Supplementary MaterialsSupplementary Information srep16842-s1

Supplementary MaterialsSupplementary Information srep16842-s1. approved by the Animal Experimentation Committee, Kyoto University. All animal experiments were performed according to the hybridization (SS-FISH). GHM-constructs transplantation Seven days after inducing MI, each rat was randomly assigned to one of the three groups: GHM-construct TX group, control-construct TX group, and sham group. In the former two groups, five-cardiovascular cell sheet constructs with or without GHMs were Beta-Cortol applied to the surface of the anterior wall of the heart as previously described16. In summary, the constructs were spread manually to cover the whole MI area and the border area and stably placed onto the surface of the heart without sutures. The chest was closed 15C20?minutes after surgery. In sham-operated group, the chest was closed 15C20?minutes after thoracotomy. Cardiac function assessment To assess global cardiac function and left ventricle (LV) size, echocardiograms were performed with the Vivid 7 system (GE Healthcare, Waukesha, WI) and an 11-MHz imaging transducer (GE 10S ultrasound probe, GE Healthcare). Echocardiograms were performed before ligation (baseline), and on day 6 (pre TX, i.e., 6 days post-MI), and 1, 2, 4, 8, and 12 weeks after TX by an independent person in a blinded fashion as previously described16,33,34. Diastolic and systolic area of LV (LVAd, LVAs), diastolic lengths of LV inner circumference (CIRCd) and those of akinetic area in diastole (SCAR) were recorded and measured with B-mode examination. Values were calculated as follows: Fractional shortening (FS) (%)?=?(LVDdCLVDs)/LVDd 100. Akinetic length Ctsk (AL) (%)?=?SCAR/CIRCd 100. Besides the experimental model (GHM-construct TX group, control TX group, or sham group), echocardiograms were performed on normal rats, which had no surgical intervention in order to quantify the normal values of the parameters of Beta-Cortol the lineage/age/weight-matched rats (n?=?5). Species-specific FISH analysis FISH probes which recognize and hybridize with sequence repeats specific for each animal species were arranged by Chromosome Science Labo (Sapporo, Japan)16,35,36. The nucleotide probes were applied to the fixed and pre-treated sections that were denatured and then hybridized. Additional IF staining for cTnT and vWF was performed on the FISH samples. Samples were examined by fluorescence microscopy (LSM 710 Laser Scanning Microscopes, Carl Zeiss, Oberkochen, Germany) and Carl Zeiss software. Histological analyses For cross-sectional observation, cardiovascular cell sheets were fixed in 4% paraformaldehyde and routinely processed into 5-m-thick paraffin-embedded sections. Hematoxylin and eosin (HE) staining was performed using conventional methods as previously described16,33,34. For cTnT-staining, sections were incubated for 60?min with primary antibody at room temperature, and then applied to LSAB2 kit/horseradish peroxidase (HRP) (diaminobenzidine; DAB) (DAKO) according to the manufacturers instructions. Hearts were immersed and perfusion fixed with 4% PFA and embedded in OCT compound (Sakura Finetek Japan, Tokyo, Japan) and frozen. Several 5-micrometer sections were made at 50-m intervals along the short axis and examined. For IF staining, sections Beta-Cortol were treated with Protein Block Serum Free (DAKO) and incubated for 60?min with primary antibodies at room temperature. The area of engraftment was calculated as double positive cells for cTnT staining and mouse signal with SS-FISH or as positive cells for Hoechst 33342. For lectin perfusion analysis, rats were Beta-Cortol received intravenous injections of 1 1.5 ml of 1 1 mg/ml DyLight 594-conjugated tomato (Lycopersicon esculentum) lectin (Dye-lectin) (Vector Labs, Burlingame, CA) in PBS into the inferior vena cava 15 min prior to sacrifice. After excision, the hearts were sectioned manually into 5-micrometer that were made at 50-micrometer intervals along the short axis and examined. All immunostained sections were photographed and calculated with Biorevo BZ-9000 or LSM 710 Laser Scanning Microscopes (Carl Zeiss, Oberkochen, Germany). Extracellular field potential measurement Extracellular field potential (EFP) of cell sheet constructs before and after TX was measured by.