Sensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing

Tóth, Zsuzsanna; Shahar, Tal; Leker, Ronen R.; Szalayova, Ildikó; Bratincsák, András; Key, Sharon; Lonyai, Anna; Németh, Krisztián; Mezey, Éva

doi:10.1016/j.yexcr.2007.02.024

Cited by 22 publications

(23 citation statements)

References 42 publications

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“…To ensure that cells expressing GFP originate from donor BM, we used FISH to determine the presence of a Y chromosome in the GFP ϩ cells (Figure 3 and Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). 18 The present findings confirmed our previously published results 18 that about 10% of cells expressing the Y chromosome were not positive for GFP (even when signal amplification was used), suggesting that when using only GFP as a marker we significantly underestimate the percentage of brain cells originating from the donor BM. This may be due to silencing of the GFP expression during neural differentiation.…”

Section: G-csf/scf Increases Influx Of Gfp ؉ Cells Into the Injured Bsupporting

confidence: 90%

The combination of granulocyte colony-stimulating factor and stem cell factor significantly increases the number of bone marrow–derived endothelial cells in brains of mice following cerebral ischemia

Tóth

Leker

Shahar

et al. 2008

Blood

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Granulocyte colony-stimulating factor (G-CSF) induces proliferation of bone marrow–derived cells. G-CSF is neuroprotective after experimental brain injury, but the mechanisms involved remain unclear. Stem cell factor (SCF) is a cytokine important for the survival and differentiation of hematopoietic stem cells. Its receptor (c-kit or CD117) is present in some endothelial cells. We aimed to determine whether the combination of G-CSF/SCF induces angiogenesis in the central nervous system by promoting entry of endothelial precursors into the injured brain and causing them to proliferate there. We induced permanent middle cerebral artery occlusion in female mice that previously underwent sex-mismatched bone marrow transplantation from enhanced green fluorescent protein (EGFP)–expressing mice. G-CSF/SCF treatment reduced infarct volumes by more than 50% and resulted in a 1.5-fold increase in vessel formation in mice with stroke, a large percentage of which contain endothelial cells of bone marrow origin. Most cells entering the brain maintained their bone marrow identity and did not transdifferentiate into neural cells. G-CSF/SCF treatment also led to a 2-fold increase in the number of newborn cells in the ischemic hemisphere. These findings suggest that G-CSF/SCF treatment might help recovery through induction of bone marrow–derived angiogenesis, thus improving neuronal survival and functional outcome.

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Section: G-csf/scf Increases Influx Of Gfp ؉ Cells Into the Injured Bsupporting

confidence: 90%

The combination of granulocyte colony-stimulating factor and stem cell factor significantly increases the number of bone marrow–derived endothelial cells in brains of mice following cerebral ischemia

Tóth

Leker

Shahar

et al. 2008

Blood

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“…All values were expressed as means Ϯ SE. therefore, be obscured as differentiation continues (31). Therefore, the NPY-pretreated MSC from male SD rats employed in this study were able to trigger the expression of cell proliferation and migration-related genes in the myocardium of female SD rats and acquire the characteristics of cardiomyocytes and endothelial cells as revealed by FISH analysis.…”

Section: Discussionmentioning

confidence: 88%

Combining neuropeptide Y and mesenchymal stem cells reverses remodeling after myocardial infarction

