Then culture medium was changed to differentiation medium from StemPro Osteogenesis Differentiation Kit (Gibco) and refreshed every 3 to 4 4 days for more than 3 weeks. neomycin resistant gene), while the additional was designed with the EF1 promoter traveling the expression of a gene and a (SV40) promoter traveling the expression of the gene (the hygromycin resistant gene). Both donor sequences were flanked by homologous DNA sequences from Ex lover1 locus (chromosome 15: nucleotides 44,710,501C44,711,401 and nucleotides 44,711,615C44,712,485, GRCh38.p2 Main Assembly). Generation of B2 M Knockout hP-iPSC Clones B2MKO hP-iPSCs were generated by two methods: double-color selection and one-shot puromycin selection. For the double-color selection, hP-iPSCs were dissociated by Accutase (Merck Millipore), washed by phosphate-buffered saline (PBS, Lonza), and resuspended in Opti-MEM I Reduced Serum Medium (Gibco) as single-cell suspension; 1 106 cells were transfected with 2.5 g pX260 plasmid and 2.5 g donor plasmid with GFP by electroporator (Nepa Gene, Chiba, Japan). Transfected solitary cells were recovered in NutriStem hPSC XF Medium (Biological Industries, Beit-Haemek, Israel) and seeded on Matrigel-coated plates. Four days after electroporation, the tradition medium was changed back to mTeSR1 medium, and DUBs-IN-2 the cells were selected by 25 g/ml Geneticin (G418 Sulfate, Gibco) for 2 weeks. Determined cells were subjected to fluorescence-activated cell sorting (FACS) for single-cell seeding, which was performed by BD FACSAria I Flow Cytometer (BD Biosciences). Determined hP-iPSCs were dissociated by Accutase as solitary cells, and the GFP positive human population was seeded as one cell per well on Matrigel-coated 96 well-plates in the NutriStem medium. Single-cell clones were expanded, genotyped by PCR and sequencing. A monoallelic knockout single-cell clone was confirmed and preceded to a second round of knockout within the additional allele. The monoallelic knockout single-cell clone was transfected with 2.5 g pX260 plasmid and 2.5 g donor plasmid with mCherry by electroporation. Transfected cells were selected by 10 g/ml Hygromycin B (Gibco) for 2 weeks and subjected to single-cell seeding as well. The double-color single-cell clones were collected, expanded, and confirmed by genotyping. For the one-shot puromycin selection, 1 106 hP-iPSCs were transfected with 5 g pX459 plasmid by electroporation and recovered in the NutriStem medium on Matrigel-coated plates overnight. Afterward, transfected cells were selected by 1 g/ml puromycin (Thermo Fisher Scientific) in the NutriStem medium for 24 h. Survival single cells were cultured in new Nutristem medium for DUBs-IN-2 3 to 4 4 days before changed back to mTeSR1 tradition. Single-cell clones were isolated and subjected to genotyping and phenotyping. B2M bad clones were confirmed from those single-cell clones. Western Blotting and Circulation Cytometry Analysis For Western blot analysis, sample proteins were extracted by lysing cells with radioimmunoprecipitation assay (RIPA) buffer (Nacalai Tesque, Kyoto, Japan), analyzed in sodium dodecyl sulfate-polyacrylamide gel DUBs-IN-2 electrophoresis gel under reducing condition, and then electroblotted to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). Rabbit Anti-B2M antibody clone EP2978Y (1:5000 dilution, Abcam, Cambridge, UK) and mouse anti–actin antibody clone GT5512 (1:1000 dilution, Rabbit polyclonal to Vang-like protein 1 Abcam) were used as main antibodies. Goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase (HRP) (1:5000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and goat anti-mouse IgG-HRP (1:2000 dilution, Santa Cruz Biotechnology) were used as secondary antibodies. The membrane was developed and visualized for chemiluminescence by MYECLImager (Thermo Fisher Scientific). For circulation cytometry analysis, iPSCs and MSCs were stained with antibodies in autoMACS Working Buffer (Miltenyi Biotec, Bergisch GladBach, Germany) and analyzed by BD Accuri C6 Circulation Cytometer (BD Biosciences). Data were analyzed by CFlow Sampler software (BD Biosciences) and antibodies in circulation cytometry assays were listed in Table S2. Southern Blotting Southern blot was performed as explained previously25. For each sample, 15 g genomic DNA was digested with 50 U HindIII-HF DUBs-IN-2 (New England Biolabs, Ipswich, MA, USA) over night. Digested DNA was loaded on a 1% agarose gel, and gel electrophoresis was performed at 40 V for 5 h. DNA was then transferred to a positively charged nylon membrane by using iBlot Dry Blotting System (Invitrogen, Thermo Fisher Scientific). The membrane was washed with 1.5 M sodium chloride/0.5 M sodium hydroxide denaturing solution and then air-dried. Ultraviolet cross-linking was performed at 130 mJ/cm2. The membrane was first prehybridized in DIG Easy Hyb (Roche Diagnostics, Basel, Switzerland) buffer for 1 hour and then DUBs-IN-2 hybridized having a DIG-labeled probe over night. Afterward, the membrane was first washed twice with 2 saline-sodium citrate (SSC)/0.1% sodium dodecyl sulfate (SDS).