Novel MicroRNA Prosurvival Cocktail for Improving Engraftment and Function of Cardiac Progenitor Cell Transplantation

Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305-5454, USA.
Circulation (Impact Factor: 14.43). 09/2011; 124(11 Suppl):S27-34. DOI: 10.1161/CIRCULATIONAHA.111.017954
Source: PubMed


Although stem cell therapy has provided a promising treatment for myocardial infarction, the low survival of the transplanted cells in the infarcted myocardium is possibly a primary reason for failure of long-term improvement. Therefore, the development of novel prosurvival strategies to boost stem cell survival will be of significant benefit to this field.
Cardiac progenitor cells (CPCs) were isolated from transgenic mice, which constitutively express firefly luciferase and green fluorescent protein. The CPCs were transduced with individual lentivirus carrying the precursor of miR-21, miR-24, and miR-221, a cocktail of these 3 microRNA precursors, or green fluorescent protein as a control. After challenge in serum free medium, CPCs treated with the 3 microRNA cocktail showed significantly higher viability compared with untreated CPCs. After intramuscular and intramyocardial injections, in vivo bioluminescence imaging showed that microRNA cocktail-treated CPCs survived significantly longer after transplantation. After left anterior descending artery ligation, microRNA cocktail-treated CPCs boost the therapeutic efficacy in terms of functional recovery. Histological analysis confirmed increased myocardial wall thickness and CPC engraftment in the myocardium with the microRNA cocktail. Finally, we used bioinformatics analysis and experimental validation assays to show that Bim, a critical apoptotic activator, is an important target gene of the microRNA cocktail, which collectively can bind to the 3'UTR region of Bim and suppress its expression.
We have demonstrated that a microRNA prosurvival cocktail (miR-21, miR-24, and miR-221) can improve the engraftment of transplanted cardiac progenitor cells and therapeutic efficacy for treatment of ischemic heart disease.

