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  • The karyotype of the BIOT EHMT

    2018-11-08

    The karyotype of the BIOT-0708-EHMT1 iPSC line was determined with Giemsa-banding, proving normal diploid 46, XX karyotype, without any detectable abnormalities (Fig. 1B). In parallel, the pathogenic NM_024757.4(EHMT1):c.3413G>A mutation was confirmed by Sanger sequencing in the newly established iPSC line (Fig. 1C).
    Materials and methods
    Resource table.
    Resource details The generation of human IPSC line i11001 was carried out using E8 based media and feeder-free conditions. EBV-transformed B-lymphocyte ctap from a 79-year old Alzheimer\'s Disease (AD) patient, generated by Coriell Institute For Medical Research, were employed. The patient had a history of progressive presenile dementia, with a parent and three siblings of the patient also being affected (iPSC line i10984). The patient was homozygous for the APOE4 allele. This was confirmed by a PCR-RFLP assay (Hixson and Vernier, 1990) carried out with the lymphoblastoid cell line (Fig. 1A). The lymphoblastoid cells (LCs) were electroporated with OriP/EBNA-1 based episomal plasmids expressing reprogramming factors OCT4, SOX2, KLF-4, L-MYC, LIN28 and a p53 shRNA (Okita et al., 2011). The APOE4/4 genotype of the generated iPS line was confirmed by sequencing (Fig. 1A). After several passages, a PCR for episomal vector backbone confirmed that exogenous episomal factors were not being expressed anymore (Fig. 1A). i11001 iPSC colonies displayed a typical ES-like colony morphology (Fig. 1B). Immunocyto-chemical analysis revealed expression of transcription factors OCT4 and SOX2, and surface markers SSEA4 and TRA-1-81, characteristic of pluripotent stem cells (Fig. 1B). The iPS cells exhibited a normal karyotype (46, XX) by G-Banding analysis (Fig. 1B). The cells also demonstrated differentiation capacity to three germ layers using an embryoid body-based directed differentiation protocol, followed by immunostaining with markers corresponding to the three germ layers i.e. ectoderm (PAX6), mesoderm (FOXC1) and endoderm (GATA4) (Fig. 1C).
    Materials and methods
    Acknowledgements This work was supported by the Alzheimer Forschung Initiative e.V. (grant agreement 15032).
    Resource table Resource details Primary dermal fibroblasts from H0012 (female, 50years, low-steatosis) were reprogrammed by transduction of a combination of two episomal-based plasmids (7F1) expressing OCT4, SOX2, NANOG, LIN28, c-Myc and KLF-4 (H0012; Yu et al., 2011). iPSCs expressed the pluripotency-associated transcription factors OCT4, NANOG, SOX2 and cell surface markers SSEA-4, TRA-1-60, TRA-1-81. Pluripotency was further demonstrated in vitro by embryoid body (EB)-based differentiation to the three germ layers endoderm, ectoderm, and mesoderm. DNA fingerprinting confirmed the origin of both iPSC lines, while karyogram revealed a 46,XX,t(7;10)(q11;p12) karyotype for S12.
    Materials and methods
    Acknowledgements J.A. acknowledges support from the medical faculty of Heinrich–Heine Universität Düsseldorf.
    Resource table.
    Resource details Fibroblasts were obtained from a 77-year-old healthy woman, who did not display any disease related symptoms. Reprogramming was performed by electroporation with 3 episomal plasmids containing hOCT4, hSOX2 and hKLF4, and hL-MYC and hLIN28 (Okita et al., 2011). The line was termed hiPSC-16423 #6. qPCR analysis with plasmid-specific primers showed that none of the plasmids were present anymore after reprogramming (Fig. 1A). qRT-PCR analysis showed that the endogenous pluripotency genes OCT4, NANOG, GABRB3, TDGF1 and DNMT38 were expressed in the same range as in the control iPSC line BIONi010-C iPSCs (Rasmussen et al., 2014) (Fig. 1B). Immunocytochemistry (ICC) analysis demonstrated the presence of the pluripotency markers OCT4, NANOG, TRA1-60, TRA1-81 and SSEA4 on protein level (Fig. 1C). In vitro differentiation followed by ICC analysis with the mesodermal marker smooth muscle actin (SMA), the endodermal marker alpha-1-fetoprotein (AFP) and the ectodermal marker ctap beta-III-Tubulin (TUJI) demonstrated the differentiation potential into all three germ layers (Fig. 1D). In addition, the iPSC-16423 #6 line maintained a normal, female karyotype after the reprogramming process (Fig. 1E).