NextSeq 500 sequencing; Immortalized cardiac cell line characterization and comparison (rat/rat-derived)
Immortalized cardiac cell line characterization and comparison (rat/rat-derived)
H9C2-CON-1_s
Cells seeded onto 6-well plates were harvested either in proliferation stage (one day before full confluence; day -1) or in differentiated stage (H9C2, AC16: day 8-10; hiPSC-CM: day 40) in Qiazol Lysis buffer (79306, Qiagen, Hilden, Germany) for RNA isolation. Human left ventricular tissue samples from free wall were taken at the time of heart explantation (avoiding scarred, fibrotic, or adipose tissue, endocardium, epicardium, or coronary vessels). Rat left ventricular tissue was collected from male Wistar rats (n=3), and mouse left ventricular tissue was collected from C57BL/6 mice (n=3) by excision of the heart after anesthesia with an intraperitoneal injection of 60 mg/kg pentobarbital (#17F015. Produlab Pharma, Raamsdonksweer, The Netherlands,). Samples were rinsed immediately in saline, blotted dry, frozen in liquid nitrogen and kept at −80°C until further processing. Rat cardiomyoblast H9C2 cell line obtained from ECACC (passage 29; CRL-1446) was maintained in growth medium at 37°C in a humidified atmosphere of 5% CO2. Cells were subcultured at 70% confluence in order to maintain morphological characteristics. Human cardiac myocyte AC16 cell line obtained from Merck (Lot: RD1606008; SCC109) was maintained in growth medium at 37°C in a humidified atmosphere of 5% CO2. Cells were subcultured until 70% confluence then divided. Mouse cardiomyocyte HL-1 cell line obtained from Merck (SCC065, Lot: RD1601001) was cultured as previously described. Briefly, cells were seeded on gelatin/fibronectin coated flasks or plates and maintained in HL-1 expansion medium at 37°C in a humidified atmosphere of 5% CO2. The XCL-1, male control pluripotent stem cell line reprogrammed from CD34+ cord blood cells by episomal vectors, were purchased from XCellScience (CAXIP-001-1V, Novato, USA). The hiPSCs were maintained under standard conditions in 5% CO2 incubator at 37°C, on Matrigel (Corning Inc, Corning, NY, USA) coated six well plates in mTeSR medium (Stemcell Technologies, Vancouver, Canada).  After H9C2 cells reached full confluence (day 0), growth medium was changed to a medium containing 1% FBS for 24 hours. On day 1, medium was supplemented with 10nM all-trans retinoic acid. Differentiation medium was changed on every two to three days.After AC16 cells reached 80% confluence (day 0), growth medium was changed to differentiation media with 2% FBS supplemented with 1× insulin-transferrin-selenite supplement and with 10nM ATRA.To perform differentiation on hiPSC into cardiomyocyte dense monolayers (75-90% confluent) of hiPSCs were used at passage 26. Differentiation was initiated by medium change to RPMI 1640 medium supplemented with insulin-free B27 containing 10 uM CHIR99021. On day 2 medium was changed to RPMI medium supplemented with insulin-free B27.The next day medium was changed to RPMI medium supplemented with insulin-free B27 containing 2.5 uM C59 Wnt inhibitor. From day 5, the medium was changed every other day with RPMI supplemented with B27 (containing insulin). First beating areas typically arised after 7 days following induction. The metabolic selection was performed by medium change to RPMI without glucose supplemented with B27 for 4 days between days 10-14. Cells and tissue samples were collected and lysed in 1 mL of QIAzol Lysis Reagent (79306, Qiagen, Hilden, Germany). Total RNA was extracted from 350 µl of the lysates using Direct-zol RNA MiniPrep System with on-column DNAse I treatment according to the manufacturer's (Zymo Research, Irvine, CA, USA) protocol. The RNA Integrity Numbers and RNA concentration were determined by RNA ScreenTape system with 2200 Tapestation (Agilent Technologies, Santa Clara, CA, USA) and RNA HS Assay Kit with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. For Gene Expression Profiling (GEx) library construction, QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen GmbH, Wien, Austria) was applied according to the manufacturer's protocol. The quality and quantity of the library was determined by using High Sensitivity DNA1000 ScreenTape system with 2200 Tapestation (Agilent Technologies, Santa Clara, CA, USA) and dsDNA HS Assay Kit with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively.