The Hypertrophic Response in C2C12 Myoblasts Recruits the G1 Cell Cycle Machinery

The Journal of Biological Chemistry. Jun, 2002  |  Pubmed ID: 11967266

Hypertrophy occurs in postmitotic muscle as an adaptive response to various physiological and pathological stresses. Studies in vascular smooth muscle cells and primary cardiomyocytes suggest that angiotensin II-mediated hypertrophy activates signaling pathways associated with cell proliferation. Regulation of cyclin-dependent kinase (Cdk)-cyclin activities is essential to cell size control in lower eukaryotes, yet their role in the hypertrophic response in muscle is incompletely understood. We describe an in vitro model of hypertrophy in C2C12 skeletal myoblasts and demonstrate that induction of hypertrophy involves transient activation of Cdk4, subsequent phosphorylation of Rb, and release of HDAC1 from the Rb inhibitory complex. We also demonstrate that E2F-1 becomes transcriptionally active yet remains associated with Rb. We propose a model whereby partial inactivation of the Rb complex leads to derepression of a subset of E2F-1 targets necessary for cell growth without division during hypertrophy.

Genome-wide Examination of Myoblast Cell Cycle Withdrawal During Differentiation

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Jan, 2003  |  Pubmed ID: 12508234

Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up-regulation of MyoD, followed by myogenin, and activation of Bcl-2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40% fused myotubes, as levels of p21(WAF1/Cip1) increased and alpha-actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome-wide screen to identify other gene products involved in this process. Using microarrays containing approximately 10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT-PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly-6A, whose expression specifically was up-regulated during cell cycle withdrawal coincident with early myoblast differentiation.

Distinct Domains of Human CDC5 Direct Its Nuclear Import and Association with the Spliceosome

Cell Biochemistry and Biophysics. 2003  |  Pubmed ID: 14515018

Genetic studies have shown that CDC5 proteins are essential for G2 progression and mitotic entry. CDC5 homologs in yeast and mammals are essential for pre-messenger ribonucleic acid (mRNA) processing. Other gene products also have been shown to play roles in both pre-mRNA splicing and cell cycle regulation, prompting the description of several models to explain the mechanism(s) linking these two processes. In this study, we demonstrate that the amino-terminus of human CDC5 directs nuclear import independent of consensus nuclear localization signals or phosphorylation, and that the carboxyl-terminus of human CDC5 preferentially associates with spliceosomal complexes in proximity of RNA transcription during interphase. hCDC5 colocalizes with Sm proteins in a cell cycle- and domain-dependent manner, and several proteins in the human CDC5-associated complex are identified that suggest potential roles for the complex in coupling pre-mRNA splicing to transcriptional activation and protein translation. These results indicate that human CDC5 may function in pre-mRNA processing and cell cycle progression through more than one mechanism.

E2F-1 Regulates the Expression of a Subset of Target Genes During Skeletal Myoblast Hypertrophy

The Journal of Biological Chemistry. Oct, 2004  |  Pubmed ID: 15304485

Cellular hypertrophy, or growth without division, is an adaptive response to various physiological and pathological stimuli in postmitotic muscle. We demonstrated previously that angiotensin II stimulates hypertrophy in C2C12 myoblasts by transient activation of the cyclin-dependent kinase 4 complex, subsequent phosphorylation of retinoblastoma protein, release of histone deacetylase 1 from the retinoblastoma protein inhibitory complex, and partial activation of the transcription factor E2F-1. These observations led us to propose a model in which partial inactivation of the retinoblastoma protein complex leads to the derepression of a subset of E2F-1 targets necessary for cell growth without division during hypertrophy. We now present data that support this model and suggest the mechanism by which E2F-1 regulates hypertrophy. We examined expression profiles of angiotensin II-stimulated myoblasts and identified a subset of E2F-1 target genes that are specifically regulated during the hypertrophic response. We showed that the expression of E2F-1 targets involved in G1/S transit, DNA replication, and mitosis is not altered during the hypertrophic response, while the expression of E2F-1-regulated genes controlling early G1 progression, cytoskeletal organization, protein synthesis, mitochondrial function, and programmed cell death is up-regulated. Furthermore, we demonstrated that activation of cytochrome c oxidase genes occurs during the development of hypertrophy and that cytochrome c oxidase IV is a direct transcriptional target of E2F-1. These studies demonstrated that E2F-1 activity at specific promoters is dependent on physiological circumstances and that E2F-1 should be considered a potential target in the treatment of pathologic hypertrophy.

