br Resource table br Resource details Spastic paraplegia

Resource table
Resource details Spastic paraplegia gene type 5 (SPG5) is an autosomal recessive subtype of hereditary spastic paraplegia caused by mutations in CYP7B1, a gene encoding for the cytochrome P-450 oxysterol 7-α-hydroxylase, essential for the liver-specific alternative pathway in bile p38 pathway synthesis (Schule et al., 2010). In this study, skin fibroblasts from a 47-year-old man were reprogrammed into iPSCs via electroporation of three episomal plasmids encoding human OCT4, SOX2, KLF4, L-MYC and LIN28 (Okita et al., 2011). To determine the quality of generated iPSCs several genotypic and functional assays were performed. The characterization included analysis of genomic integrity given by comparative SNP analysis of parental fibroblasts and the respective iPSC line (Fig. 1A), resequencing of the mutation-site (Fig. 1B) and excluding the integration of episomal plasmids by PCR (Fig. 1C). Furthermore, the expression of stem cell marker on protein and RNA level were assessed by alkaline phosphatase staining (ALP) (Fig. 2A), immunocytochemical stainings of OCT4 and NANOG (Fig. 2B) and qRT-PCR analysis of OCT4, SOX2, KLF4, c-MYC, NANOG, DNMT3B and TDGF1 in comparison to human embryonic stem cell lines (HuES-H6, HuESH1 and HuES-H9) and fibroblasts (Fig. 2C). To prove the functionality of the generated iPSCs, iPSC-derived embryoid bodies (EBs) were differentiated into cells of all three germ layers. Generated iPSCs were able to differentiate into neurons expressing β-III-tubulin, muscle cells positive for α-smooth muscle actin (SMA), and early endodermal structures like hepatic precursors detectable by antibodies against α-fetoprotein (AFP) (Fig. 2D).
Materials and methods
Acknowledgements This study was supported by the European Union Seventh Framework Programme through funding for the NEUROMICS network (F5-2012-305121 to L.S.), the Marie Curie International Outgoing Fellowship (grant PIOF-GA-2012-326681 to R.S. and L.S.), the E-Rare Network NEUROLIPID (01GM1408B to RS), and the DZNE intersite project (grant to L.S.). We thank the patient for participation.
Resource table. Resource details In this study fibroblasts were isolated and reprogrammed from a 48-year-old female patient carrying compound heterozygous mutations (c.610+364G>A and c.1311A>G) in OPA1. The combination of a deep intronic mutation and a mutation in an intragenic modifier leads to a severe early-onset optic atrophy phenotype complicated by ataxia and pyramidal signs (Behr syndrome; OMIM #210000) (Bonifert et al., 2014) (Chao de la Barca et al., 2016). The iPSC line iPS-OPA1-BEHR was established by nucleofection of episomal plasmids carrying the sequence of OCT4, SOX2, KLF4, L-MYC and LIN28 (Okita et al., 2011). To prove genomic integrity upon reprogramming, whole-genome single nucleotide polymorphism (SNP) genotyping of original fibroblasts and the generated iPSC line iPS-OPA1-BEHR was performed (Fig. 1A). Additionally both mutation-sites were resequenced (Fig. 1B) and genomic integration of episomal plasmids was excluded (Fig. 1C). Pluripotency of generated iPS-OPA1-BEHR was analysed via alkaline phosphatase staining (ALP) (Fig. 2A), staining of pluripotency markers OCT4, TRA-1-81 and SSEA-4 (Fig. 2B), comparative qRT-PCR analysis of pluripotency markers (OCT4, NANOG, KLF4, C-MYC, SOX2, REX1, DNMT3B and TDGF1) with human embryonic stem cells and fibroblasts (Fig. 2C) as well as by the capability to differentiate into cells of all three germ layers (ectoderm, mesoderm, endoderm) (Fig. 2D).
Materials and methods
Acknowledgements This study was supported by the German Federal Ministry of Education and Research (BMBF) to mitoNET (01GM1113E to L.S.). We thank the patient for participation.
Resource table. Resource details Here, we targeted exon 2 of TLE1 using CRISPR-Cas9 based genome engineering (Ran et al. 2013) to generate a TLE1 homozygous knockout hESC line, TLE1-464-G04. This approach generated a 1bp biallelic insertion in exon 2 of TLE1 (Fig. 1A). This insertion was predicted to shift the TLE1 full-length reading frame, resulting in loss of TLE1 expression. To this end, the hESC line WA01 was transfected with constructs expressing Cas9 nuclease coupled with puromycin resistance and an sgRNA targeting exon 2 of TLE1. Cells were then selected with puromycin and plated at clonal density to allow for targeted colonies to emerge. We manually isolated a total of 94 individual colonies, which were expanded in 96-well plates and then duplicated for cell freezing and extraction of genomic DNA. To detect the presence of frame-shift mutations, the region flanking the predicted cut site of exon 2 was PCR amplified, barcoded and deep-sequenced. The putative TLE1 homozygous clone, TLE1-464-G04 (TLE1), and a paired wild-type TLE1 clone, TLE1-464-A01 (TLE1), were expanded and banked for further analyses.