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  • br Materials and methods br Acknowledgments We

    2018-11-08


    Materials and methods
    Acknowledgments We thank Dr. Keisuke Okita and Prof. Shinya Yamanaka for providing the reprogramming plasmids. Additionally, we would like to thank Tina Christoffersen and Birgit Kassow for skillful technical assistance. We acknowledge SHARE\'s Cross Faculty PhD Initiative (University of Copenhagen) for financial support. We also 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), the European Union seventh Framework Programme (PIAP-GA-2012-324451-STEMMAD) and Innovation Fund Denmark, BrainStem.
    Resource details Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by a CAG-repeat expanding mutation of the gene ATXN3 encoding ataxin-3. CAG-repeat length of mutated order T-5224 can vary from 45 to 87 repeats (Matos et al. 2011). In the current study, dermal fibroblasts (H249) were derived from a skin biopsy of a 58-year-old man with spinocerebellar ataxia type 3 (SCA3) with 78 CAG-repeats in the disease allele of ATXN3. Patient fibroblasts were reprogrammed to iPSCs by electroporation with three episomal plasmids encoding human L-MYC and LIN28, SOX2 and KLF4, and OCT4 combined with a short hairpin RNA for P53 (shP53). The iPSC line described in this publication was termed SCA3.B11. An additional iPSC line termed SCA3.B1 was derived from the same patient and characterized (data not shown). SCA3.B11 had a numerically and structurally normal karyotype (46, XY) (Fig. 1A) and no integration of reprogramming plasmids (Fig. 1B). The pluripotency genes OCT4, NANOG, SOX2 and LIN28 were upregulated in iPSCs compared to patient fibroblasts (Fig. 1C) and the gene expression levels were comparable to those of a previously described positive control iPSC line (Rasmussen et al., 2014). In agreement, all SCA3.B11 iPSCs expressed the pluripotency proteins OCT4, NANOG, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4 (Fig. 1D), illustrating the purity of the iPSC line. Pluripotency was supported by the ability of SCA3.B11 to differentiate to cells from the three germ layers in vitro, as shown by fluorescent immunocytochemistry displaying expression of the endodermal marker α-fetoprotein (AFP), the mesodermal marker smooth muscle actin (SMA) and the ectodermal marker β-III-tubulin (βIIItub) (Fig. 1E). The identity of SCA3.B11 was confirmed by demonstrating expression of both normal and expanded ataxin-3 proteins by western blot (Fig. 1F). Furthermore, the lengths of the CAG-repeats in the two ataxin-3 alleles were determined to 14 and 74 repeats in both patient iPSCs and fibroblasts by fragment length analysis (data not shown).
    Materials and methods
    Acknowledgments
    Resource table
    Resource details RCe007-A (RC-3) was shown to be pluripotent by expression of Oct-4, Nanog, Sox-2, Tra-1-60 and Tra-1-81, but not SSEA-1, using immunocytochemistry (Table 1, Fig. 1). By flow cytometric analysis, the expression of pluripotency makers Oct-4 and SSEA-4 was 86.2% and 96.8%, respectively, whereas low expression of the differentiation marker SSEA-1 (5.9%) was observed (Fig. 2). Differentiation to the three germ layers, endoderm, ectoderm and mesoderm, was demonstrated using embryoid body formation and expression of the germ layer markers α-fetoprotein, β-tubulin and muscle actin (Fig. 3). HLA Class I and II typing is available and blood group genotyping gave the blood group O1O1 (Table 2).
    Verification and authentication The cell line was analysed for genome stability by G-banding (Fig. 4) and showed a normal 46XX female genotype. The cell line is free from mycoplasma contamination as determined by RT-qPCR.
    Materials and methods
    Acknowledgements Research culminating in the derivation of this line was funded by a grant (PM07321) from Scottish Enterprise Economic Development Agency to PDS, MB, and AC.