Isolation of c-kit+VEGFR-2+ MSCs
Isolation of MSCs from bone marrow of Sprague-Dawley (SD) rats (2−4 weeks) was performed as described previously  with minor modification. The protocol was permitted by the law of the People’s Republic of China on the Protection of Wildlife and approved by the Institutional Animal Care Committee of Fudan University. Briefly, bone marrow cells in the femurs and tibias were flushed out with PBS supplemented with 5 mM ethylene diamine tetraacetic acid (EDTA). The mononuclear cells in the bone marrow cells were isolated by Percoll (GE Healthcare, Leics, UK) gradient centrifugation, and then incubated with Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 15% fetal bovine serum (FBS; Carlsbad, CA, USA) for 24 hrs. The non-adherent cells were discarded, the adherent cells were used as MSCs. The cells at the second passage were digested using 0.25% trypsin–EDTA (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at 4oC and then resuspended in 1% bovine serum albumin (BSA, Amresco, Solon, OH, USA). After centrifugation, the cells were incubated with mouse anti-rat c-kit antibody (1:100) and polyclonal rabbit anti-rat VEGFR-2 antibody (1:200, Santa Cruz Biotechnology, Dallas, TX, USA) for 50 min at 4 oC. The cells were washed with 0.5% BSA and then incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:100) and Alexa Fluor 647 goat anti-mouse IgG (1:400; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 30 min at 4oC. After washing twice with PBS, the cells were suspended with DMEM containing 2.5% FBS, and c-kit+VEGFR-2+ MSCs were sorted by a fluorescence-activated cell sorter (Beckman Coulter, Fullerton, CA, USA). The sorted cells were incubated with the complete medium, and the medium was changed every 3 days. c-kit+VEGFR-2+ MSCs at passage 3−6 were used in the following experiments.
For examining biologic characteristics of c-kit+VEGFR-2+ MSCs, total RNA of c-kit+VEGFR-2+ and c-kit+VEGFR-2− MSCs was extracted using the TRIzol Reagent Kit (Invitrogen) respectively. The samples were processed following the manufacturer’s instructions (BGI Tech Solutions, Shenzhen, China). In brief, the target RNA was obtained after purification by mRNA enrichment using Oligo (dT) magnetic beads. Fragment target RNA was reverse transcribed into double-strand cDNA. After end repair and bubble adapter ligation, cDNA was amplified. PCR product was denatured by heat and cyclized to establish DNA library. Primary sequencing data (raw data) was acquired by Illumina HiSeq 2000 (Illumina, Santiago, CA, USA). After filtering raw data to clean data, reads were aligned to genome reference. Bowtie2 was used to map clean reads to Rattus norvegicus. Differentially expressed genes were screened using Possion distribution with the fragments assigned per kilobase of target per million mapped reads (FPKM) method. Gene ontology (GO) was used to recognize the main biological functions of the two subpopulations of c-kit+ cells. Moreover, GO functional annotation and enrichment analysis was used to compare the molecular functions and biological processing of the cells in two subpopulations. The pathways enriched with differentially expressed genes were made with Database of Kyoto Encyclopedia of Genes and Genomes (KEGG) .
Examination of proliferation and migration of the cells
c-kit+VEGFR-2+ MSCs were divided into vehicle, VEGF (vascular endothelial growth factor), SCF and VEGF + SCF groups. In VEGF and SCF groups, 10 ng/ml VEGF (Peprotech, Rocky Hill, NJ, USA) and 50 ng/ml SCF (Peprotech) were added in the medium respectively. After treatment with the growth factors for 24 hrs, the cells were incubated with rabbit anti-rat ki67 antibody (1:200; Abcam, Cambridge, UK), followed by Alexa Fluor 594-labelled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Ki67-positive cells were counted with a fluorescent microscope. Migration of the cells was assessed by transwell assay. The experimental grouping is the same as above. The diameter of the pores of the polyethylene terephthalate membrane in the transwell (Becton Dickinson, Franklin Lakes, NJ, USA) is 8 µm. The cells were seeded on the upper chamber, and the medium supplemented with the growth factors was added into the lower chamber. After incubation for 12 h, the cell insert was taken out and stained with 10% Giemsa. The cells on the upper chamber were wiped with tissue paper, and then the cells migrated into the lower chamber were counted. Three repeated experiments were performed.
