br Materials and methods br Acknowledgments We

Materials and methods
Acknowledgments We would like to thank Dr. Keisuke Okita and Prof. Shinya Yamanaka for providing the oleuropein for reprogramming. We thank I Bente Smith Thorup for excellent technical assistance in the cell culture. We thank Jeannette Schoene and Dirk A. Pflumm for cytogenetic technical assistance. We thank the following agencies for financial support: The Danish Agency for Science, Technology and Innovation (6114-00003B-768138), the People Programme (Marie Curie Actions) of the European Union\'s Seventh Framework programme FP7 under REA grant agreement (STEMMAD, grant no. PIAPP-GA-2012-324451), Innovation Fund Denmark (BrainStem – Stem cell Centre of Excellence in Neurology, grant no. 4108-00008B).
Resource details Primary fibroblasts from steatosis patient H0008 (male, 61years, high grade -steatosis) were reprogrammed by transduction of retroviruses encoding for OCT4, SOX2, KLF-4 and c-MYC (H0008; Jozefczuk et al., 2012). The pluripotency-associated transcription factors OCT4, NANOG, SOX2 and cell surface markers SSEA-4, TRA-1-60, TRA-1-81 were expressed. 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 the iPSC line, while karyogram revealed a 46,XY karyotype.
Materials and methods
Acknowledgements J.A. acknowledges support from the medical faculty of Heinrich-Heine Universität Düsseldorf.
Resource table.
Resource details Human foreskin fibroblasts were reprogrammed by lipotransfection with oriP/EBNA1 (Epstein–Barr nuclear antigen-1)-based episomal vectors (Yu et al., 2009). Plasmids contain reprogramming factors OCT4, SOX2, NANOG, KLF4, L-MYC, Lin28, shRNA-p53, and miR302/367 cluster, along with the GFP marker (System Biosciences) for efficient pluripotency induction (Takashi et al., 2007, Yu et al., 2007, Kawamura et al., 2009, Anokye-Danso et al., 2011). Obtained cell line ULEIi001-A was characterised and its differentiation potential was demonstrated. No GFP expression was observed 30days after transfection. Obtained iPSCs are morphologically similar to embryonic stem cells (Fig. 1A) and express pluripotency markers: OCT4, SOX2, NANOG, SSEA4, Claudin4 (Fig. 1B). They are able to differentiate into three germ layers (Fig. 1C), and into hepatocyte-like cells (Fig. 1D). Furthermore, this cell line has normal karyotype (Fig. 1E).
Materials and methods
Karyotype analysis
Acknowledgement We thank Dr. Heidrun Holland (Team Leader, Authentication, Stability, and Identity of Cells, SIKT and Faculty of Medicine Leipzig University) for karyotype analysis and Katja Hofmann for fibroblasts isolation. This work was funded by the EU Marie Sklodowska-Curie Actions BIOART Project. grant no. 316690, EUFP7-PEOPLE-ITN-2012.
Resource Table: Resource details Previously, we have generated an induced pluripotent stem cell (iPSC) line (H237 C3) from a symptomatic, 59-year-old woman carrying an R406W mutation in microtubule-associated protein tau (MAPT) gene. Reprogramming was performed by electroporation with three episomal plasmids encoding hOCT4, hSOX2, hKLF4, hL-MYC, and hLIN28 (Okita et al., 2011; Takahashi et al., 2007). This cell line, termed H237 C3, has previously been described (Rasmussen et al., 2016). We have generated a gene-corrected clone of H237 C3 using CRISPR/Cas9 technology, where the mutated triplet TGG (tryptophan) was corrected to the wild-type triplet CGG (arginine) using an ssODN (single stranded oligodeoxynucleotide) as homologous template (Fig. 1A). We confirmed by sequencing analysis that the mutated triplet was corrected without further deletions or insertions at the CRISPR cutting site (Fig. 1B). Finally, we confirmed that the cells were still pluripotent after gene-correction (Fig. 1C, D and E) and that they showed a normal karyotype (Fig. 1F).
Materials and methods