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  • The karyotype of the BIOT LOAD

    2018-11-08

    The karyotype of the BIOT-4828-LOAD iPSC line was determined with Giemsa-banding, proving normal diploid 46, XX karyotype, without any detectable abnormalities (Fig. 1B). Expression of pluripotency markers was examined by immunocytochemistry staining, using pdgfr against human OCT3/4, E-CADHERIN, and NANOG (Fig. 1A). The in vitro spontaneous differentiation potential towards the three germ layer of the BIOT-4828-LOAD iPSC line was demonstrated by the expression of ectodermal (βIII-TUBULIN), mesodermal (BRACHYURY) and endodermal (GATA4) markers (Fig. 1A) (Itskovitz-Eldor et al., 2000; Carpenter et al., 2003).
    Materials and methods
    Acknowledgement We would like to thank for Dr. Mária Judit Molnár and Dr. Viktória Reményi (Institute of Genomic Medicine and Rare Disorders, Semmelweis University, Budapest, Hungary) for collecting the patient blood samples and providing clinical data. This work was supported by grants from EU FP7 projects (STEMCAM, PIAP-GA-2009-251186; STEMMAD, PIAPP-GA-2012-324451; EpiHealth, HEALTH-2012-F2-278418; EpiHealthNet, PITN-GA-2012-317146); a national research project RG-IPI-2013_TP7/026 and Research Centre of Excellence – 9878-3/2016/FEKUT.
    Resource table
    Resource details To generate the BIOT-0630-LOAD iPSC line (Fig. 1A) the four “Yamanaka reprogramming factors” OCT3/4, SOX2, KLF4, and C-MYC were delivered into PBMCs using the integration-free Sendai virus gene-delivery method (Yang et al. 2008; Fusaki et al. 2009). The iPSC-like colonies were picked after 20–27days post-transduction. Beginning from passage 5 of the iPSCs the absence/presence of Sendai virus vector was analysed by RT-PCR using Sendai virus vector (SeV) - specific primers (Table 1). After 7 passages, the elimination of the reprogramming vector was confirmed in BIOT-0630-LOAD iPSC line which was selected for further analysis (Fig. 1B). The karyotype of the BIOT-0630-LOAD iPSC line was determined with Giemsa-banding, proving normal diploid 46, XY karyotype, without any detectable abnormalities (Fig. 1B). Expression of pluripotency markers was examined by immunocytochemistry staining, using antibodies against human OCT3/4, E-CADHERIN, and NANOG (Fig. 1A). The in vitro spontaneous differentiation potential towards the three germ layer of the BIOT-0630-LOAD iPSC line was demonstrated by the expression of ectodermal (βIII-TUBULIN), mesodermal (BRACHYURY) and endodermal (GATA4) markers (Fig. 1A) (Itskovitz-Eldor et al. 2000; Carpenter et al. 2003).
    Materials and methods
    Acknowledgement We would like to thank for Dr. Mária Judit Molnár and Dr. Viktória Reményi (Institute of Genomic Medicine and Rare Disorders, Semmelweis pdgfr University, Budapest, Hungary) for collecting the patient blood samples and providing clinical data. This work was supported by grants from EU FP7 projects (STEMCAM, PIAP-GA-2009-251186; STEMMAD, PIAPP-GA-2012-324451; EpiHealth, HEALTH-2012-F2-278418; EpiHealthNet, PITN-GA-2012-317146); a national research project RG-IPI-2013_TP7/026 and Research Centre of Excellence – 9878-3/2016/FEKUT.
    Resource table
    Resource details To generate the BIOT-0726-LOAD iPSC line (Fig. 1A) the four “Yamanaka reprogramming factors” OCT3/4, SOX2, KLF4, and C-MYC were delivered into PBMCs using the integration-free Sendai virus gene-delivery method (Yang et al. 2008; Fusaki et al. 2009). The iPSC-like colonies were picked after 20–27days post-transduction. Beginning from passage 5 of the iPSCs the absence/presence of Sendai virus vector was analysed by RT-PCR using Sendai virus vector (SeV) - specific primers (Table 1). After 7 passages, the elimination of the reprogramming vector was confirmed in BIOT-0726-LOAD iPSC line which was selected for further analysis (Fig. 1B). The karyotype of the BIOT-0726-LOAD iPSC line was determined with Giemsa-banding, proving normal diploid 46, XX karyotype, without any detectable abnormalities (Fig. 1B).