Tube formation assay
The incorporation capacity of the c-kit+VEGFR-2+ cells into tube-like structures formed by pulmonary microvascular endothelial cells was accessed using previous method . The lungs of SD rats (two-weeks old) were removed and put into pre-iced HBSS. After removing the pleura, the tissue near the border of the lung was cut into 1 mm3 pieces. The tissue masses were incubated with DMEM in gelatin-coated wells for one week. The migrated endothelial cells were harvested, and then incubated in 24-well plate coated with Matrigel (3:2 in DMEM; BD Biosciences, San Jose, CA, USA). After capillary-like structures were formed, c-kit+VEGFR-2+ cells labelled with Dil (Beyotime Biotech, Haimen, Jiangsu, China) were added, and continued to be incubated for 4 hrs. c-kit+VEGFR-2+ cells incorporated into the capillary-like structures were counted with a fluorescent microscope. Five fields were selected randomly in each group. The experiment was repeated for four times. In VEGF group, c-kit+VEGFR-2+ cells were pretreated with 10 ng/ml VEGF for 2 hrs.
Reverse transcription-polymerase chain reaction (RT-PCR)
For accessing differentiation of c-kit+VEGFR-2+ MSCs towards endothelial cells, smooth muscle cells and cardiomyocytes, the cells were divided into VEGF, TGF-β (transforming growth factor-β) and BMP-2 groups. Total RNA was collected with the TRIzol Reagent Kit. After mRNA were reverted to cDNA using Primescript RT Reagent Kit with gDNA Eraser (Takara Biotechnology, Otsu, Japan), cDNA was amplified with PCR assay. In VEGF group, the cells were treated with 10 ng/ml VEGF for 2 weeks. Expression of CD31 and vWF (von Willebrand factor) in the cells was analyzed by 1.2% agarose gel electrophoresis and visualized under ultraviolet light. In TGF-β group, the cells were treated with 2 ng/ml TGF-β (Peprotech) for 2 weeks. Expression of α-SMA (α-smooth muscle actin) and CNN1 (calponin 1) in the cells was analyzed. mRNAs of the femoral artery from SD rat were used as positive controls in VEGF and TGF-β groups. In BMP-2 group, the cells were treated with 10 ng/ml BMP-2 (Peprotech) for 2 weeks. Expression of Nkx2.5 and GATA-4 in the cells was detected. mRNAs of the myocardium from SD rat were used as positive controls. The experiment was repeated for three times. The sequences of the primers were shown in Table S1.
Differentiation of c-kit+VEGFR-2+ MSCs was also examined with immunostaining. The cells induced with VEGF were incubated with mouse anti-rat CD31 antibody (1:200; Abcam) overnight, and then incubated with Alexa Fluor 594 goat anti-mouse IgG (1:400; Jackson ImmunoResearch Laboratories). The cells induced with TGF-β were incubated with mouse anti-rat α-SMA (1:200) and rabbit anti-rat desmin (1:100; Abcam) overnight, followed with Alexa Fluor 594 goat anti-mouse IgG (1:400) and Alexa Fluor 488 goat anti-rabbit IgG (1:200; Jackson ImmunoResearch Laboratories) respectively. After induction with BMP-2, the cells were incubated with mouse anti-rat cTnT (cardiac troponin T; 1:200) and rabbit anti-rat Cx43 (connexin-43; 1:200; Santa Cruz Biotechnology). The differentiated cells were viewed with a fluorescence microscope.
Enzyme-linked immunosorbent assay (ELISA)
Cytokine secretion of c-kit+VEGFR-2+ MSCs after treatment with hypoxia was detected with ELISA. The cells were incubated in a hypoxia chamber with 1% O2, 5% CO2 and 94% N2 for 12 hrs. After the supernatants were collected, the cytokines were detected using VEGF, SCF and SDF-1α (stromal cell-derived factor-1α) ELISA kits (Boster, Wuhan, China) according to the manufacturer’s protocols. Absorbance was measured with a microplate reader (Tecan Infinite 200, Mannedorf, Switzerland) at 450 nm. Moreover, paracrine of VEGF, SCF and SDF-1α in the peri-infarct region of the left ventricle (LV) was also examined using ELISA at 1 week after cell transplantation. In brief, LV tissues were cut into pieces and ground into tissue homogenate with homogenizer. After centrifugation, the supernatants were collected and stored at -20 oC. The cytokines were detected with ELISA kits. The experiment was repeated for thrice.
