Illumina HiSeq 2500 paired end sequencing; Atrial Myxomas Arise From Multipotent Myxoid Cardiac Stem Cells
Atrial Myxomas Arise From Multipotent Myxoid Cardiac Stem Cells
Myxoid and non-myxoid human tissue samples were obtained from patients undergoing heart surgery and were collected only when removal of tissue was required for surgical reasons. Collection of myxoid tissues samples was approved by the local ethics committee at the University of Campania "L. Vanvitelli" of Naples. Written informed consent was obtained from every patient before cardiac surgery. All patient data were kept anonymous. Myxoma polypoid samples, with irregular or smooth appearance, were obtained from right or left atria from patients during cardiac surgery. We examined 19 left and 4 right atrial myxomas obtained from 8 male and 15 female patients 60±10 years old. In the last 6 consecutive tumours, freshly excised samples were equally divided to be fixed for immunohistochemistry analysis or immediately processed for cell harvesting as below described.Human myocardial samples were obtained, after written consent, from right or left atria of patients undergoing cardiac surgery at the University of Magna Graecia of Catanzaro and were used as controls (63± 4 years old). All patient data were kept anonymous. Both types of human samples were stored in saline on ice until ready to process (~1hr). All steps were performed at 4°C unless stated otherwise. Briefly, cardiac tissue was minced then digested with collagenase II (0.3mg/ml; Worthington Laboratories) in Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich) at 37°C in a series of sequential digestions for 3 minutes each. Enzymatically released cells were filtered through a 40μm cell strainer (Becton Dickinson, BD) and collected in enzyme quenching media (DMEM + 10% FBS). The isolated cardiac cells were collected by centrifugation at 400g for 10 min, resuspended in incubation media (PBS, 0.5% BSA, 2 mM EDTA).For the isolation of c-kitpos/CD45neg/CD31neg CSCs, first the small cardiac cells were depleted of CD45pos and CD31pos cells by immunolabelling with anti-human CD45 and CD31 magnetic immunobeads (Miltenyi) diluted (1:10) in incubation media for 30 minutes at 4°C with agitation. After antibody binding, the CD45pos/CD31pos cells were depleted from the preparation using magnetic activated cell sorting (MACS; Miltenyi). The elution cells, which were CD45neg/ CD31neg were then enriched for c-kitpos cardiac cells through incubation with anti-human CD117 immunobeads (Miltenyi) (1:10) for 30 minutes at 4°C with agitation and again sorted using MACS according to the manufacturer's instructions. Libraries were prepared as described in Torella et al., (3). In detail , indexed libraries were prepared using 1 ug of total RNA as starting material, with TruSeq Stranded Total RNA Sample Prep Kit (Illumina Inc.). Libraries were sequenced (paired-end,2x100 cycles) at a concentration of 8 pM/lane on the HiSeq 2500 platform (Illumina Inc).
Experimental Factor: disease
normal