br Materials and methods br Author disclosure statement

Materials and methods
Author disclosure statement
5. Acknowledgments This work was supported by the UK Medical Research Council grants G0701172 and G0801061. We thank Dr. Yacoub Khalaf, director of the Assisted Conception Unit of Guy\'s and St. Thomas\' NHS Foundation Trust and his staff for supporting the research program. We are especially indebted to Prof. Peter Braude and to the patients who donated embryos.
Resource table
Materials and methods
Verification and authentication
Human embryonic stem cell (hESC) line HES-415 was derived from abnormal E-64d donated by G6PD deficiency patient after informed consent. Sequencing analysis confirmed a heterozygous missense mutation c.1376G>T (p.R459L) of in the cells(A). This mutation was already documented in the main databases such as HGMD () and ClinVar (), indicating a pathogenic effect of this nucleotide change. The result is consistent with that of the proband. These cells expressed pluripotency-related genes NANOG, POU5F1, SOX2 (B) and were positive for pluripotent markers POU5F1, NANOG, TRA-1-81 and TRA-1-60 as well as alkaline phosphatase (C). During long-term culture on the mitotically inactivated mouse embryonic fibroblasts (MEFs), this cell line maintained a stable karyotype 46, XX (D). The differentiation capacity of this cell line was confirmed through and assays. The cells from embryoid bodies expressed the key genes related with the development of main organs from all three germ layers, such as ectoderm markers (PAX6, SOX1, KRT17), mesoderm markers (T, FLK1, RUNX1) and endoderm markers (FOXA2, CXCR4, GATA4) (B), while the teratoma contained a complex pattern of differentiation to three germ layers was detected in teratoma (E). Materials and methods
Verification and authentication Karyotyping and Sequencing analysis were performed at Reproductive & Genetic Hospital of CITIC-Xiangya. All 15 metaphase cells observed had the normal karyotype 46, XX. PCR-Sanger sequencing confirmed a heterozygous missense mutation c.1376G>T (p.R459L) of G6PD in chHES-415 cells which is consistent with that of the proband.
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 IVF team of the Reproductive & Genetic Hospital of CITIC-Xiangya for their assistance.
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Resource details The generation of the human iPSC line, LND554SV.3, was carried out using non-integrative Sendai viruses containing the reprogramming factors, OCT3/4, SOX2, cMYC, KLF4 (Takahashi et al., 2007). For this purpose, fibroblasts from a described patient with Leigh syndrome, an inherited devastating neurodegenerative disorder, were employed (Monlleo-Neila et al., 2013). The patient\'s fibroblasts carried a heteroplasmic mitochondrial DNA (mtDNA) mutation in the MT-ND5 gene (m.13513G>A; p.D393N) with a mutant mtDNA load of 55%. The presence of this mutation in the iPSCs was confirmed (Fig. 1A). Interestingly, the percentage of mutant mtDNA in the LND554SV.3 line was only 32% due to spontaneous segregation of the heteroplasmic mtDNA content (Fig. 1A). LND554SV.3 iPSC colonies displayed a typical ES-like colony morphology and growth behavior (Fig. 1B) and they stained positive for alkaline phosphatase activity (Fig. 1C). We confirmed the clearance of the vectors and the exogenous reprogramming factor genes by RT-PCR after eight culture passages (Fig. 1D). The endogenous expression of the pluripotency associated transcription factors OCT4, SOX2, KLF4, NANOG, CRIPTO and REX1 was evaluated by quantitative real time polymerase chain reaction (qPCR) (Fig. 1E). Immunofluorescence analysis revealed expression of transcription factors OCT4, NANOG, SOX2 and surface markers SSEA3, SSEA4, TRA1-60 and TRA1-81 characteristics of pluripotent ES cells (Fig. 1F). Promoters of the pluripotency associated genes, OCT4 and NANOG, heavily methylated in the original fibroblasts were almost demethylated in the LND554SV.3 line suggesting an epigenetic reprogramming to pluripotency (Fig. 1G). The iPSC line has been adapted to feeder-free culture conditions and displays a normal karyotype (46, XY) after more than twenty culture passages (Fig. 1H). We also confirmed by DNA fingerprinting analysis that the line LND554SV.3 was derived from the patient\'s fibroblasts (Fig. 1I). Finally, the capacity of the generated iPSC line to differentiate into the three germ layers (endoderm, mesoderm and ectoderm) was tested in vitro using an embryoid body based assay (Fig. 1J).