Detection of autophagy and apoptosis
To determine optimal preconditioning time of hypoxia and serum deprivation for c-kit+VEGFR-2+ MSCs, the degrees of autophagy and apoptosis of the cells were assessed with Western blotting and flow cytometry respectively. The cells were treated with hypoxia (1% O2) and serum deprivation (3% FBS) for 1 hrs, 2 hrs, 4 hrs, 6 hrs and 8 hrs. After treatment, total protein in each group was extracted with RIPA buffer (Beyotime, Beijing, China) and separated in a 15% SDS-polyacrylamide gel. The lanes were incubated with polyclone rabbit anti-rat LC3 (microtubule-associated protein 1 light chain 3) antibody (1:500; Novus, Littleton, CO, USA) and mouse anti-rat β-actin monoclonal antibody (1:4000; Proteintech, Rosemont, IL, USA) at 4 oC overnight. Then, they were incubated with anti-rabbit IgG HRP-linked antibody (1:4000; Cell Signaling, Danvers, MA, USA) and anti-mouse IgG HRP-linked antibody (1:4000; Cell Signaling) at room temperature for 1 hr. After being transferred onto a polyvinylidene fluoride membrane, the protein bands were monitored using Substrate Chemiluminescence Kit (Thermo, Rockford, IL, USA). The ratio of LC3-II/β-actin was analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA). The experiment was repeated for four times. For examining apoptosis of the hypoxia-treated cells, the cells were labeled with FITC Annexin V Apoptosis Detection Kit (BD Biosciences). The percentage of the apoptotic cells was determined by flow cytometry. The experiment was repeated for thrice.
Implantation of the cells into abdominal pouches
To assess the survival of the cells preconditioned with hypoxia and serum deprivation in the ischaemic tissue, the cells were implanted into abdominal subcutaneous pouches of SD rats. The pouches were prepared as previous method . To mimic the ischaemic tissue, the subcutaneous vessels at the pouch were ligated. After treatment with 5 µM DiI for 20 min, the cells were suspended with 20 ng/mL fibrinogen and 50 IU/mL thrombin, and seeded on the poriferous polyethylene terephthalate membrane (pore size, 8 µm) removed from the transwell. For formation of fibrin gel, the cell-loaded membranes were incubated for 20 min. Then, the membranes were implanted into the abdominal pouches. At 24 hrs after implantation, the membranes were harvested, and the survived cells (DiI-labelled cells) were counted with a fluorescent microscope. Five fields of each membrane were selected randomly. The experiment was repeated for three times.
Scanning and transmission electron microscopies
The fibrin gel formed by mixing 500 µL of the fibrinogen (10 mg/mL) and 500 µL of thrombin (10 IU/mL; Sigma-Aldrich, St. Louis, MO, USA) in a 35-mm dish. The cells were seeded on the fibrin gel and incubated for 1 hr. Then, the specimens were pre-fixed with 1.25% glutaraldehyde and post-fixed with 1% buffered osmium tetroxide. After dehydration and substitution, the specimens were coated with gold-palladium and then viewed with a scanning electron microscope (Hitachi SU8010, Tokyo, Japan). In transmission electron microscopy, the specimens were fixed with 2.5% glutaraldehyde, post-fixed with 1% buffered osmium tetroxide and dehydrated with graded ethanol and acetone. The ultrathin sections were stained with uranyl acetate and lead citrate. The cells within the fibrin gel were viewed using a transmission electron microscope (CM120; Philips, Eindhoven, Holland). Moreover, compatibility of fibrin with the implanted cells and myocardium was examined by transmission electron microscopy at 2 hrs after transplantation.