Stem Cell Antigen-1 is Necessary for Cell-cycle Withdrawal and Myoblast Differentiation in C2C12 Cells

Journal of Cell Science. Dec, 2004  |  Pubmed ID: 15546912

Extracellular signaling pathways regulating myoblast differentiation and cell-cycle withdrawal are not completely understood. Stem cell antigen-1 (Sca-1/Ly-6A/E) is a glycosylphosphatidylinositol-anchored membrane protein known for its role in T-cell activation, and recently described as a marker for regeneration-competent myoblasts. We previously determined that expression of Sca-1/Ly-6A is transiently upregulated during myocyte cell-cycle withdrawal; however, a specific function for Sca-1 in myogenesis has not been described. Here, we show that Sca-1 expression on the surface of a subpopulation of differentiating C2C12 myoblasts is maximal at the time of cell-cycle withdrawal, and that blocking Sca-1 with monoclonal antibodies or downregulating Sca-1 expression by antisense both promotes proliferation and inhibits myotube formation. Downregulating Sca-1 expression derepresses Fyn at the time of myoblast cell-cycle withdrawal, and dominant-negative and constitutively active Fyn mutants rescue and recapitulate the Sca-1 antisense phenotype, respectively. This suggests a Fyn-mediated mechanism for Sca-1 action. Thus, we demonstrate an unprecedented role for Sca-1 in early myogenesis in C2C12 cells, and propose a novel pathway from the myoblast cell surface to intracellular signaling networks controlling proliferation versus differentiation in mammalian muscle. These findings suggest that, beyond its role as a marker for muscle progenitors, Sca-1 may be an important therapeutic target for promoting muscle regeneration.

CDC2/CDK1 Expression in Esophageal Adenocarcinoma and Precursor Lesions Serves As a Diagnostic and Cancer Progression Marker and Potential Novel Drug Target

The American Journal of Surgical Pathology. Mar, 2005  |  Pubmed ID: 15725809

Esophageal adenocarcinoma arises through well-defined precursor lesions (Barrett esophagus), although only a subset of these lesions advances to invasive adenocarcinoma. The lack of markers predicting progression in Barrett esophagus, typical presentation at advanced stage, and limitations of conventional chemotherapy result in >90% mortality for Barrett-associated adenocarcinomas. To identify potential prognostic markers and therapeutic targets, we compared gene expression profiles from Barrett-associated esophageal adenocarcinoma cell lines (BIC1, SEG1, KYAE, OE33) and normal esophageal epithelial scrapings utilizing the Affymetrix U133_A gene expression platform. We identified 560 transcripts with >3-fold up-regulation in the adenocarcinoma cell lines compared with normal epithelium. Utilizing tissue microarrays composed of normal esophageal squamous mucosa (n = 20), Barrett esophagus (n = 10), low-grade dysplasia (n = 14), high-grade dysplasia (n = 27), adenocarcinoma (n = 59), and node metastases (n = 27), we confirmed differential up-regulation of three proteins (Cdc2/Cdk1, Cdc5, and Igfbp3) in adenocarcinomas and Barrett lesions. Protein expression mirrored histologic progression; thus, 87% of low-grade dysplasias had at least focal surface Cdc2/Cdk1 and 20% had >5% surface staining; 96% of high-grade dysplasias expressed abundant surface Cdc2/Cdk1, while invasive adenocarcinoma and metastases demonstrated ubiquitous expression. Esophageal adenocarcinoma cell lines treated with the novel CDC2/CDK1 transcriptional inhibitor, tetra-O-methyl nordihydroguaiaretic acid (EM-1421, formerly named M4N) demonstrated a dose-dependent reduction in cell proliferation, paralleling down-regulation of CDC2/CDK1 transcript and protein levels. These findings suggest a role for CDC2/CDK1 in esophageal adenocarcinogenesis, both as a potential histopathologic marker of dysplasia and a putative treatment target.

