Allophycocyanin (APC) conjugated anti-mouse Flk-1, Phycoerythrin (PE) conjugated anti-mouse Sca-1 and PE-CD44 antibodies were purchased from eBioscience (San Diego, California). Anti-APC microbeads were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). PE conjugated anti-mouse CD11b, PE-CD45, PE-CD117(c-kit), PE-CD31, PE-CXCR4, Fluorescein isothiocyanate (FITC) conjugated anti-mouse CD34, FITC-CD90 and CD31 monoclonal antibodies were purchased from BD Pharmingen (San Diego, California). PE-SSEA1 antibody was purchased from R&D Systems (Minneapolis, Minnesota). PE-VE-cadherin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, California). Alexa Fluor® 555 conjugated E-Cadherin antibody was purchased from Cell Signaling Technology (Danvers, Massachusetts). PKH26 Red Fluorescent Cell Linker Kit was purchased from SIGMA-ALDRICH (St Louis, Missouri).
Germline competent mouse iPS cell lines "iPS-MEF-Ng-20D-17" generated from mouse embryonic fibroblasts by introducing the four factors(Oct3/4, Sox2, Klf4 and the c-Myc mutant c-Myc(T58A) with the use of retroviral vectors were provided by Riken Cell Bank with the permission of Dr. S. Yamanaka [10, 27]. iPS-MEF-Ng-20D-17 cell is carrying Nanog promoter-driven GFP/IRES/puromycin-resistant gene (Nanog-iPS cells). iPS cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Van Allen Way Carlsbad, California) containing 10% Knockout Serum Replacement (KSR)(Invitrogen), 1% fetal bovine serum (FBS), nonessential amino acids, 5.5 mmol/L 2-mercaptoethanol, 50 U/mL penicillin, and 50 mg/mL streptomycin on feeder layers of mytomycin-C-treated mouse embryonic fibroblast cells stably releasing leukemia inhibitory factor (LIF). Cell differentiation was induced as described previously. In brief, differentiation medium (DM) (α-minimum essential medium (Invitrogen) supplemented with 10% FBS and 5 × 10-5 mol/L 2-mercaptoethanol) was used for iPS cell differentiation. Flk-1+ mesodermal cells were induced by 96 to 108 hr culture of iPS cells (plated at 1.7 × 103 cells/cm2) in DM in the absence of LIF on type IV collagen-coated dishes (ASAHI GLASS CO., LTD, Tokyo, Japan).
Cell separation and analysis
Cultured cells were harvested after induction of Flk-1+ cells by 96 to 108 hr culture in DM on type IV collagen-coated dishes. Induced cells were stained with APC conjugated anti-mouse Flk-1 antibody. Flk-1+ cells were sorted with a magnetic cell separation system and purity was confirmed by flow cytometric analysis (BD FACS Canto, BD, Franklin Lakes, New Jersey). For the detailed characterization, induced cells were stained with APC conjugated anti-mouse Flk-1 antibody and additional antibody (CD11b, CD44, CD45, CD117, Sca-1, SSEA-1, E-cadherin, CXCR4, CD31, VE-cadherin, CD34 and CD90). Stained cells were analyzed by BD FACS Canto.
Tube formation assay and incorporation of Flk-1+ iPS cells
The formation of vascular-like structures by Flk-1+ iPS cells on growth factor-reduced Matrigel (BD Biosciences, Bedford, Massachusetts) was performed as described previously. Briefly, iPS positive cells (labeled with the PKH26 Red Fluorescent Cell Linker Kit [SIGMA-ALDRICH, St Louis, Missouri]) and HUVEC(Cambrex Bio Science Walkersville, Inc., Charles City, Iowa) were seeded at a ratio of 1:1 on coated plates at 3 × 104 cells/cm2 in EBM-2 medium containing EGM-2(LONZA, Basel, Switzerland) and incubated at 37°C for 24 h. Network formation and Flk-1+ iPS cell incorporation were assessed using an inverted phase contrast microscope (Biozero BZ8000, KEYENCE Japan, Osaka, Japan).
Mouse model of hind limb ischemia and cell transplantation
Male KSN athymic nude mice were used for this study. Study protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Nagoya University School of Medicine. Mice, ages 8 to 12 weeks, were subjected to operative unilateral hind limb ischemia under anesthesia with sodium pentobarbital (50 mg/kg i.p.). In this model, the entire left femoral artery and vein were excised surgically . Before surgery and on postoperative days three, seven, 14, and 21, body weight and systolic blood pressure were determined using a tail-cuff pressure analysis system in the conscious state. Flk-1+ cells (2 × 103, 2 × 104 or 6 × 104 cells/mouse) or PBS as a control were injected into four different sites of adductor muscles in the ischemic limb on postoperative day one. To trace transplanted cells in the ischemic tissues, sorted Flk1+ cells were labeled with a PKH26 Red Fluorescent Cell Linker Kit and then injected into ischemic adductor muscles. Implanted cells were evaluated by immunohistochemical analysis 21 days after cells implantation. The signals were detected and analyzed by fluorescence microscopy.