Establishment of MI model and cell transplantation
Twenty-four adult female SD rats (200 ± 20 g) were anesthetized with ketamine (80 mg/kg) and xylazine (3 mg/kg). After endotracheal intubation, the heart was exposed, and the left anterior descending coronary artery (LAD) was ligated . Two rats died immediately after LAD ligation. The rest rats were divided into sham (n = 5), control (n = 5), cell (n = 6) and precondition (n = 6) groups. In control group, 40 µl fibrinogen (10 mg/ml) and 40 µl thrombin (10 IU/ml) were injected simultaneously with a Duploject syringe into the peri-infarcted region at four points. In the cell or precondition (1% O2, 3% FBS) groups, 1 × 106 cells were suspended in fibrinogen.
Echocardiograms were recorded with Vevo 2100 Imaging System (VisualSonics Inc., Toronto, ON, Canada) before MI, at 1 week after MI (before transplantation) and 4 weeks after transplantation respectively. The rats were anesthetized with isoflurane and fixed on the metal plate for detection. After adequate two-dimensional images had been obtained, the M-mode cursor was set to the parasternal long axis at the level of the papillary muscles. The LV end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD) were measured from at least three consecutive cardiac cycles. To evaluate the systolic function, the LV end-diastolic volume (LVEDV), the LV end-systolic volume (LVESV), the ejection fraction (EF = LVEDV-LVESV/LVEDV×100%) and fractional shortening (FS = LVEDD − LVESD/LVEDD × 100%) were examined. Successful establishment of MI model was confirmed by EF of < 50% and FS of < 30%. Three measurements at least were taken and averaged for each parameter.
Masson’s trichrome staining
To evaluate myocardial repair of the infarcted region, the cryosections were performed with Masson’s trichrome. At 4 weeks after transplantation, the hearts were removed and perfused with 4% paraformaldehyde solution. Then, the hearts were cut into upper and lower parts along the cross-section, and continued to be fixed using 4% paraformaldehyde solution. After washing with DPBS, the tissue was treated with 16% and 30% sucrose gradient to dehydration, and then embedded with Tissue-Tek OCT (Sakura Finetek, Torrance, CA, USA). The cryosections (5-µm thickness) were prepared and stained. Scar tissue with plentiful collagen was stained blue, while myocardial tissue was stained red. The scar area was defined as percentage of circumference of infarct region in the whole LV wall circumference, and measured by Image J 1.46r (National Institutes of Health, Bethesda, MD, USA). The thickness of the LV wall at the infarcted region was measured at the thinnest part of the region. At least 5 independent sections and 2 fields (20 x) on each section were selected randomly.
Immunostaining of the myocardium
To determine angiogenesis of the peri-infarcted and infarcted regions, cryosections were incubated with mouse anti-rat CD31 antibody (1:200), followed by Alexa Fluor 594 conjugated goat anti-mouse IgG (1:400). Myocardial regeneration in the infarct region was assessed by double-labelled immunostaining using mouse anti-rat cTnT antibody (1:200) and rabbit anti-rat Cx43 antibody (1:100).
To trace differentiation of the transplanted cells into endothelial cells, smooth muscle cells and cardiomyocytes, the cells were labelled with GFP (green fluorescent protein) and co-localized with CD31, α-SMA and cTnT respectively. The gap junction between new cardiomyocytes differentiated from the transplanted cells and resident myocardium was determined by immunostaining of GFP, cTnT and Cx43. The cryosections were incubated with chicken anti-rat GFP antibody (1:200; Novus), mouse anti-rat cTnT antibody (1:200) and rabbit anti-rat Cx43 antibody (1:100) overnight, followed with DyLight 488 goat anti-chicken lgG (H + L) (1:200; Novus), DyLight 594 goat anti-rabbit IgG (1:400; Abcam) and Alexa Fluor 647 goat anti-mouse IgG (1:400; ImmunoResearch Laboratories) respectively.
Data were presented as means ± standard deviation in the experiments above. Statistical analysis was conducted by SPSS 17.0 software (SPSS, Chicago, IL, USA) using t-test and one-way ANNOVA. p ˂ 0.05 were regarded as the statistically significance.