Stem Cell Antigen-1 Regulates the Tempo of Muscle Repair Through Effects on Proliferation of Alpha7 Integrin-expressing Myoblasts

Experimental Cell Research. Mar, 2008  |  Pubmed ID: 18073129

Skeletal muscle repair occurs through a programmed series of events including myogenic precursor activation, myoblast proliferation, and differentiation into new myofibers. We previously identified a role for Stem cell antigen-1 (Sca-1) in myoblast proliferation and differentiation in vitro. We demonstrated that blocking Sca-1 expression resulted in sustained myoblast cell division. Others have since demonstrated that Sca-1-null myoblasts display a similar phenotype when cultured ex vivo. To test the importance of Sca-1 during myogenesis in vivo, we employed a myonecrotic injury model in Sca-1(-/-) and Sca-1(+/+) mice. Our results demonstrate that Sca-1(-/-) myoblasts exhibit a hyperproliferative response consisting of prolonged and accelerated cell division in response to injury. This leads to delayed myogenic differentiation and muscle repair. These data provide the first in vivo evidence for Sca-1 as a regulator of myoblast proliferation during muscle regeneration. These studies also suggest that the balance between myogenic precursor proliferation and differentiation is critical to normal muscle repair.

MicroRNA Regulation of Cell Lineages in Mouse and Human Embryonic Stem Cells

Cell Stem Cell. Mar, 2008  |  Pubmed ID: 18371447

Cell fate decisions of pluripotent embryonic stem (ES) cells are dictated by activation and repression of lineage-specific genes. Numerous signaling and transcriptional networks progressively narrow and specify the potential of ES cells. Whether specific microRNAs help refine and limit gene expression and, thereby, could be used to manipulate ES cell differentiation has largely been unexplored. Here, we show that two serum response factor (SRF)-dependent muscle-specific microRNAs, miR-1 and miR-133, promote mesoderm formation from ES cells but have opposing functions during further differentiation into cardiac muscle progenitors. Furthermore, miR-1 and miR-133 were potent repressors of nonmuscle gene expression and cell fate during mouse and human ES cell differentiation. miR-1's effects were in part mediated by translational repression of the Notch ligand Delta-like 1 (Dll-1). Our findings indicate that muscle-specific miRNAs reinforce the silencing of nonmuscle genes during cell lineage commitment and suggest that miRNAs may have general utility in regulating cell-fate decisions from pluripotent ES cells.

Stem Cell Antigen-1 Localizes to Lipid Microdomains and Associates with Insulin Degrading Enzyme in Skeletal Myoblasts

Journal of Cellular Physiology. Oct, 2008  |  Pubmed ID: 18506847

Stem cell antigen-1 (Sca-1, Ly6A/E) is a glycosylphosphotidylinositol-anchored protein that identifies many tissue progenitor cells. We originally identified Sca-1 as a marker of myogenic precursor cells and subsequently demonstrated that Sca-1 regulates proliferation of activated myoblasts, suggesting an important role for Sca-1 in skeletal muscle homeostasis. Beyond its functional role in regulating proliferation, however, little is known about the mechanism(s) that drive Sca-1-mediated events. We now report that lipid microdomain organization is essential for normal myogenic differentiation, and that Sca-1 constitutively localizes to these domains during myoblast proliferation and differentiation. We also demonstrate that Sca-1 associates with insulin degrading enzyme (IDE), a catalytic protein responsible for the cleavage of mitogenic peptides, in differentiating myoblasts. We show that chemical inhibition of IDE as well as RNAi knockdown of IDE mRNA recapitulates the phenotype of Sca-1 interference, that is, sustained myoblast proliferation and delayed myogenic differentiation. These findings identify the first signaling protein that physically and functionally associates with Sca-1 in myogenic precursor cells, and suggest a potential pathway for Sca-1-mediated signaling. Future efforts to manipulate this pathway may lead to new strategies for augmenting the myogenic proliferative response, and ultimately muscle repair.

