Lentiviral Vectors and Protocols for Creation of Stable hESC Lines for Fluorescent Tracking and Drug Resistance Selection of Cardiomyocytes

Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and alpha-myosin heavy chain (alphaMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function.The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications

Patterns of Blood Protein Concentrations of ELGANs Classified by Three Patterns of Respiratory Disease in the First 2 Postnatal Weeks

We examined the association between elevated concentrations of 25 blood proteins in blood spots collected on postnatal days 1, 7, and 14 from infants < 28 weeks gestation who survived to 24 months and the risk of two patterns of early lung disease i.e., early and persistent pulmonary dysfunction (EPPD), and normal early pulmonary function followed by pulmonary deterioration (PD). 38% (N=347) of our cohort had PD, and 43% (N=383) had EPPD. On postnatal day 14, elevated concentrations of two proteins (RANTES and VEGF) were associated with reduced risk of PD. Similarly, the risk of EPPD was also reduced if three proteins had elevated concentrations on postnatal day 14 (RANTES, MMP-1, and VEGF). In contrast, the risk of EPPD was increased if on day 14 two proteins had elevated concentrations (IL-8 and ICAM-1). Inflammation might influence the risk of EPPD and PD, or be a consequence of lung damage or therapies to minimize lung dysfunction

Ambient pollutants, polymorphisms associated with microRNA processing and adhesion molecules: the Normative Aging Study

<p>Abstract</p> <p>Background</p> <p>Particulate air pollution has been associated with cardiovascular morbidity and mortality, but it remains unclear which time windows and pollutant sources are most critical. MicroRNA (miRNA) is thought to be involved in cardiovascular regulation. However, little is known about whether polymorphisms in genes that process microRNAs influence response to pollutant exposure. We hypothesized that averaging times longer than routinely measured one or two day moving averages are associated with higher soluble intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1) levels, and that stationary and mobile sources contribute differently to these effects. We also investigated whether single nucleotide polymorphisms (SNPs) in miRNA-processing genes modify these associations.</p> <p>Methods</p> <p>sICAM-1 and sVCAM-1 were measured from 1999-2008 and matched to air pollution monitoring for fine particulate matter (PM_2.5) black carbon, and sulfates (SO₄^2-). We selected 17 SNPs in five miRNA-processing genes. Mixed-effects models were used to assess effects of pollutants, SNPs, and interactions under recessive inheritance models using repeated measures.</p> <p>Results</p> <p>723 participants with 1652 observations and 1-5 visits were included in our analyses for black carbon and PM_2.5. Sulfate data was available for 672 participants with 1390 observations. An interquartile range change in seven day moving average of PM_2.5(4.27 μg/m³) was associated with 3.1% (95%CI: 1.6, 4.6) and 2.5% (95%CI: 0.6, 4.5) higher sICAM-1 and sVCAM-1. Interquartile range changes in sulfates (1.39 μg/m³) were associated with 1.4% higher (95%CI: 0.04, 2.7) and 1.6% (95%CI: -0.4, 3.7) higher sICAM-1 and sVCAM-1 respectively. No significant associations were observed for black carbon. In interaction models with PM_2.5, both sICAM-1 and sVCAM-1 levels were lower in rs1062923 homozygous carriers. These interactions remained significant after multiple comparisons adjustment.</p> <p>Conclusions</p> <p>PM_2.5seven day moving averages are associated with higher sICAM-1 and sVCAM-1 levels. SO₄^-2seven day moving averages are associated with higher sICAM-1 and a suggestive association was observed with sVCAM-1 in aging men. SNPs in miRNA-processing genes may modify associations between ambient pollution and sICAM-1 and sVCAM-1, which are correlates of atherosclerosis and cardiovascular disease.</p