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Resource table
Resource details The generation of the human iPSC line, Oex2054SV.4, 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 patient with an optic atrophy ‘plus’ phenotype were obtained (Amati-Bonneau et al., 2008). The patient\'s fibroblasts carried a heterozygous mutations in the OPA1 gene (c.1861C>T; p.Gln621Ter). The presence of this mutation in the iPSC line was evaluated and confirmed by Sanger sequencing (Fig. 1A). Oex2054SV.4 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 also evaluated by RT-PCR (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 BV6 (Fig. 1F). Promoters of the pluripotency associated genes, OCT4 and NANOG, heavily methylated in the original fibroblasts were almost demethylated in the Oex2054SV.4 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 Oex2054SV.4 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).
Materials and methods
Author disclosure statement
Acknowledgments This work was supported by grants from the “Centro de Investigación Biomédica en Red en enfermedades raras” (CIBERER) (Grant 13-717/132.05 to RG), the “Instituto de Salud Carlos III” [Fondo de Investigación Sanitaria and European Regional Development Fund (ERDF/FEDER) funds PI10/0703 and PI13/00556 to RG and PI15/00484 to MEG], “Comunidad Autónoma de Madrid” (grant number S2010/BMD-2402 to R.G); T.G-M. receives grant support from the Universidad Autónoma de Madrid, FPI-UAM and F.Z-D from the Ministerio de Educación, Cultura y Deporte, grant number FPU13/00544. M.E.G. is staff scientist at the “Centro de Investigación Biomédica en Red en Enfermedades Raras” (CIBERER).
Resource table. Resource details Bernard Soulier syndrome (BSS) is an inherited autosomal recessive rare platelet disorder caused by mutations in the genes coding for the membrane glycoprotein complex GPIb-IX-V (Berndt and Andrews, 2011). In this study we generated an iPSC line from PBMCs of a BSS patient containing the mutation p.Trp71Arg in the GPIX gene (PBMCs-BSS1) (Sánchez-Guiu et al., 2014). This new iPSC line was named BSS1-PBMC-iPS4F4. We used CytoTune iPS 2.0 Reprograming System (Life Technologies, Invitrogen) that contains the vectors used for delivering and expressing the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc to reprogram PBMCs-BSS1 (Takahashi et al., 2007; Yu et al., 2007). Sequencing analysis of the GPIX locus confirmed the presence of c. 259 T>C change in exon 3 of the GPIX gene corresponding to a homozygous p.W71R mutation, identical to PBMCs-BSS1 (Fig. 1A). Additionally, Short Tandem Repeat (STR) profiling confirmed same genetic identity between both samples (Table 1). This iPSC line silenced the expression of exogenous reprogramming transgenes after 8 passages (Fig. 1B) and showed normal karyotype (46, XX) (Fig. 1C). BSS1-PBMC-iPS4F4 colonies displayed typical round shape morphology and they were positive for alkaline phosphatase activity (Fig. 1D).