Cell Cycle-dependent Phosphorylation of Human CDC5 Regulates RNA Processing

Cell Cycle (Georgetown, Tex.). Jun, 2008  |  Pubmed ID: 18583928

CDC5 proteins are components of the pre-mRNA splicing complex and essential for cell cycle progression in yeast, plants and mammals. Human CDC5 is phosphorylated in a mitogen-dependent manner, and its association with the spliceosome is ATP-dependent. Examination of the amino acid sequence suggests that CDC5L may be phosphorylated at up to 28 potential consensus recognition sequences for known kinases, however, the identity of actual phosphorylation sites, their role in regulating CDC5L activity, and the kinases responsible for their phosphorylation have not previously been determined. Using two-dimensional phosphopeptide mapping and nanoelectrospray mass spectrometry, we now show that CDC5L is phosphorylated on at least nine sites in vivo. We demonstrate that while CDC5L is capable of forming homodimers in vitro and in vivo, neither homodimerization nor nuclear localization is dependent on phosphorylation at these sites. Using an in vitro splicing assay, we show that phosphorylation of CDC5L at threonines 411 and 438 within recognition sequences for CDKs are required for CDC5L-mediated pre-mRNA splicing. We also demonstrate that a specific inhibitor of CDK2, CVT-313, inhibits CDC5L phosphorylation in both in vitro kinase assays and in vivo radiolabeling experiments in cycling cells. These studies represent the first demonstration of a regulatory role for phosphorylation of CDC5L, and suggest that targeting these sites or the implicated kinases may provide novel strategies for treating disorders of unguarded cellular proliferation, such as cancer.

Subpopulations of Human Embryonic Stem Cells with Distinct Tissue-specific Fates Can Be Selected from Pluripotent Cultures

Stem Cells and Development. Dec, 2009  |  Pubmed ID: 19254177

Directed differentiation of human embryonic stem cells (hESCs) has generated much interest in the field of regenerative medicine. While subpopulations of hESCs within pluripotent cultures have been identified based on expression of specific surface antigens, their significance and fates are not well understood. To determine whether such subpopulations indicate specific tissue fates or represent stochastic antigen distributions within proliferating cultures, we isolated CD133(+) or CD135(+) hESCs from proliferating cultures constitutively expressing enhanced green fluorescent protein (GFP), and co-cultured these with unselected GFP(-) hESCs. After passage in culture, GFP(+) hESCs reanalyzed for the persistence of CD133 or CD135 expression, as well as other surface antigens (Tra-1-60, SSEA-4, FGFR-1), demonstrated that these two subpopulations continued to express CD133 or CD135 over serial passage, and that CD133(+) hESCs were enriched for SSEA-4 expression as well. Upon differentiation in vitro, CD133(+)GFP(+) hESCs gave rise solely to ectoderm, as detected by expression of nestin. Tissues representing endoderm (alpha-fetoprotein(+)) and mesoderm (smooth muscle actin(+)) were not seen among GFP(+) tissues. In contrast, selection against CD133 gave rise almost exclusively to mesoderm and endoderm. In contrast, CD135(+)GFP(+) hESCs gave rise to tissues representing all three embryonic germ layers, and were virtually indistinguishable from CD135(-)-derived tissues. Similar results were obtained by in vivo differentiation in teratomas. These data establish that subpopulations of proliferating hESCs whose tissue fate is predetermined exist, and challenge the notion that all cells within proliferating hESC cultures are truly "pluripotent." This co-culture approach also will enable identification of other distinct hESC subpopulations, and selection for these should prove valuable in generating tissue-specific reagents for cell-based therapy.

Usefulness of Various Plasma Biomarkers for Diagnosis of Heart Failure in Children with Single Ventricle Physiology

The American Journal of Cardiology. Nov, 2009  |  Pubmed ID: 19840577

Children with single ventricle physiology have increased ventricular work and are at greater risk of developing heart failure than other children with congenital heart disease. However, the diagnosis of heart failure is difficult because few objective measures have been validated for this cohort. Plasma proteins have been identified as biomarkers of heart failure in adults with structurally normal hearts. However, whether these correlate similarly with heart failure in children with single ventricle physiology is unknown, because the etiology of adult heart failure is typically ischemic heart disease, but heart failure in these children is presumed to be due to primary myocardial dysfunction. We conducted a single-site, cross-sectional observational study of young, single-ventricle patients. Clinical heart failure was defined as a Ross score >2. The association of several candidate biomarkers with heart failure was assessed using logistic regression analysis and receiver operating characteristic curves. Of the 29 included children, 9 (31%) were in clinical heart failure. A doubling of plasma B-type natriuretic peptide was associated with an odds ratio for heart failure of 2.17. The area under the receiver operating characteristic curve was 80.3%. A threshold value of > or =30 pg/ml showed both sensitivity and specificity for heart failure. Three other candidate biomarkers were not associated with clinical heart failure in this sample. In conclusion, plasma B-type natriuretic peptide is a sensitive biomarker for clinical heart failure in young children with single-ventricle heart disease. The use of this plasma biomarker might facilitate detection of heart failure in these complex patients.

