br Materials and methods br

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. B) and ICC with pluripotency markers (Fig. D).
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 (project number 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) and Innovation Fund Denmark, BainStem.
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Resource details Skin fibroblasts were collected from a 58-year old male with familial Alzheimer\'s disease (AD) caused by a c.449T>C mutation in the PSEN-1 gene and electroporated with episomal plasmids of human OCT4, SOX2, L-MYC, KLF4, NANOG, LIN28, and shRNA for TP53 using a Neon™ electroporation device (Invitrogen). Sequencing analysis of the iPSCs confirmed a heterozygous c.449T>C mutation in PSEN-1, which results in lysine being substituted by proline (L150P) (Fig. 1A). The absence of the three reprograming plasmids in the glutathione peroxidase was verified by quantitative PCR after 10 passages (Fig. 1B). The iPSCs expressed the pluripotency markers NANOG, OCT4, TDGF1, DNMT3B, GABRB3, and GDF3 (International Stem Cell Banking Initiative 2009) in the same range as BION010-A iPSC, a control iPSC line described previously (Rasmussen et al. 2014; Fig. 1C). Expression of the pluripotency markers, NANOG, OCT4, SSEA4, and TRA-1-60 at the protein level was confirmed by immunofluorescence staining (Fig. 1D). In vitro differentiation followed by ICC analysis with the ectodermal marker beta-III-Tubulin (TUJ1), the mesodermal marker smooth muscle actin (SMA) and the endodermal marker alpha-feto protein (AFP) demonstrated the differentiation potential into cells representing all three germ layers (Fig. 1E). In addition, the iPSCs presented a normal karyotype (46, XY) (Fig. 1F).
Materials and methods
Verification and authentication Karyotyping was performed at the Cell Guidance Systems, England. 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, XY karyotype, free of any discernible abnormalities (Fig. 1F). iPSC line identity and purity were furthermore confirmed by sequencing of the PSEN-1 gene (Fig. 1A) and ICC with pluripotency markers (Fig. 1C).
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. The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union Seventh Framework Programme FP7/2007-2013/under REA grant agreement n°PIAPP-GA-2012-324451 (STEMMAD).
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Resource details Lymphoblast cells (AD2-2), derived from a 65-year-old AD patient expressing the TREM2 missense p.R47H mutation (Cuyvers et al., 2014), were reprogrammed employing oriP/EBNA-1-based episomal plasmids expressing OCT4, SOX2, KLF4, C-MYC, L-MYC, LIN28 and a p53 shRNA. After picking the clones, clone A of Lymph2-AD2-iPS cell line retained traces of EBNA1 (Fig. 1A) but was diluted out after several passages (data not shown). Clone B was negative for EBNA-1 and oriP (Fig. 1A), but both clones were positive for the pluripotency-associated genes OCT4, SOX2, NANOG and TGDF1 (Fig. 1A). Pluripotency was confirmed by (i) expression of OCT4, SOX2, NANOG, TRA-1-60 and TRA-1-81 and SSEA4 (Fig. 1B) and (ii) embryoid body (EB)-based spontaneous differentiation into cell types representative of the three germ layers, namely ectoderm (Nestin, PAX6), mesoderm (SMA — smooth muscle actin) and endoderm (AFP — α-feto protein, SOX17) (Fig. 1C).