Isolation and Identification of Human ASCs
Subcutaneous abdominal adipose tissues were obtained from a healthy male who underwent a liposuction surgery. Adipose tissue was washed in phosphate-buffered saline (PBS) and minced, followed by digestion in 5 ml of type I Collagenase (1 mg/ml in 1% bovine serum albumin(BSA)/Hank’s balanced saline solution; Life Technologies Japan) for 40 minutes at 37°C using a gentle MACS Dissociator (MiltenyiBiotec K.K., Tokyo, Japan) according to the manufacturer’s instructions. The digested tissue was filtered through a 40-μm cell strainer (BD Falcon, Tokyo, Japan) and centrifuged at 450g for 10 minutes. The supernatant containing adipocytes and debris were discarded. Pelleted cells were rinsed twice with PBS, and then planted on the petri dishes with a density of ~2.0×105/cm2. ASCs were cultured according to the standard protocol, with our modiﬁed media in Dulbecco’s modified Eagle’s medium (DMEM)-F12 (Gibco), supplemented with 10% FBS (HyClone), 100U/ml penicillin, and 100μg/ml streptomycin (Biolot). ASCs were subcultured after attaining 70-80% conﬂuency. Passage 3-4 cells were used in the study.
ASCs were identified with flowcytometry analysis by incubation with primary antibodies for 40 min at 4 °C in phosphate-buffered saline (PBS) supplemented with 2% FBS and 2mM ethylenediaminetetraacetic acid (EDTA). The following direct conjugated antibodies (BD Pharmingen™) were used: anti-human (PE-CD34, PerCP-CD45, PE-CD90, APC-CD44, PE-CD73, and PE-CD105). All staining was controlled with appropriate isotope control antibodies. Analysis was performed on a SORP LSRII (Becton Dickinson) equipped with five lasers and data were collected with FACS DIVA software. Analysis was performed using FlowJo™ 10.0.8 (Treestar, Ashland, OR). All measurements were performed with three biological replicates.
Linage differentiation tests for osteogenesis, adipogenesis and chondrogenesis was performed as described before [18, 19]. Osteogenic differentiation was evaluated by cellular alkaline phosphatase (ALP) activity (Alkaline Phosphatase kit, 86R; Sigma-Aldrich). Adipogenesis was confirmed by Oil red O staining of intracellular lipids, and chondrogenesis was confirmed by Alcian blue staining .
Hypoxia Precondition of ASCs
Hypoxic treatment protocol was referred as previously reported with some modification . A total of 3 x 106 ASCs (P3) were seeded on a T75 flask and cultured at 2% O2 (hypoxia) in a ProOx-C-chamber system (Biospherix, Redfield, NY), in comparation with the ambient oxygen tension 21% O2 (normoxia). Seventy-two hours later, cells were harvested for RNA expression analysis (Supplement Table 1).
Cord blood mononuclear cells (CBMNCs) Isolation
Isolation of CBMNCs was performed as reported before . In brief, 35ml of fresh cord blood was collected from a healthy woman with an uncomplicated delivery. The blood was mixed with 7ml (1:5) Hetastarch solution (HetaSep™, STEM CELL TECH, US), and incubated at 37℃ for 30 min. Then, the cells in the floating layer were transferred to another tube, mixed with normal saline (NS) to 50ml, and centrifuged for 10min (1500rpm). After having been washed for 2 times, the cord blood cells were mixed with a 5ml lymphocyte separation medium (50494 LSM®, CappelTM, Shanghai), and centrifuged for 20min (1500rpm). The thin layer of mononuclear cells was extracted carefully and washed 2 times. Cell viability was evaluated with a trypan blue exclusion test, and cell concentration was brought to 1.5-2.5×106 cells/ml and used within 30 min.
Surgical Procedure and Transplantation
Female Wistar rats (250–300g) were intubated under general anesthesia with 4% chloral hydrate (4 mg/kg, intraperitoneally injection) and ventilated with room air by a small animal ventilator (SAR-830/AP, CWE, US). Myocardial infarction was induced by permanent ligation of the left anterior descending coronary artery with a 6–0 silk suture. Successful performance of coronary occlusion was verified by blanching of the myocardium distal to the coronary ligation . Stem cells or PBS were transplanted immediately after ligation of the left anterior descending coronary artery. Transplanted groups were divided into 5 groups: (1) HP-ASCs group (n=10, 1x106ASCs); (2) HP-ASCs + CBMNCs group (n=10, 0.5×106 ASCs+0.5×106 EPCs); (3) CBMNCs group (n=10, 1×106 ASCs); (4) Control group (n=10, 40μL PBS); (5) Sham group (n=10). A total amount of 40μL mixture was directly injected into 5 spots around the ischemic region, except the sham group, which only underwent the thoracotomy without coronary artery ligament or injection.
Measurement of Heart Function
Echocardiography (Vevo770; Visualsonics, Toronto, ON, Canada) was performed at 0d and 30d after myocardial ischemia. Rats were anesthetized with 4% chloral hydrate (40 mg/kg, intraperitoneally), and imaged in the supine position at the fourth and fifth intercostal space with a 710B transducer. Both 2D and M-mode images were used for measurements, and images were later analyzed by a trained blind reader using the cardiac analysis software (VisualSonics, version 2.2.3). The following variables are measured: ejection fraction (EF), fractional shortening (FS), stroke volume (SV), heart rate (HR), and left ventricle posterior wall thickening (LVPW) were measured.
Infarct Size Measurement
All the hearts were harvested at 30d and embedded in optimal cutting temperature (OCT) compound (Sakura Finetek USA Inc, Torrance, Calif). The infarct and peri-infarct regions were cut into three transverse sections then and stained by Masson trichrome and hematoxylin–eosin (HE). The stained sections were measured and calculated for the average ratio of fibrosis area (blue) to the entire LV area (percentage of fibrosis area), and the average ratio of the reduced LV wall thickness in the scarred area to the intact LV wall thickness from three different sites in each wall (LV wall thinning %). For each slice, 10 randomly selected fields were captured (× 100) and images were digitized and analysed with a digital image analyser (MIQAS, Qiuwei Co, China).
Living ASCs Cell Count and Vessel Count in Necrotic Area
To count the living ASCs in heart sections, we used anti-human SRY antibody (Cat#MA5-17181, 1:200, Invitrogen, US) to trace the ASCs cells (ASCs used were only from human male). Six fields per heart section were randomly chosen and photographed under 40× magnification with a fluorescent microscope, and live cells (cells/mm2) were counted in every tenth heart section across the entire region of interest.
For vascular counting, we used anti-human CD31 (Cat#ab32457, 1:200, Abcam, Shanghai) to stain the newly formed capillaries. The number of capillaries was counted under a light microscope (magnification × 250, OLYMPUS BH2, Japan) for 10 random fields in each transverse slice and presented as the mean number of blood vessels per unit area (number/mm2). Both of these performances were repeated in 8 separate sections per heart. Two independent observers were blinded to the identity of the tissues.
Statistics were performed using SPSS 17.0 Software. Data are expressed as Mean ± Standard Error. Statistical comparisons were made using an unpaired t-test or one-way analysis of variance followed by Bonferroni multiple comparison post hoc tests where appropriate. The results were considered statistically significant when P<0.05.