Mutations in Potassium Channel Kir2.6 Cause Susceptibility to Thyrotoxic Hypokalemic Periodic Paralysis

Cell. Jan, 2010  |  Pubmed ID: 20074522

Thyrotoxic hypokalemic periodic paralysis (TPP) is characterized by acute attacks of weakness, hypokalemia, and thyrotoxicosis of various etiologies. These transient attacks resemble those of patients with familial hypokalemic periodic paralysis (hypoKPP) and resolve with treatment of the underlying hyperthyroidism. Because of the phenotypic similarity of these conditions, we hypothesized that TPP might also be a channelopathy. While sequencing candidate genes, we identified a previously unreported gene (not present in human sequence databases) that encodes an inwardly rectifying potassium (Kir) channel, Kir2.6. This channel, nearly identical to Kir2.2, is expressed in skeletal muscle and is transcriptionally regulated by thyroid hormone. Expression of Kir2.6 in mammalian cells revealed normal Kir currents in whole-cell and single-channel recordings. Kir2.6 mutations were present in up to 33% of the unrelated TPP patients in our collection. Some of these mutations clearly alter a variety of Kir2.6 properties, all altering muscle membrane excitability leading to paralysis.

Labeling Human Embryonic Stem Cell-derived Cardiomyocytes with Indocyanine Green for Noninvasive Tracking with Optical Imaging: an FDA-compatible Alternative to Firefly Luciferase

Cell Transplantation. 2010  |  Pubmed ID: 20370988

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have demonstrated the ability to improve myocardial function following transplantation into an ischemic heart; however, the functional benefits are transient possibly due to poor cell retention. A diagnostic technique that could visualize transplanted hESC-CMs could help to optimize stem cell delivery techniques. Thus, the purpose of this study was to develop a labeling technique for hESCs and hESC-CMs with the FDA-approved contrast agent indocyanine green (ICG) for optical imaging (OI). hESCs were labeled with 0.5, 1.0, 2.0, and 2.5 mg/ml of ICG for 30, 45, and 60 min at 37 degrees C. Longitudinal OI studies were performed with both hESCs and hESC-CMs. The expression of surface proteins was assessed with immunofluorescent staining. hESCs labeled with 2 mg ICG/ml for 60 min achieved maximum fluorescence. Longitudinal studies revealed that the fluorescent signal was equivalent to controls at 120 h postlabeling. The fluorescence signal of hESCs and hESC-CMs at 1, 24, and 48 h was significantly higher compared to precontrast data (p < 0.05). Immunocytochemistry revealed retention of cell-specific surface and nuclear markers postlabeling. These data demonstrate that hESCs and hESC-CMs labeled with ICG show a significant fluorescence up to 48 h and can be visualized with OI. The labeling procedure does not impair the viability or functional integrity of the cells. The technique may be useful for assessing different delivery routes in order to improve the engraftment of transplanted hESC-CMs or other stem cell progenitors.

Timed Inhibition of P38MAPK Directs Accelerated Differentiation of Human Embryonic Stem Cells into Cardiomyocytes

Cytotherapy. Oct, 2010  |  Pubmed ID: 20586669

Heart failure therapy with human embryonic stem cell (hESC)-derived cardiomyocytes (hCM) has been limited by the low rate of spontaneous hCM differentiation. As others have shown that p38 mitogen-activated protein kinase (p38MAPK) directs neurogenesis from mouse embryonic stem cells, we investigated whether the p38MAPK inhibitor, SB203580, might influence hCM differentiation.

Cardiac Regeneration Using Human Embryonic Stem Cells: Producing Cells for Future Therapy

Regenerative Medicine. Sep, 2010  |  Pubmed ID: 20868331

Directed differentiation of human embryonic stem cells (hESCs) has generated much interest in the field of regenerative medicine. Because of their ability to differentiate into any cell type in the body, hESCs offer a novel therapeutic paradigm for myocardial repair by furnishing a supply of cardiomyocytes (CMs) that would ultimately restore normal myocardial function when delivered to the damaged heart. Spontaneous CM differentiation of hESCs is an inefficient process that yields very low numbers of CMs. In addition, it is not clear that fully differentiated CMs provide the benefits sought from cell transplantation. The need for new methods of directed differentiation of hESCs into functional CMs and cardiac progenitors has led to an explosion of research utilizing chemical, genetic, epigenetic and lineage selection strategies to direct cardiac differentiation and enrich populations of cardiac cells for therapeutic use. Here, we review these approaches and highlight their increasingly important roles in stem cell biology and cardiac regenerative medicine.