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    • "Recently, it was demonstrated that in mouse and human hematopoietic stem and progenitor cells, low expression of Bim or Bmf provokes a similar effect to overexpression of Bcl-2 and that their downregulation inhibits apoptosis, favoring HSC long-term engraftment (Labi et al., 2013). Furthermore , Bim is an important target for a miRNA cocktail (miR-21, miR-24, and miR-221) that significantly improves survival of untreated Sca1 + CPCs (Hu et al., 2011). In agreement with these data, our results suggest that miR-133 protects CPCs from oxidative stress-induced apoptosis, at least in part, through targeting of Bmf and Bim. "
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    ABSTRACT: miR-133a and miR-1 are known as muscle-specific microRNAs that are involved in cardiac development and pathophysiology. We have shown that both miR-1 and miR-133a are early and progressively upregulated during in vitro cardiac differentiation of adult cardiac progenitor cells (CPCs), but only miR-133a expression was enhanced under in vitro oxidative stress. miR-1 was demonstrated to favor differentiation of CPCs, whereas miR-133a overexpression protected CPCs against cell death, targeting, among others, the proapoptotic genes Bim and Bmf. miR-133a-CPCs clearly improved cardiac function in a rat myocardial infarction model by reducing fibrosis and hypertrophy and increasing vascularization and cardiomyocyte proliferation. The beneficial effects of miR-133a-CPCs seem to correlate with the upregulated expression of several relevant paracrine factors and the plausible cooperative secretion of miR-133a via exosomal transport. Finally, an in vitro heart muscle model confirmed the antiapoptotic effects of miR-133a-CPCs, favoring the structuration and contractile functionality of the artificial tissue. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
    Stem Cell Reports 11/2014; 3(6). DOI:10.1016/j.stemcr.2014.10.010 · 5.37 Impact Factor
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    • "Various approaches have been tested to enhance the survival of transplanted cells in the myocardium. These include the preconditioning of cells prior to injection with various pro-survival factors (cytokines, growth factors, drugs, microRNAs) [50], [69] or physical stimuli (hypoxia, heat shock) [70], [71], transplantation of cell sheets [72] or cells embedded in tissue-engineered 3D matrices [47], instead of direct injection of enzymatically dissociated cells. In addition, genetic engineering of cells to stably express pro-survival or angiogenic factors [51], [73] and use of more immature cells for transplantation that display higher proliferation rates and increased hypoxia resistance than mature cells [22] were used. "
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    ABSTRACT: Cell loss after transplantation is a major limitation for cell replacement approaches in regenerative medicine. To assess the survival kinetics of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) we generated transgenic murine iPSC lines which, in addition to CM-specific expression of puromycin N-acetyl-transferase and enhanced green fluorescent protein (EGFP), also constitutively express firefly luciferase (FLuc) for bioluminescence (BL) in vivo imaging. While undifferentiated iPSC lines generated by random integration of the transgene into the genome retained stable FLuc activity over many passages, the BL signal intensity was strongly decreased in purified iPS-CM compared to undifferentiated iPSC. Targeted integration of FLuc-expression cassette into the ROSA26 genomic locus using zinc finger nuclease (ZFN) technology strongly reduced transgene silencing in iPS-CM, leading to a several-fold higher BL compared to iPS-CM expressing FLuc from random genomic loci. To investigate the survival kinetics of iPS-CM in vivo, purified CM obtained from iPSC lines expressing FLuc from a random or the ROSA26 locus were transplanted into cryoinfarcted hearts of syngeneic mice. Engraftment of viable cells was monitored by BL imaging over 4 weeks. Transplanted iPS-CM were poorly retained in the myocardium independently of the cell line used. However, up to 8% of cells survived for 28 days at the site of injection, which was confirmed by immunohistological detection of EGFP-positive iPS-CM in the host tissue. Transplantation of iPS-CM did not affect the scar formation or capillary density in the periinfarct region of host myocardium. This report is the first to determine the survival kinetics of drug-selected iPS-CM in the infarcted heart using BL imaging and demonstrates that transgene silencing in the course of iPSC differentiation can be greatly reduced by employing genome editing technology. FLuc-expressing iPS-CM generated in this study will enable further studies to reduce their loss, increase long-term survival and functional integration upon transplantation.
    PLoS ONE 09/2014; 9(9):e107363. DOI:10.1371/journal.pone.0107363 · 3.23 Impact Factor
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    • "However, we observed that knockdown of CHOP but not any other gene we tested above attenuated the increased apoptosis induced by miR-24 inhibitor (Fig.3C). Since increased Bim protein levels appeared to be a major mediator of apoptosis upon miR-24 inhibition in other cell types [35], [40], we tested whether CHOP could regulate Bim in murine cardiomyocytes. Indeed, CHOP knockdown resulted in a decrease in Bim mRNA and protein levels, suggesting that CHOP normally upregulates Bim as it promotes apoptosis (Fig. 3 D and E). "
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    ABSTRACT: Numerous cardiac diseases, including myocardial infarction (MI) and chronic heart failure, have been associated with cardiomyocyte apoptosis. Promoting cell survival by inhibiting apoptosis is one of the effective strategies to attenuate cardiac dysfunction caused by cardiomyocyte loss. miR-24 has been shown as an anti-apoptotic microRNA in various animal models. In vivo delivery of miR-24 into a mouse MI model suppressed cardiac cell death, attenuated infarct size, and rescued cardiac dysfunction. However, the molecular pathway by which miR-24 inhibits cardiomyocyte apoptosis is not known. Here we found that miR-24 negatively regulates mouse primary cadiomyocyte cell death through functioning in the intrinsic apoptotic pathways. In ER-mediated intrinsic pathway, miR-24 genetically interacts with the CEBP homologous gene CHOP as knocking down of CHOP partially attenuated the induced apoptosis by miR-24 inhibition. In mitochondria-involved intrinsic pathway, miR-24 inhibits the initiation of apoptosis through suppression of Cytochrome C release and Bax translocation from cytosol to mitochondria. These results provide mechanistic insights into the miR-24 mediated anti-apoptotic effects in murine cardiomyocytes.
    PLoS ONE 01/2014; 9(1):e85389. DOI:10.1371/journal.pone.0085389 · 3.23 Impact Factor
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