Supplementary MaterialsSupplementary Data. genes owned by the potassium voltage-gated channel family or solute carrier family, partially explaining the irregular Ca2+ transients and contractility in ethanol-treated hiPSC-CMs. RNA-seq also showed significant upregulation in the expression of genes associated with collagen and extracellular matrix modeling, and downregulation of genes involved in cardiovascular system development and actin filament-based process. These results suggest that hiPSC-CMs can be a novel and physiologically relevant system for the study of alcohol-induced cardiac toxicity. 2016 and an open?access website (http://pga.mgh.harvard.edu/primerbank/). Thermocycler reaction was set up as follows using the iTaq SyBr green grasp mix: Initial denaturation step at 95C for 10?min, 40 cycles of m-Tyramine hydrobromide 2 actions with 15?s of denaturation at 95C followed by 1?min of annealing m-Tyramine hydrobromide at 60C using Applied Biosystems 7500 real-time PCR systems. All samples were normalized to the level of the housekeeping gene GAPDH. Relative expression levels compared with control samples were presented as fold changes calculated m-Tyramine hydrobromide by the 2 2?Ct method. Data are presented as mean?SD. Statistical significance was analyzed using paired Mouse monoclonal to CD22.K22 reacts with CD22, a 140 kDa B-cell specific molecule, expressed in the cytoplasm of all B lymphocytes and on the cell surface of only mature B cells. CD22 antigen is present in the most B-cell leukemias and lymphomas but not T-cell leukemias. In contrast with CD10, CD19 and CD20 antigen, CD22 antigen is still present on lymphoplasmacytoid cells but is dininished on the fully mature plasma cells. CD22 is an adhesion molecule and plays a role in B cell activation as a signaling molecule values? .05 were considered significant. Cell purity assay Cardiac spheres were dissociated using 0.25% trypsin-EDTA and plated onto a Matrigel-coated 96-well culture plate at a density of 5104 cells/well and cultured for 2?days to recover spontaneous beating. Treatment groups were maintained in ethanol-containing medium for 5?days and the medium was covered with mineral oil to prevent ethanol evaporation. On day 5, ethanol-containing medium and mineral oil were aspirated and cells were washed with D-PBS and fixed in paraformaldehyde (Sigma) and permeabilized in cold methanol. The cells were then blocked with 5% NGS in D-PBS at room heat for 1?h and incubated with the primary antibody for NKX2-5 overnight at 4C. After the incubation with the primary antibody, the cells were washed 3 times with D-PBS and incubated with the corresponding conjugated secondary antibodies followed by 3 times wash with D-PBS. The nuclei were counterstained with Hoechst in warm buffer and imaged using an ArrayScan XTI Live High Content Platform (Life Technologies). Images of NKX2-5 positive cells and Hoechst were acquired and quantitatively analyzed using ArrayScan XTI Live High Content Platform. Twenty fields/well were selected and 5 replicate wells per condition were imaged using a 10 objective. Acquisition software Cellomics Scan (ThermoFisher Scientific) was used to capture images, and m-Tyramine hydrobromide data analysis was performed using Cellomics View Software (ThermoFisher Scientific). Images were analyzed with mask modifier for Hoechst and NKX2-5-positive cells restricted to the nucleus. Percentage of NKX2-5-positive cells and mean average fluorescence intensity of NKX2-5 in each treatment were used as readout. Calcium imaging Human-induced pluripotent stem cell-derived CMs were dissociated with 0.05% trypsin-EDTA, seeded onto Matrigel-coated 25?25 mm glass coverslips and cultured for 2C3?days until they recovered beating. The cells were then treated with 0, 17, and 50?mM of ethanol for 5?days in sealed chambers to prevent ethanol evaporation (Polikandriotis system to explore the molecular underpinnings of prenatal alcohol exposure. In addition, this cell model can be leveraged for high-throughput screening of potential drugs to treat alcohol-induced cardiac defects. For example, the irregular Ca2+ transients observed in ethanol-treated hiPSC-CMs may be served as a functional readout for screening antiarrhythmic drugs using high-throughput Ca2+ imaging. Such a study will not only advance the application of hiPSCs in drug discovery for treatment of alcohol exposure-induced heart disease but also facilitate establishment of hiPSC models for studies of alcohol-induced damages in other organs because hiPSCs have the ability to differentiate into various cell types. Moreover, hiPSC lines of variable genetic backgrounds (eg, different activities of enzymes for detoxification) may also facilitate investigation on the effects of genetic variation around the susceptibility to alcohol-induced injury as well as the development of effective treatments. Supplementary Material Supplementary DataClick here for additional data file.(1.0M, pdf) ACKNOWLEDGMENTS We thank the m-Tyramine hydrobromide staff at the high-throughput DNA Sequencing Core of the Parker H. Petit Institute for Bioengineering & Bioscience, Georgia Institute of Technology for their help with the preparation of the RNA-seq library. FUNDING Center for Pediatric Technology Center at Emory/Georgia Tech, NIH/NIEHS (P30ES019776), Woodruff.