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  • br Materials and methods br Verification

    2018-11-12


    Materials and methods
    Verification and authentication Karyotyping was performed at the Center for Applied Human Molecular Genetics, Kennedy Center, Glostrup, Denmark. At least 10 metaphases were analysed per sample with an approximate resolution of 550 to 600 bands per haploid genome. The results showed a normal 46, XX karyotype, free of any discernible abnormalities (Fig. 1F). iPSC line identity and purity was confirmed by sequencing of the MAPT gene (Fig. 1B) and ICC with pluripotency markers (Fig. 1D).
    Acknowledgments We would like to thank Dr. Keisuke Okita and Prof. Shinya Yamanaka for providing the plasmids. Furthermore, we would like to thank Ida Jørring, Bente Smith Thorup and Ulla Bekker Poulsen for the excellent technical assistance. We thank the following for financial support: The Danish National Advanced Technology Foundation (047-2011-1) (patient-specific stem cell-derived models for Alzheimer\'s disease) and the European Union 7th Framework Program (PIAP-GA-2012-324451-STEMMAD).
    Resource table
    Resource details Fibroblasts were obtained from a 59-year old woman heterozygous for a R406W mutation in microtubule-associated protein tau (MAPT). The patient was clinically diagnosed with frontotemporal dementia at age 52, displaying atrophy of the temporal lobes on magnetic resonance imaging and reduction in glucose metabolism bilaterally in the temporal pole and adjacent lateral and medial temporal dpp-4 inhibitor including the anterior sections of the hippocampi and the amygdalae using 18-fluoro-deoxyglucose positron emission tomography (Lindquist et al., 2008). Reprogramming was performed by electroporation with three episomal plasmids containing hOCT4 with or without a short hairpin to TP53 (shp53), hSOX2 and hKLF4, and hL-MYC and hLIN28 (Okita et al., 2011). This method had previously been used to establish integration-free iPSC from an 18-year old healthy male (Rasmussen et al., 2014). Four weeks after reprogramming, an average of 64 colonies per 1×105 fibroblasts (0.06%) emerged with the inclusion of shp53, whereas, no colonies were observed without shp53. Integration analysis with plasmid-specific primers showed that hOCT4, hSOX2 and hLIN28, present on each of the three plasmids, had not integrated into the genome (Fig. 1A) and sequencing confirmed the presence of a c.1216C>T substitution in one of the alleles of exon 13 in the MAPT gene corresponding to a R406W mutation (Fig. 1B). Pluripotency analysis showed that transcription from the endogenous pluripotency genes NANOG, POU5F1 (OCT4), TDGF1, DNMT3B, GABRB3 and GDF3 was between 100 and 10,000 times upregulated compared with fibroblasts (Fig. 1C) and immunocytochemical (ICC) analysis demonstrated the presence of the pluripotency markers OCT4, NANOG, TRA1-60, TRA1-81, SSEA3 and SSEA4 at the protein level (Fig. 1D). Finally, in vitro differentiation followed by ICC analysis with the mesodermal marker smooth muscle actin (SMA), the endodermal marker alpha-feto protein (AFP) and the ectodermal marker beta-III-Tubulin (TUJI) demonstrated the differentiation potential into all three germ layers (Fig. 1E).
    Materials and methods
    Verification and authentication Karyotyping was performed at the Center for Applied Human Molecular Genetics, Kennedy Center, Glostrup, Denmark. At least 10 metaphases were analyzed per sample with an approximate resolution of 550 to 600 bands per haploid genome. The results showed a normal 46, XX karyotype, free of any discernible abnormalities (Fig. 1F). iPSC line identity and purity was confirmed by sequencing of the MAPT gene (Fig. 1B) and ICC with pluripotency markers (Fig. 1D).
    Acknowledgments We would like to thank Dr. Keisuke Okita and Prof. Shinya Yamanaka for providing the plasmids. Furthermore, we would like to thank Ida Jørring, Bente Smith Thorup and Ulla Bekker Poulsen for excellent technical assistance. We thank the following for financial support: The Danish National Advanced Technology Foundation (Patient-specific stem cell-derived models for Alzheimer\'s disease) (047-2011-1) and the European Union 7th Framework Program (PIAP-GA-2012-324451-STEMMAD).