteros T. Andreu Z. Hortigüela R. Lie D.C. Mira H. BMP and WNT signalling cooperate through LEF1 in the neuronal specification of adult hippocampal neural stem and progenitor cells.Sci. Rep. 2018; 8: 9241Crossref PubMed Scopus (19) Google Scholar,18Zhang Y. Yu J. Shi C. Huang Y. Wang Y. Yang T. Yang J. Lef1 contributes to the differentiation of bulge stem cells by nuclear translocation and cross-talk with the Notch signaling pathway.Int. J. Med. Sci. 2013; 10: 738-746Crossref PubMed Scopus (23) Google Scholar Recent studies have focused on the functions of LEF1 related to stem cells and cardiogenesis. High expression of LEF1 between the mesoderm and cardiac progenitor cell stage has been identified in various studies, and it has been suggested that the gene plays an important role between these stages.19Li Y. Lin B. Yang L. Comparative transcriptomic analysis of multiple cardiovascular fates from embryonic stem cells predicts novel regulators in human cardiogenesis.Sci. Rep. 2015; 5: 9758Crossref PubMed Scopus (15) Google Scholar,20Liu Q. Jiang C. Xu J. Zhao M.T. Van Bortle K. Cheng X. Wang G. Chang H.Y. Wu J.C. Snyder M.P. Genome-Wide Temporal Profiling of Transcriptome and Open Chromatin of Early Cardiomyocyte Differentiation Derived From hiPSCs and hESCs.Circ. Res. 2017; 121: 376-391Crossref PubMed Scopus (54) Google Scholar Moreover, direct and temporal contribution of LEF1 in mouse heart maturation has also been reported. It has been demonstrated that the gene is mainly expressed in MSCs in the valvular region during the murine heart development,21Ye B. Li L. Xu H. Chen Y. Li F. Opposing roles of TCF7/LEF1 and TCF7L2 in cyclin D2 and Bmp4 expression and cardiomyocyte cell cycle control during late heart development.Lab. Invest. 2019; 99: 807-818Crossref PubMed Scopus (8) Google Scholar but its function in mouse and human MSCs (hMSCs) needs additional study. Despite this positive potential in cell proliferation, survival, and cardiac differentiation, the cardio-protective effects from MI in stem cell therapy have not been demonstrated yet. In parallel, diverse gene introduction systems, such as viruses, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR and CRISPR-associated 9 (Cas9), have been developed.22Gaj T. Gersbach C.A. Barbas 3rd, C.F. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.Trends Biotechnol. 2013; 31: 397-405Abstract Full Text Full Text PDF PubMed Scopus (2109) Google Scholar, 23Carroll D. Genome engineering with zinc-finger nucleases.Genetics. 2011; 188: 773-782Crossref PubMed Scopus (550) Google Scholar, 24Joung J.K. Sander J.D. TALENs: a widely applicable technology for targeted genome editing.Nat. Rev. Mol. Cell Biol. 2013; 14: 49-55Crossref PubMed Scopus (907) Google Scholar, 25Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (8230) Google Scholar Since 2012, when the CRISPR system was initially demonstrated, it has been improved to reduce off-target mutations25Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (8230) Google Scholar and has been widely adopted as the simplest and most efficient gene-integration systems in various cells and organisms, including human, rat, and mouse.26Hsu P.D. Lander E.S. Zhang F. Development and applications of CRISPR-Cas9 for genome engineering.Cell. 2014; 157: 1262-1278Abstract Full Text Full Text PDF PubMed Scopus (2969) Google Scholar, 27Sander J.D. Joung J.K. CRISPR-Cas systems for editing, regulating and targeting genomes.Nat. Biotechnol. 