cell type: induced pluripotent stem cells derived from exosomes age: 15y gender: male
Treatment protocol
HHH-DPCs were cultured in MSCGM medium. When they became 70 to 80% confluent, they were washed three times with PBS and medium was replaced by serum free-DMEM (SIGMA-ALDRICH, Tokyo, Japan) and cultured for 24 hours. Then, the medium was collected and filtrated 0.45μm and 0.22μm syringe filters (Minisart, Goettingen, FRG), before ultracentrifugation at 90,000 rpm for 3h at 4℃. The pellet was resuspended in PBS and stored at 80℃. HHH-iPSCs cultured on SNL feeder cells were first replated to 6cm culture dish coated by laminin 5-1-1 (BioLamina, SUNDBYBERG, SWEDEN), replated onto another 6cm dish, and then half of the medium was replaced by fresh medium and the medium was collected after 24 hours. Exosomes were collected with the same manner with HHH-DPCs.
Growth protocol
Using a protocol approved by the Institutional Review Board of Gifu University, we collected normal human third molars at the Gifu University Medical Hospital after having obtained informed consent from each patient. (Takeda et al. 2008). We isolated DPCs from more than 300 patients, mostly from young patients with maturing wisdom teeth. Three HHH-DPCs lines (DP74, 16-year-old-female; DP94,16-year-old-female; and DP263, 15-year-old-male) were picked up. Briefly, the pulp tissues were minced into small clumps and digested with 1 mg/mL collagenase type I (Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at 37°C. We also established iPSC lines from DP74 and DP94 using episomal or lenti virus vector, respectively (Tamaoki et al. 2010; Okita et al. 2011). In this report, following the guidelines for the generation of human iPSCs approved by the Institutional Review Board of Gifu University, we used DP263 for generating iPSCs within 10 passages. Using a Sendai viral vector kit (ID Pharma, Tokyo, Japan), we generated iPSCs as reported previously (Takeda-Kawaguchi et al. 2014 ). Briefly, we first introduced Sendai viruses carrying OCT3/4, SOX2, KLF4, and c-MYC into HHH-DPCs according to the manufacturer's instruction. HHH-DPCs were seeded at 5.0×105cells/well in a 6 well plate and cultured in MSCGM medium. From the day after infection, the culture media were changed daily up to day 7. Then, the cells were dissociated using 0.05% trypsin-EDTA (Life Technologies, Tokyo, Japan), and HHH-DPCs (5.0×104 cells/6 cm dish) were seeded onto mitomycin C-treated SNL feeder layers (SNL cell line obtained from Sanger Institute, Cambridge, UK) in Primate ES cell medium (ReproCell, Tokyo, Japan) supplemented with 4 ng/ml basic fibroblast growth factor (bFGF, Wako, Osaka, Japan), and cultured for a further 14 days until colonies were ready to be picked up. Afterwards, HHH-iPSCs (iPS74, iPS94, iPS263) were cultured on mitomycin C-treated SNL feeder layers in Primate ES cell medium supplemented with 4 ng/mL bFGF at 37℃ in a humidified atmosphere containing 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Exosomes prepared from DP74, DP94, and DP263 and iPSCs derived from these cells were subjected for miRNA isolation using miRCURY™ RNA Isolation Kit- Cells&Plant content（EXIQON, Woburn, USA) Agilent miRNA labeling system was used for RNA expression analyses.
Label
Cy3
Label protocol
After dephosphorylation and denaturation, 100 ng of total RNA was labeled with cyanine 3-pCp.
Hybridization protocol
hybridized to an Agilent Microarray Sure Print G3 Human miRNA 8 × 60 K miRBase 16.0 array with the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Tokyo, Japan).
Scan protocol
After hybridization for 20 h, the slides were washed with the Gene Expression Wash Buffer Kit and scanned by an Agilent Scanner.
Description
exosomes purified from iPS263
Data processing
The images were processed and analyzed with Agilent Feature Extraction Software. The raw data were normalized by quantile normalization and then analyzed in GeneSpring GX software 12.5 (Agilent Technologies).
