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    • Several genetic and functional assays were

    2018-10-31

    Several genetic and functional assays were performed to determine the quality of the BSS2-PBMC-iPS4F24 line. Sequencing analysis of the GPIX locus confirmed the presence of c. 182 A>G change in exon 3 of the GPIX gene corresponding to a homozygous p.Asn45Ser mutation, identical to PBMCs-BSS2 (Fig. 1A). In addition, BSS2-PBMC-iPS4F24 line silenced the expression of exogenous reprogramming transgenes after 8 passages (Fig. 1B) and showed normal karyotype (46, XX) (Fig. 1C). Additionally, Short Tandem Repeat (STR) profiling confirmed same genetic identity between both samples (Table 1). Pluripotency was assessed by alkaline phosphatase staining (ALP) (Fig. 1D), qRT-PCR analysis of endogenous pluripotent transcription factors OCT-4, SOX-2, NANOG and REX1 (Fig. 1E) and flow cytometry analysis of protein stem cell markers SSEA3, SSEA4, Tra1-60, Tra1-81 and Oct3/4 (Fig. 2A). Finally, we assessed the functionality of the BSS2-PBMC-iPS4F24 ace inhibitor by differentiating them into the three germ layers both in vitro (by embryoid body (EB) formation) and in vivo (teratoma formation). Both EBs (Fig. 2B) and teratomas (Fig. 2C) showed expression of representative markers of ectoderm, mesoderm and endoderm.
    Materials and methods
    Author disclosure statement
    Resource table. Resource details Myoclonic epilepsy associated with ragged-red fibers (MERRF) is a multisystem disorder caused by a mutation in the mitochondrial DNA (mtDNA) gene MT-TK. The most common pathogenic variant is an A-to-G transition at nucleotide 8344 (m.8344A>G). However, the occurrence of “heteroplasmy” can result in varying tissue distribution of mutated mtDNA, and lead to varying symptoms (Hammans et al., 1993; Enriquez et al., 1995). In this study, PBMCs were obtained from an 11-years-old MERRF patient with m.8344A>G mutation in the MT-TK allele. Using Sendai virus containing the reprogramming factors Oct3/4, Sox2, cMyc, Klf4, the human MERRF iPSC lines were generated from the PBMCs non-integratively (Fig. 1A). The iPSC line described in this publication was named iMERRF-C7. Five additional clones from the same patient were also isolated and characterized (data not shown). All iMERRF-C7 iPSCs stained positive for the pluripotency markers SSEA-4, TRA-1-60 and TRA-1-81 at passage of 10 (Fig. 1B), illustrating the purity of the iPSC line. Accordingly, the expression of the other pluripotency genes OCT4, SOX2, C-MYC, NANOG and TERT was upregulated in iPSCs compared to patient PBMCs, and the gene expression levels were comparable to those of a characterized positive control H9 line (Fig. 1C). Pluripotency was supported by the capability of iMERRF-C7 to differentiate into the three germ layers in vivo by teratoma assay (Fig. 1D). iMERRF-C7 had a normal karyotype (46, XX), and had no copy number variation larger than 300kb analyzed by SNP-array (Fig. 1E). The co-existence of the m.8344A>G mutation and wild type allele in both iMERRF-C7 and patient PBMCs was confirmed by Sanger sequencing. The heteroplasmic level of iMERRF-C7 was measured by pyrosequencing the mitochondrial DNA flanking m.8344 loci, which is about the same level compared to the primary patient PBMCs (Fig. 1F). Additionally, there are variations of the m.8344 heteroplasmic levels in the other iPSC lines generated from the same patient, including one line with no mutant allele (Table S1), which is similar with the previous study (Fujikura et al., 2012).
    Materials and methods
    Acknowledgements We are grateful to the patient for participating in this study. The study was supported by the National Natural Science Foundation of China (81500244), the Natural Science Foundation of Jiangsu Province of China (BK20150081) and the Project on Social development of Jiangsu Province (BE2015614).
    Resource table. Resource details Purified patient\'s PBMCs were transduced with Sendai virus particles (Yang et al., 2008–2012), delivering the four Yamanaka factors (OCT4, SOX2, KLF4, and c-Myc; Takahashi et al., 2007). Colonies with typical iPSC-like morphology appeared 10–12d after transduction and some of them were picked 16–18d later. Clones were expanded and further characterized. One of the isolated clones gave rise to the MMD ISCH#6 iPSC line, characterized by homogeneous colonies, normal diploid 46, XX karyotype, without detectable abnormalities, and stable expression of the pluripotency markers OCT4, Nanog, SOX2 and TRA-1-60, verified by both immunofluorescence, RT-PCR and western blot (Fig. 1A–C) (Itskovitz-Eldor et al., 2000; Carpenter et al., 2003). Absence of residual episomal Sendai virus was confirmed at passage 10 (Fusaki et al., 2009), suggesting that the expression of OCT4, SOX2, KLF4, and c-Myc was indicative of a successful complete reprogramming process (Fig. 1B).