2014; 32: 347-355Crossref PubMed Scopus (1832) Google Scholar, 28de la Fuente-Núñez C. Lu T.K. CRISPR-Cas9 technology: applications in genome engineering, development of sequence-specific antimicrobials, and future prospects.Integr. Biol. 2017; 9: 109-122Crossref Scopus (29) Google Scholar Taken together, the objective of this study is to examine the therapeutic efficacy of the hUCB-MSCs stably expressing the LEF1 by CRISPR/CAS9-mediated gene integration (LEF1/hUCB-MSCs) in MI. We first investigated autocrine effects of overexpressing LEF1 in hUCB-MSCs on cell proliferation and survival of hUCB-MSCs in both normal and oxidative stress conditions and then survival-promoting effects of the cells in the MI region in vivo. Moreover, paracrine effects via stimulation of growth factor and cytokine secretion by LEF1 were also analyzed to explain the recovery of cardiac function in the MI animal model by LEF1. To find the target gene for hUCB-MSC therapy, we examined multiple transcriptome analyses from 4 different studies that were associated with cardiomyocyte differentiation from pluripotent cells, since the genes that exhibit cell-specific expression from mesoderm to cardiac progenitor cell stage promote self-renewal to maintain the stage or differentiation to cardiac progenitor cells.29Tateno H. Hiemori K. Hirayasu K. Sougawa N. Fukuda M. Warashina M. Amano M. Funakoshi T. Sadamura Y. Miyagawa S. et al.Development of a practical sandwich assay to detect human pluripotent stem cells using cell culture media.Regen Ther. 2017; 6: 1-8Crossref PubMed Scopus (4) Google Scholar, 30Lian X. Hsiao C. Wilson G. Zhu K. Hazeltine L.B. Azarin S.M. Raval K.K. Zhang J. Kamp T.J. Palecek S.P. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.Proc. Natl. Acad. Sci. USA. 2012; 109: E1848-E1857Crossref PubMed Scopus (882) Google Scholar, 31Witman N. Sahara M. Cardiac Progenitor Cells in Basic Biology and Regenerative Medicine.Stem Cells Int. 2018; 2018: 8283648Crossref PubMed Scopus (13) Google Scholar We focused on genes that are enriched in these stages. From 8 different analyses, we first examined the 100 most strongly upregulated genes in these stages in comparison to human pluripotent stem cells and then listed them individually in supplemental data (Table S1). From these gene lists, the 11 genes that were commonly enriched in 5 or more analyses were selected (Figure S1A). Among these, 36% (4 out of 11) of the genes are known to function in cardiac differentiation, e.g., PLXNA2, TBX3, and BMP4, and in MSC proliferation, e.g., LIX1, and were excluded from further target selection, finally leaving only 7 target genes. The 7 target genes selected from the in silico literature surveys were subjected to conventional PCR (Figure S1B) and qRT-PCR to examine the gene expression in hUCB-MSCs and expression patterns during the cardiomyocyte differentiation. Various patterns were observed (Figure S2), but the aim of this study was to enhance the protection efficacy of hUCB-MSCs by insertion of target genes, so we focused on the genes that maintained a constant low expression (LEF1, ZEB2). However, the effects of LEF1 on cell proliferation, as well as the direct and temporal contributions to heart development, have been reported.13Huang C. Qin D. Role of Lef1 in sustaining self-renewal in mouse embryonic stem cells.J. Genet. Genomics. 2010; 37: 441-449Crossref PubMed Scopus (17) Google Scholar,21Ye B. Li L. Xu H. Chen Y. Li F. Opposing roles of TCF7/LEF1 and TCF7L2 in cyclin D2 and Bmp4 expression and cardiomyocyte cell cycle control during late heart development.Lab. Invest. 2019; 99: 807-818Crossref PubMed Scopus (8) Google Scholar,32Reya T. O’Riordan M. Okamura R. Devaney E. Willert K. Nusse R. Grosschedl R. Wnt signaling regulates B lymphocyte proliferation through a LEF-1 dependent mechanism.Immunity. 2000; 13: 15-24Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar Consequently, all additional studies proceeded with LEF1 (Figures 1A and 1B ). Since low-proliferative capacity is one of the limits in the use of differentiated stem cells,33Segers V.F. Lee R.T. Stem-cell therapy for cardiac disease.Nature. 