Letter by Lowenthal Et Al Regarding Article, "BNP Levels Predict Outcome in Pediatric Heart Failure Patients: Post Hoc Analysis of the Pediatric Carvedilol Trial"

Circulation. Heart Failure. Nov, 2010  |  Pubmed ID: 21081737

An Engineered Cardiac Reporter Cell Line Identifies Human Embryonic Stem Cell-derived Myocardial Precursors

PloS One. 2011  |  Pubmed ID: 21245908

Unlike some organs, the heart is unable to repair itself after injury. Human embryonic stem cells (hESCs) grow and divide indefinitely while maintaining the potential to develop into many tissues of the body. As such, they provide an unprecedented opportunity to treat human diseases characterized by tissue loss. We have identified early myocardial precursors derived from hESCs (hMPs) using an α-myosin heavy chain (αMHC)-GFP reporter line. We have demonstrated by immunocytochemistry and quantitative real-time PCR (qPCR) that reporter activation is restricted to hESC-derived cardiomyocytes (CMs) differentiated in vitro, and that hMPs give rise exclusively to muscle in an in vivo teratoma formation assay. We also demonstrate that the reporter does not interfere with hESC genomic stability. Importantly, we show that hMPs give rise to atrial, ventricular and specialized conduction CM subtypes by qPCR and microelectrode array analysis. Expression profiling of hMPs over the course of differentiation implicate Wnt and transforming growth factor-β signaling pathways in CM development. The identification of hMPs using this αMHC-GFP reporter line will provide important insight into the pathways regulating human myocardial development, and may provide a novel therapeutic reagent for the treatment of cardiac disease.

Treatment of Pulmonary Arterial Hypertension with Circulating Angiogenic Cells

American Journal of Physiology. Lung Cellular and Molecular Physiology. Jul, 2011  |  Pubmed ID: 21398496

Despite advances in the treatment of pulmonary arterial hypertension, a truly restorative therapy has not been achieved. Attention has been given to circulating angiogenic cells (CACs, also termed early endothelial progenitor cells) because of their ability to home to sites of vascular injury and regenerate blood vessels. We studied the efficacy of human CAC therapy in the treatment of pulmonary arterial hypertension at two different stages of disease severity. Cells were isolated from peripheral blood and administered to nude rats on day 14 ("early") or day 21 ("late") after monocrotaline injection. The control group received monocrotaline but no cell treatment. Disease progression was assessed using right heart catheterization and echocardiography at multiple time points. Survival differences, right ventricular hypertrophy (RVH), and vascular hypertrophy were analyzed at the study endpoint. Quantitative PCR was performed to evaluate cell engraftment. Treatment with human CACs either at the early or late time points did not result in increased survival, and therapy did not prevent or reduce the severity of disease compared with control. Histological analysis of RVH and vascular muscularization showed no benefit with therapy compared with control. No detectable signal was seen of human transcript in transplanted lungs at 14 or 21 days after cell transplant. In conclusion, CAC therapy was not associated with increased survival and did not result in either clinical or histological benefits. Future studies should be geared toward either earlier therapeutic time points with varying doses of unmodified CACs or genetically modified cells as a means of delivery of factors to the pulmonary arterial circulation.

The Promise of Human Embryonic Stem Cells in Aging-associated Diseases

Aging. May, 2011  |  Pubmed ID: 21566262

Aging-associated diseases are often caused by progressive loss or dysfunction of cells that ultimately affect the overall function of tissues and organs. Successful treatment of these diseases could benefit from cell-based therapy that would regenerate lost cells or otherwise restore tissue function. Human embryonic stem cells (hESCs) promise to be an important therapeutic candidate in treating aging-associated diseases due to their unique capacity for self-renewal and pluripotency. To date, there are numerous hESC lines that have been developed and characterized. We will discuss how hESC lines are derived, their molecular and cellular properties, and how their ability to differentiate into all three embryonic germ layers is determined. We will also outline the methods currently employed to direct their differentiation into populations of tissue-specific, functional cells. Finally, we will highlight the general challenges that must be overcome and the strategies being developed to generate highly-purified hESC-derived cell populations that can safely be used for clinical applications.

