p-Cresyl sulfate br Resource table br Resource details Urine

Resource table
Resource details Urine epithelial cells underwent the reprogramming procedure using the CytoTune®-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, USA) containing reprogramming vectors with the four Yamanaka factors, Oct3/4, Sox2, Klf4 and c-Myc. These factors have been repeatedly shown to be sufficient for efficient reprogramming (Lieu et al., 2013; Takahashi et al., 2007). The identity of derived human iPS cell line was confirmed by immunocytochemistry, using the following p-Cresyl sulfate for pluripotency markers, TRA-1-60, Sox2, Oct3/4 and SSEA4 (Fig. 1a). Sendai virus transgenes were undetectable from passage 7 (Fig. 1c). To confirm trilineage differentiation potential, in vitro embryonic body (EB) formation assay was performed (Fig. 3a). Spontaneous differentiation induced the transcription of the following genes: AFP (endoderm), MSX1 (mesoderm) and Pax6 (ectoderm) (Fig. 3b). Additionally, the formation of the three germ layers was confirmed at the protein level by immunocytochemistry, which showed the expression of Nestin, TUJ1, SMA and AFP (Fig. 3c). Ploidy of the derived iPS cell line was analyzed by low-pass whole-genome sequencing (Fig. 2) (Wells et al., 2014).
Materials and methods
Resource table. Resource details Skin biopsy was obtained from a 63-year-old man (anonymized as H256) and an induced pluripotent stem cell (iPSC) line, H256 clone (C) 6, was generated using an episomal vector system carrying transcripts for human OCT4, SOX2, KLF4, L-MYC, LIN28, and small hairpin RNA for TP53 (Okita et al., 2011; Rasmussen et al., 2014). We then generated a heterozygous CHMP2B point-mutation (pathogenic G-to-C transition in the 5′ acceptor splice site of exon 6) in this iPSC line via the CRISPR-Cas9 system (Ran et al., 2013). The mutation was validated by DNA sequencing (Fig. 1A). Subsequently, we confirmed that the gene modified clone of H256 C6, termed CHMP2B IVS5AS GG-GC, remained truly pluripotent. This was demonstrated via expression analyses of key pluripotency markers on protein level (Fig. 1B). Additionally, CHMP2B IVS5AS GG-GC retained the potential to differentiate into cell types of all three germ layers upon embryoid body formation (Fig. 1C). More importantly, the gene editing process introduced no genetic chromosomal aberrations and the cells exhibit a normal karyotype (Fig. 1D). In summary, we have generated a disease-specific heterozygous CHMP2B mutant human iPSC line. Together with the original isogenic healthy control, they will serve as an ideal study tool for in vitro disease modeling and pathological study of FTD3 (Skibinski et al., 2005), independent of the familial background and thereby completely focused on the biological effect of the particular CHMP2B mutation.
Materials and methods
Acknowledgments We would like to thank Dr. Keisuke Okita and Prof. Shinya Yamanaka for providing the plasmids for reprogramming and Dr. Feng Zhang for providing the plasmids for gene editing. We thank the following agencies for financial support: 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 Center of Excellence in Neurology, grant No. 4108-00008B) and the China Scholarship Council.
Resource table. Resource details Skin biopsy was obtained from a 63-year-old man (anonymized as H256) and an induced pluripotent stem cell (iPSC) line, H256 clone (C) 6, was generated using an episomal vector system carrying transcripts for human OCT4, SOX2, KLF4, L-MYC, LIN28, and small hairpin RNA for TP53 (Okita et al., 2011; Rasmussen et al., 2014). We then generated a homozygous CHMP2B point-mutation (pathogenic G-to-C transition in the 5′ acceptor splice site of exon 6) in this iPSC line via the CRISPR-Cas9 system (Ran et al., 2013). The mutation was validated by DNA sequencing (Fig. 1A).