2008; 451: 937-942Crossref PubMed Scopus (904) Google Scholar we first examined if introducing LEF1 would enhance hUCB-MSC proliferation. The hUCB-MSCs transfected with LEF1 showed significantly increased numbers of cells, 72 h after incubation, without any aberrant changes in morphology (Figures 1C and 1D). The activation of canonical Wnt/β-catenin signaling by LEF1 transfection was confirmed by RT-PCR of β-catenin (CTNNB1) and c-Myc, as well as LEF1 itself. LEF1 significantly increased the expression of CTNNB1 and c-Myc. Cyclin D1 expression was also dramatically increased, representing stimulated cell cycles by LEF1 (Figures 1E and 1F). This result was confirmed in protein levels, as western blot and densitometry analysis clearly showed that Wnt/β-catenin signaling and cyclin D1 were upregulated by LEF1 (Figures 1G and 1H). We then presented the evidence for the proliferative fate of hUCB-MSCs enhanced in LEF1 transfection. Cell-cycle analysis using propidium iodide (PI) staining, combined with an automated fluorescence cell counter, showed cell fates shifted up from G0/G1 to S phases in the LEF1-transfected hUCB-MSC population. Approximately 40% cell-cycle enhancement was observed within the hUCB-MSCs transfected with LEF1 (Figures 1I and 1J). A major issue in stem cell therapy for ischemic heart diseases, including MI, is the low survival of transplanted cells in the ischemic region. It is reported that most hMSCs implanted onto ischemic hearts died within 4 days after transplantation.34Toma C. Pittenger M.F. Cahill K.S. Byrne B.J. Kessler P.D. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart.Circulation. 2002; 105: 93-98Crossref PubMed Scopus (1909) Google Scholar Therefore, we examined the protective function of LEF1 from the hydrogen peroxide-induced cell death of hUCB-MSCs in vitro. Apoptosis was induced by 500 μM H2O2 treatment for 48 h in hUCB-MSCs, whereas hUCB-MSCs expressing LEF1 still survived at a significantly higher number of cells (Figure 2A). Slightly more cells were counted in LEF1-transfected hUCB-MSCs than in the control hUCB-MSCs under normal conditions. Yet, the number of cells remained drastically higher in the LEF1-expressing hUCB-MSC group (80% survival) than the control hUCB-MSC group (30% survival) under severe oxidative stress conditions induced by H2O2 (Figure 2B). Western blot analysis for Bax and Bcl-2 proteins revealed that LEF1 blocked proapoptotic Bax but induced anti-apoptotic Bcl-1, even without oxidative stress, thus presenting a reduced level of Bax but an increased level of Bcl-2 in both normal and severe oxidative stress conditions (Figures 2C–2E). These results demonstrate that the cells had a protective effect upon H2O2 treatment. We next demonstrated that LEF1 could protect hUCB-MSCs from apoptosis under severe oxidative stress using flow cytometry with Annexin V/PI labeling. As shown in Figures 2F and 2G, a drastic increase in the apoptotic cells was observed in control hUCB-MSCs upon H2O2 treatment (∼25%). However, a remarkable reduction in the apoptotic cell ratio was shown in LEF1-transfected hUCB-MSCs under both normal (∼4%) and oxidative stress conditions (∼10%). Altogether, these results indicate that the LEF1 plays an important role in anti-apoptosis of hUCB-MSCs under oxidative stress. A number of studies have demonstrated that stem cell therapy is effective in myocardial regeneration via enhancement of angiogenesis in the region of MI.33Segers V.F. Lee R.T. Stem-cell therapy for cardiac disease.Nature. 2008; 451: 937-942Crossref PubMed Scopus (904) Google Scholar Various types of stem cells, different treatments or gene editing, and transplantation methods have been developed and tested to improve therapeutic efficacy.35Tompkins B.A. Natsumeda M. Balkan W. Hare J.M. What Is the Future of Cell-Based Therapy for Acute Myocardial Infarction.Circ. Res. 2017; 120: 252-255Crossref PubMed Scopus (15) Google Scholar In the present study, we first constructed LEF1/hUCB-MSCs that steadily express LEF1 using the CRISPR/Cas9 system, followed by homologous recombination on the AAVS1 genomic safe harbor site (Figure 3A). Successful integration of LEF1 on AAVS1 was confirmed by genomic PCR spanning from LEF1 to the AAVS1 site (Figures 3B and 3C). LEF1 protein was stably expressed by LEF1/hUCB-MSCs