br Materials and methods br Verification and

Materials and methods
Verification and authentication Karyotyping and PCR-RFLP analysis were performed at Reproductive & Genetic Hospital of CITIC-Xiangya. Twenty metaphase cells were observed and all had a normal karyotype of 46, XX. The results of PCR-RFLP assay confirmed a homozygous deletion of SMN1 exon 7 in chHES-427 cells.
Acknowledgments This work was supported by grants from the National Basic Research Program of China (973 program, 2011CB964901 and 2012CB944901), and the National Natural Science Foundation of China (81222007 and 31101053). We thank the postdoctoral research station of Central South University for their support. We thank the IVF team of the Reproductive & Genetic Hospital of CITIC-Xiangya for their assistance.
Resource table
Resource details The study was approved by the “De Videnskabsetiske Komiteer for Region Hovedstaden” (protocol number H-4-2011-157), Copenhagen, Denmark and written informed consent was obtained in all cases. To protect patient family privacy, no personal patient information is presented here. A skin biopsy was obtained from a 48-year-old woman carrying a heterozygous mutation in exon 4 of presenilin 1 (PSEN1) gene causing a change in amino atp gamma s Supplier A79V. Mutations in PSEN1 are the most common known causes of inherited Alzheimer\'s disease (AD). Her mother is carrying the same mutation and she has severe, late-stage AD whereas the 48-year-old daughter was presymptomatic at the time of biopsy. Episomal plasmids carrying gene sequences for hOCT4, hSOX2, hKLF4, hL-MYC, hLIN28 and a short hairpin against TP53 (Okita et al., 2011) were used to reprogram the fibroblasts into iPSCs. This technique successfully established integration and feeder-free iPSCs, as previously reported by Rasmussen et al. (2014). 24days after reprogramming, several iPSC colonies were picked for further selection and expansion as single cells. Episomal plasmid integration was analyzed by qPCR with DNA from iPSCs at passage 10 with plasmid-specific primers, using human fibroblasts as control. The analysis confirmed that the reprogramming genes hOCT4, hSOX2 and hLIN28 were absent and had, consequently, not integrated into the genome (Fig. 1A). The clones were karyotypically normal and sequencing confirmed by the presence of a c.236 C>T change in exon 4 of the PSEN1 gene corresponding to a heterozygous A79V mutation (Fig. 1B). qPCR was performed to analyze the expression of pluripotency markers at mRNA level, which showed that the endogenous pluripotency genes OCT4, DMNT3B, GABRG3, NANOG, TDGF1, GDF3, SOX2 and ZFP42 were present at levels comparable to human embryonic stem cells (hESCs) (Fig. 1C). At the protein level, immunocytochemical (ICC) analysis confirmed the expression of the pluripotency markers NANOG, SSEA3, SSEA4, TRA-1-60 and TRA-1-81 (Fig. 1D). Furthermore, in vitro differentiation followed by ICC analysis of the endodermal marker α-feto protein (AFP), the mesodermal marker smooth muscle actin (SMA) and the ectodermal marker β-III tubulin (TUJI) confirmed the ability the iPSCs to differentiate into all three germ layers (Fig. 1E).
Materials and methods
Verification and authentication Karyotyping was performed by Cell Guidance Systems (UK) and a minimum of 20 metaphases were analyzed. The results showed a normal 46, XX karyotype, without any detectable abnormalities (Fig. 1F). A79V-iPSC line identity and purity was confirmed by sequencing of PSEN1 (Fig. 1B) and ICC for pluripotency genes expression (Fig. 1D).
Acknowledgments We would like to thank Dr. Keisuke Okita and Prof. Shinya Yamanaka for providing the plasmids. Furthermore, we would like to thank Tina Christoffersen and Anita Pacht for excellent technical assistance. We thank the following agencies for financial support: the European Union 7th Framework Program (PIAP-GA-2012-324451-STEMMAD), Innovation Fund Denmark \"Brainstem\" (4108-00008B) and the UCPH Excellence Programme for Interdisciplinary Research supporting the PhD program of Carlota Pires.