Wang¹,

Zhang²,

Ashraf³

et al. 2010

American Journal of Physiology-Heart and Circulatory Physiology

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. Combining neuropeptide Y and mesenchymal stem cells reverses remodeling after myocardial infarction. Am J Physiol Heart Circ Physiol 298: H275-H286, 2010. First published November 6, 2009 doi:10.1152/ajpheart.00765.2009.-Neuropeptide Y (NPY) induced reentry of differentiated rat neonatal and adult cardiomyocytes into the cell cycle. NPY also induced differentiation of bone marrow-derived mesenchymal stem cells (MSC) into cardiomyocytes following transplantation into infarcted myocardium. Rat neonatal and adult cardiomyocytes were treated in vitro with vehicle, NPY, fibroblast growth factor (FGF; 100 ng/ml), or FGF plus NPY. DNA synthesis, mitosis, and cytokinesis were determined by immunocytochemistry. NPY-induced MSC gene expression, cell migration, tube formation, and endothelial cell differentiation were analyzed. Male rat green fluorescent protein-MSC (2 ϫ 10 6 ), pretreated with either vehicle or NPY (10 Ϫ8 M) for 72 h, were injected into the border zone of the female myocardium following left anterior descending artery ligation. On day 30, heart function was assessed, and hearts were harvested for histological and immunohistochemical analyses. NPY increased 5-bromo-2Ј-deoxy-uridine incorporation and promoted both cytokinesis and mitosis in rat neonatal and adult myocytes. NPY also upregulated several genes required for mitosis in MSC, including aurora B kinase, FGF-2, cycline A2, eukaryotic initiation factor 4 E, and stromal cell-derived factor-1␣. NPY directly induced neonatal and adult cardiomyocyte cell-cycle reentry and enhanced the number of differentiated cardiomyocytes from MSC in the infarcted myocardium, which corresponded to improved cardiac function, reduced fibrosis, ventricular remodeling, and increased angiomyogenesis. It is concluded that a combined treatment of NPY with MSC is a novel approach for cardiac repair. deoxyribonucleic acid synthesis FETAL AND NEONATAL CARDIAC myocytes are capable of undergoing DNA synthesis and cell division. However, their ability to divide diminishes progressively during postnatal development (2). Adult mammalian cardiomyocytes are considered terminally differentiated, incapable of proliferation and irreversibly withdrawn from the cell cycle soon after birth (28, 29). The permanent loss of cardiomyocytes following myocardial infarction (MI) often results in heart failure. Potential therapies could include stimulating the myocytes to divide and adding new cells via pharmacological and genetic manipulations or delivering stem cells to multiply and subsequently differentiate into cardiomyocytes (7). Recent reports have shown that adult cardiac myocytes can be induced to reenter into cell cycle with periostin (17), p38 MAP kinase inhibitor (8), cyclin D1/CDK4 (27), cyclin A2 (3), and transforming growth factor-␤ (4).Although the adult heart has a limited capacity for myocyte proliferation, increasing the number of remaining cardiomyocytes by activating their proliferative potential via extrinsic factors could result in the repair of damaged myocardium. Therefor...

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“…A significant number of these studies relied solely on green fluorescent protein (GFP) to track cell fate following transplantation, its continued presence being necessary as a marker of engraftment. Given that GFP labeling can diminish and that the transgene construct might be silenced as cells proliferate and differentiate, detection of differentiation could be obscured (30,31). Conversely, in cases where multiple cell tagging techniques (fluorescent dyes, nanoparticles, sex mistmatch) were used, strong evidence of engraftment and cardiomyocyte formation has been shown (6,24).…”

Section: Discussionmentioning

confidence: 99%

Allogeneic mesenchymal stem cells restore cardiac function in chronic ischemic cardiomyopathy via trilineage differentiating capacity

Quevedo

Hatzistergos

Oskouei

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

523

413

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The mechanism(s) underlying cardiac reparative effects of bone marrow-derived mesenchymal stem cells (MSC) remain highly controversial. Here we tested the hypothesis that MSCs regenerate chronically infarcted myocardium through mechanisms comprising long-term engraftment and trilineage differentiation. Twelve weeks after myocardial infarction, female swine received catheterbased transendocardial injections of either placebo (n ‫؍‬ 4) or male allogeneic MSCs (200 million; n ‫؍‬ 6). Animals underwent serial cardiac magnetic resonance imaging, and in vivo cell fate was determined by co-localization of Y-chromosome (Y pos ) cells with markers of cardiac, vascular muscle, and endothelial lineages. MSCs engrafted in infarct and border zones and differentiated into cardiomyocytes as ascertained by co-localization with GATA-4, Nkx2.5, and ␣-sarcomeric actin. In addition, Y pos MSCs exhibited vascular smooth muscle and endothelial cell differentiation, contributing to large and small vessel formation. Infarct size was reduced from 19.3 ؎ 1.7% to 13.9 ؎ 2.0% (P < 0.001), and ejection fraction (EF) increased from 35.0 ؎ 1.7% to 41.3 ؎ 2.7% (P < 0.05) in MSC but not placebo pigs over 12 weeks. This was accompanied by increases in regional contractility and myocardial blood flow (MBF), particularly in the infarct border zone. Importantly, MSC engraftment correlated with functional recovery in contractility (R ‫؍‬ 0.85, P < 0.05) and MBF (R ‫؍‬ 0.76, P < 0.01). Together these findings demonstrate long-term MSC survival, engraftment, and trilineage differentiation following transplantation into chronically scarred myocardium. MSCs are an adult stem cell with the capacity for cardiomyogenesis and vasculogenesis which contribute, at least in part, to their ability to repair chronically scarred myocardium.cardiac chimerism ͉ cellular cardiomyoplasty ͉ heart failure ͉ catheter delivery

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Sensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing

Cited by 22 publications

References 42 publications

The combination of granulocyte colony-stimulating factor and stem cell factor significantly increases the number of bone marrow–derived endothelial cells in brains of mice following cerebral ischemia

The combination of granulocyte colony-stimulating factor and stem cell factor significantly increases the number of bone marrow–derived endothelial cells in brains of mice following cerebral ischemia

Combining neuropeptide Y and mesenchymal stem cells reverses remodeling after myocardial infarction

Allogeneic mesenchymal stem cells restore cardiac function in chronic ischemic cardiomyopathy via trilineage differentiating capacity

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