until 14 days after transfection (Figure 3D). To determine the cardioprotective potential of LEF1/hUCB-MSCs in ischemic heart disease, we conducted an experiment using transplantation of LEF1/hUCB-MSCs and control hUCB-MSCs in a post-MI rat model. This experiment included a group of sham rats (surgery without MI) and MI rats (nontreat with MI) as controls (Figure 3E). The cell transplantation of each group was administered as a cell patch using the UpCell system (Figure 3F). A total of 20 rats, 5 rats in each group, was initially designed and subjected to MI surgery. The rats were randomized to the following groups: operation + non-MI, MI + nontreat, MI + hUCB-MSCs, MI + LEF1/hUCB-MSCs. Each cell sheet was transplanted 30 min after visual inspection of the infarction. However, 6 out of 11 rats with MI died within 2 weeks after the operation (45.5% survival). Three out of 8 (62.5% survival) died in the MI + hUCB-MSC group and 1 out of 6 in the MI + LEF1/hUCB-MSC group (83.3% survival) (Figure 3G). To measure the combined therapy in MI, we performed echocardiography at 1 week and 4 weeks after MI surgery (Figure 4A). A successful induction of MI in the rat model was confirmed by echocardiography showing drastic reduction of left-ventricular ejection fraction (EF) (sham: 89.01 ± 2.56% versus MI: 36.33 ± 5.11%) and fraction shortening (FS) (sham: 60.48 ± 3.82% versus MI: 16.24 ± 1.41%) when comparing the sham and MI group at 1 week postsurgery (Figures 4B and 4C). Damage of the heart muscle was also measured by an increase of the left ventricle (LV) inner diameter at diastole (LVIDd) (sham: 4.33 ± 0.41 mm versus MI: 8.77 ± 0.27 mm) and LV inner diameter at systole (LVIDs) (sham: 2.26 ± 0.33 mm versus MI: 7.60 ± 0.41 mm) (Figures 4D and 4E). To evaluate functional improvement of hUCB-MSCs and LEF1 overexpression in hUCB-MSCs on the MI, we also measured these 4 values at 4 weeks postsurgery (Figures 4B–4E). The MI + hUCB-MSC group tended to have some protective effects when compared with MI, but there was no significant difference between the values taken 1 and 4 weeks postsurgery: EF (43.56 ± 3.62%), FS (21.6 ± 1.88%), LVIDd (8.68 ± 0.37 mm), and LVIDs (7.64 ± 0.18 mm). Of note, in the comparison among the three MI groups (MI alone, MI + hUCB-MSCs, and MI + LEF1/hUCB-MSCs), LEF1 expressing hUCB-MSCs significantly improved in all 4 functional values in 4 weeks: EF (63.53 ± 4.34%), FS (37.11 ± 2.78%), LVIDd (6.29 ± 0.19 mm), and LVIDs (4.72 ± 0.34 mm). These results demonstrate the protective effects of LEF1/hUCB-MSCs in MI when compared with the MI-alone group and with the MI + hUCB-MSC group as well: EF (29.18 ± 5.13%), FS (12.36 ± 2.21%), LVIDd (9.30 ± 0.31 mm), and LVIDs (7.71 ± 0.42 mm). Transplantation of LEF1/hUCB-MSCs reduced MI size and fibrosis and protected the left-ventricular wall from thinning (Figure 5). The improvement of cardiac function was observed from a histological analysis. Masson’s trichrome staining on the rat heart, 4 weeks after sham surgery, presented heart muscle in red (Sham in Figure 5A). The other three hearts with induced MI produced different ranges of regions stained with blue, which represents fibrosis in the LV. A very thin, blue-stained wall fiber was detected in the MI-induced control model. No live heart muscle was stained in the MI region. Instead, a little thicker LV was noted in the hUCB-MSC-treated MI group, but large fibrosis was still stained. Some live heart cells stained in red could be seen in the region of fibrosis. This may indicate that hUCB-MSC itself has a minor protective effect (MI + hUCB-MSCs in Figure 5A). In particular though, a clear improvement in the anti-fibrosis effect was detected in the group treated with LEF1/hUCB-MSCs (MI + LEF/hUCB-MSCs in Figure 5A). The region of fibrosis stained blue was greatly reduced and the number of heart muscle cells remaining (stained with red) in the fibrotic region were more readily observed in the magnified image (Figure 