Usefulness of B-Type Natriuretic Peptide and N-Terminal Pro-B-Type Natriuretic Peptide As Biomarkers for Heart Failure in Young Children With Single Ventricle Congenital Heart Disease

The American Journal of Cardiology. Dec, 2011  |  Pubmed ID: 22196786

Children with single ventricle (SV) physiology have increased ventricular work and are at risk of heart failure (HF). However, a HF diagnosis is especially difficult, because few objective measures of HF have been validated in this cohort. We have previously shown that plasma B-type natriuretic peptide (BNP) levels are sensitive and specific for detecting HF in a small, heterogeneous SV cohort. The aim of the present study was to define the effect of SV morphology and stage of palliation on the correlation between BNP and HF. We also examined the utility of N-terminal pro-BNP (NT-proBNP), a more stable product of pre-BNP processing, as a biomarker of HF in these patients. A cross-sectional observational study of SV children aged 1 month to 7 years was conducted. The presence of HF was defined as a Ross score >2. The association of BNP or NT-proBNP with HF was assessed using logistic regression analysis and receiver operating characteristic curves. Of the 71 included children, 22 (31%) had clinical HF. A doubling of BNP was associated with an odds ratio for HF of 2.20 (95% confidence interval 1.36 to 3.55, p = 0.001) with a c-statistic >75%, yielding a detection threshold of ≥45 pg/ml. This threshold was preserved when patients were stratified by the right ventricular morphology or stage of surgical palliation. Similarly, a doubling of NT-proBNP was associated with an odds ratio for HF of 1.92 (95% confidence interval 1.17 to 3.14, p = 0.009). In contrast to BNP, the threshold value of NT-proBNP for predicting HF decreased with the stage of palliation. In conclusion, plasma BNP and NT-proBNP are reliable tests for clinical HF in young children with SV physiology, specifically those with right ventricular morphology, regardless of the stage of palliation.

High-throughput Tracking of Pluripotent Human Embryonic Stem Cells with Dual Fluorescence Resonance Energy Transfer Molecular Beacons

Stem Cells and Development. Mar, 2011  |  Pubmed ID: 20624034

Pluripotent human embryonic stem cells (hESCs) provide an unprecedented opportunity for the study of human tissue development, and the development of cell-based therapies for human disease. To realize these potential advances, however, methods for monitoring expression of intracellular proteins in live hESCs without altering cellular properties are needed. Molecular beacons are single-stranded oligonucleotides that have been employed to assay gene expression. To test their potential for high-throughput isolation of hESCs, we developed a dual fluorescence resonance energy transfer (FRET) molecular beacon system using fluorescence-activated cell sorting (FACS) with Oct4 as a target. We demonstrate that Oct4 can be detected by FRET using confocal microscopy, that this can be applied in a high-throughput manner to the identification and isolation of Oct4-expressing hESCs by FACS, that FRET-positive hESCs demonstrate pluripotency in culture and in vivo, and that hESCs transfected with molecular beacons demonstrate normal growth rates and oligonucleotide extinction over time. These studies demonstrate that FRET-based FACS using molecular beacons provides a useful tool for isolating Oct4-expressing pluripotent hESCs, and may also be adapted to selecting differentiating hESCs at specific developmental time points determined by transcription factor expression without functional or genomic alteration. As such, it provides an important new method for high-throughput isolation of hESC-derived tissue-specific precursors for analytic and therapeutic purposes.

Sca-1 Cardiosphere-derived Cells Are Enriched for Isl1-expressing Cardiac Precursors and Improve Cardiac Function After Myocardial Injury

PloS One. 2012  |  Pubmed ID: 22272337

Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.

Myocardial Improvement with Human Embryonic Stem Cell-derived Cardiomyocytes Enriched by P38MAPK Inhibition

Cytotherapy. Feb, 2012  |  Pubmed ID: 22040108

We have shown previously that inhibition of the p38 mitogen-activated protein kinase (p38MAPK) directs the differentiation of human embryonic stem cell (hESC)-derived cardiomyocytes (hCM). We investigated the therapeutic benefits of intramyocardial injection of hCM differentiated from hESC by p38MAPK inhibition using closed-chest ultrasound-guided injection at a clinically relevant time post-myocardial infarction (MI) in a mouse model.