Laser Doppler blood flow analysis
Hindlimb blood flow was measured using a laser Doppler blood flow (LDBF) analyzer (Moor LDI; Moor Instruments, Devon, United Kingdom). LDBF analysis was performed on legs and feet. Blood flow was displayed as changes in the laser frequency using different color pixels. After scanning, stored images were analyzed to quantify blood flow. To avoid data variations due to ambient light and temperature, hindlimb blood flow was expressed as the ratio of left (ischemic) to right (nonischemic) LDBF in a same mouse according to previous studies [26, 29–33]. In addition, in this study, the each ratio of left (ischemic) to right (nonischemic) LDBF was normalized to the each day-1(pre surgery) LDBF value.
Reverse transcriptase-polymerase chain reaction and real-time Reverse transcriptase-polymerase chain reaction
Total RNA was isolated from cultured iPS cells using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA). Total RNA from ischemic muscles was extracted using the of FastRNA Pro Green Kit. (MP Biomedicals, Solon, Ohio). The cDNA was produced using oligo-dT primers and superscript II reverse transcriptase (superscript II, Invitrogen). The cDNA was diluted with DNase-free water at a concentration of 10 ng/μl. RT-PCR was performed using the Ex-Taq PCR kit (Takara, Otsu, Japan) according to the manufacturer's instructions.
Real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) was performed using 1 μg cDNA in the Mx3000P Real-Time PCR System (Stratagene, Agilent Technologies, Santa Clara, California) using SYBR Green I as a double-stranded DNA-specific dye according to the manufacturer's instructions (Applied Biosystem, Foster City, California).
Primers were as follows: forward 5'-CAGGCTGCTGTAACGATGAA-3' (Location: Exon3, Melting Temperature:60.01°C) and reverse 5'-GCATTCACATCTGCTGTGCT-3' (Location: Exon4, Melting Temperature:60.02°C), Product size: 140 bp for murine VEGF.; forward 5'-GGCGGTGGTGACAGTATCTT-3' (Location: Exon3, Melting Temperature:60.00°C) and reverse 5'-GTCACTGACAGAGGCGATGA-3' (Location: Exon4, Melting Temperature:59.99°C), Product size: 162 bp for murine Flk-1.;forward 5'-CCAATCAGCTTGGGCTAGAG-3' (Location: Exon5, Melting Temperature:59.97°C) and reverse 5'-CTGGGAAAGGTGTCCCTGTA-3' (Location: Exon6, Melting Temperature:59.96°C), Product size: 129 bp for murine Oct3/4.;forward 5'-AAGTACCTCAGCCTCCAGCA-3' (Location: Exon3, Melting Temperature:60.01°C) and reverse 5'-GGGGATAGCTGCAATGGATG-3' (Location: Exon5, Melting Temperature:62.66°C), Product size: 199 bp for murine Nanog.;forward 5'-TCCTGTACCTCGTCCGATTC-3' (Location: Exon2, Melting Temperature:60.07°C) and reverse 5'-GGTTTGCCTCTTCTCCACAG-3' (Location: Exon3, Melting Temperature:59.84°C), Product size: 195 bp for murine c-myc.;forward 5'-AGTGTGACGTTGACATCCGT-3' (Location: Exon5, Melting Temperature:59.02°C) and reverse 5'-GCAGCTCAGTAACAGTCCGC-3' (Location: Exon7, Melting Temperature:61.15°C), Product size: 298 bp for murine beta actin.; forward 5'-AACTTTGGCATTGTGGAAGG-3' (Location: Exon3, Melting Temperature:59.97°C), and reverse 5'-ACACATTGGGGGTAGGAACA-3'(Location: Exon3, Melting Temperature:60.09°C), Product size: 223 bp for murine GAPDH.; forward 5'-AGCGGCTCTACTGCAAGAAC-3'(Location: Exon1, Melting Temperature:59.79°C), and reverse 5'-GCCGTCCATCTTCCTTCATA-3'(Location: Exon4, Melting Temperature:60.04°C), Product size: 183 bp for murine FGF2.; forward 5'-GGCAGCTATAAAGGGACGGTA-3' (Location: Exon5, Melting Temperature:60.46°C), and reverse 5'-CTTCTTCCCCTCGAGGATTT-3'(Location: Exon6, Melting Temperature:59.65°C), Product size: 154 bp for murine HGF.
Analyses of mRNA levels of VEGF were normalized to GAPDH as the internal control and expressed relative to the quantity of VEGF mRNA at day three in ischemic adductor muscle injected with PBS (day three control).
All data were obtained from at least three independent experiments. Statistical analysis of the data was performed using Student's t test or two-way ANOVA. P < 0.05 was considered significant. All data are shown as means ± SEM.