5A). For the quantitation of efficacy in cardiac function, three factors, MI-size, fibrosis, and wall thickness, were measured by analyzing the stained areas. MI-size and fibrosis were dramatically decreased in the LEF1/hUCB-MSC group when compared with both the MI and MI + hUCB-MSC groups (Figures 5B and 5C). Furthermore, the decrease of LV wall thickness resulting from MI was significantly protected from the LEF1/hUCB-MSC group, whereas the LV wall thickness tends to appear drastically reduced in the hUCB-MSC group, which was not significantly different from the MI group (Figure 5D). These results suggest that LEF1 abundantly expressed in hUCB-MSCs not only has an autocrine effect (enhancing the survival of hUCB-MSCs) but also somehow has paracrine effects, possibly via growth factors and cytokines, which can protect and regenerate damaged rat heart cells in the region of MI. LEF1 expression was confirmed by immunohistochemical staining in MI-induced rat heart tissues. Fibrous heart structure was found in the MI-only group with few cells stained with methyl green (MI in Figure 6A). There were some cells that survived in the patch of hUCB-MSCs, but no LEF1 expression was detected in the MI + hUCB-MSC group. LEF1 expression was stained only in the tissues from the cell patch of LEF1/hUCB-MSCs attached on the MI region (MI + LEF1/hUCB-MSCs in Figure 6A). This suggests that there was no endogenous expression of LEF1 in the patched hUCB-MSCs. In addition, substantially more cells were counted in the LEF1/hUCB-MSC group than the hUCB-MSC group, 4 weeks after surgery. This might contribute to the thicker LV in the MI region with LEF1/hUCB-MSCs. Quantification of the LEF1 immunohistochemistry (IHC) data successfully displayed prolonged survival of LEF1/hUCB-MSCs (Figure 6B). To distinguish implanted human stem cells from rat heart cells, IHC with anti-lamin A + C antibody was performed in the MI region. We took a picture from the edge of the MI region to include rat heart cells as a control. No lamin A + C-positive cells were detected from rat heart cells in the MI group (Figure 6C). Only a thin layer of human cells was stained with lamin A + C-positive cells in the hUCB-MSC groups, whereas a large number of lamin A + C-positive, implanted human stem cells were detected in the MI + LEF1/hUCB-MSC groups. Strikingly, rat heart myocardium still remained thicker at 4 weeks after MI induction following LEF1/hUCB-MSC treatment (Figure 6D). These results suggest that LEF1 expression in hUCB-MSCs has positive effects, not only on the survival of stem cells but also on protecting rat myocardial cells from MI via paracrine effects. Growth factors and cytokines, such as VEGF, insulin-like growth factor (IGF), HGF, interleukin-1β (IL-1β), and IL-6, have been investigated as therapeutic targets in various cell types and diseases.8Cho H.M. Kim P.H. Chang H.K. Shen Y.M. Bonsra K. Kang B.J. Yum S.Y. Kim J.H. Lee S.Y. Choi M.C. et al.Targeted Genome Engineering to Control VEGF Expres" @default.
• W3003060822 created "2020-01-30" @default.
• W3003060822 creator A5003678279 @default.
• W3003060822 creator A5013654282 @default.
• W3003060822 creator A5024446116 @default.
• W3003060822 creator A5024801112 @default.
• W3003060822 creator A5026328968 @default.
• W3003060822 creator A5048780355 @default.
• W3003060822 creator A5061255806 @default.
• W3003060822 creator A5083241424 @default.
• W3003060822 date "2020-03-01" @default.
• W3003060822 modified "2023-10-18" @default.
• W3003060822 title "Transplantation of hMSCs Genome Edited with LEF1 Improves Cardio-Protective Effects in Myocardial Infarction" @default.
• W3003060822 cites W1974595623 @default.
• W3003060822 cites W1976930659 @default.
• W3003060822 cites W2000292756 @default.
• W3003060822 cites W2001811125 @default.
• W3003060822 cites W2005749710 @default.
• W3003060822 cites W2023043128 @default.
• W3003060822 cites W2029049465 @default.
• W3003060822 cites W2039705224 @default.
• W3003060822 cites W2050051758 @default.
• W3003060822 cites W2059681758 @default.
• W3003060822 cites W2061184966 @default.
• W3003060822 cites W2061260618 @default.
• W3003060822 cites W2064815984 @default.
• W3003060822 cites W2067335193 @default.
• W3003060822 cites W2069262306 @default.
• W3003060822 cites W2086267296 @default.
• W3003060822 cites W2087293545 @default.
• W3003060822 cites W2091395040 @default.
• W3003060822 cites W2093147991 @default.
• W3003060822 cites W2096261947 @default.
• W3003060822 cites W2101995250 @default.
• W3003060822 cites W2109069210 @default.
• W3003060822 cites W2112444085 @default.
• W3003060822 cites W2119075044 @default.
• W3003060822 cites W2123196886 @default.
• W3003060822 cites W2150902176 @default.
• W3003060822 cites W2150947445 @default.
• W3003060822 cites W2166305181 @default.
• W3003060822 cites W2265874208 @default.
• W3003060822 cites W2330571432 @default.
• W3003060822 cites W2433697453 @default.
• W3003060822 cites W2536181362 @default.
• W3003060822 cites W2557537068 @default.
• W3003060822 cites W2572321150 @default.
• W3003060822 cites W2580346263 @default.
• W3003060822 cites W2585080692 @default.
• W3003060822 cites W2588798559 @default.
• W3003060822 cites W2753051611 @default.
• W3003060822 cites W2785329501 @default.
• W3003060822 cites W2797844455 @default.
• W3003060822 cites W2808361199 @default.
• W3003060822 cites W2856215326 @default.
• W3003060822 cites W2883065529 @default.
• W3003060822 cites W2888900815 @default.
• W3003060822 cites W2893588482 @default.
• W3003060822 cites W2913275818 @default.
• W3003060822 cites W4247265457 @default.
• W3003060822 cites W4255009698 @default.
• W3003060822 doi "https://doi.org/10.1016/j.omtn.2020.01.007" @default.
• W3003060822 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7019046" @default.
• W3003060822 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/32069701" @default.
• W3003060822 hasPublicationYear "2020" @default.
• W3003060822 type Work @default.
• W3003060822 sameAs 3003060822 @default.
• W3003060822 citedByCount "22" @default.
• W3003060822 countsByYear W30030608222020 @default.
• W3003060822 countsByYear W30030608222021 @default.
• W3003060822 countsByYear W30030608222022 @default.
• W3003060822 countsByYear W30030608222023 @default.
• W3003060822 crossrefType "journal-article" @default.
• W3003060822 hasAuthorship W3003060822A5003678279 @default.
• W3003060822 hasAuthorship W3003060822A5013654282 @default.
• W3003060822 hasAuthorship W3003060822A5024446116 @default.
• W3003060822 hasAuthorship W3003060822A5024801112 @default.
• W3003060822 hasAuthorship W3003060822A5026328968 @default.
• W3003060822 hasAuthorship W3003060822A5048780355 @default.
• W3003060822 hasAuthorship W3003060822A5061255806 @default.
• W3003060822 hasAuthorship W3003060822A5083241424 @default.
• W3003060822 hasBestOaLocation W30030608221 @default.
• W3003060822 hasConcept C104317684 @default.
• W3003060822 hasConcept C126322002 @default.
• W3003060822 hasConcept C141231307 @default.
• W3003060822 hasConcept C164705383 @default.
• W3003060822 hasConcept C2911091166 @default.
• W3003060822 hasConcept C500558357 @default.
• W3003060822 hasConcept C54355233 @default.
• W3003060822 hasConcept C71924100 @default.
• W3003060822 hasConcept C86803240 @default.
• W3003060822 hasConceptScore W3003060822C104317684 @default.
• W3003060822 hasConceptScore W3003060822C126322002 @default.
• W3003060822 hasConceptScore W3003060822C141231307 @default.
• W3003060822 hasConceptScore W3003060822C164705383 @default.
• W3003060822 hasConceptScore W3003060822C2911091166 @default.
• W3003060822 hasConceptScore W3003060822C500558357 @default.
• W3003060822 hasConceptScore W3003060822C54355233 @default.

• next