FRAX597 manufacturer The pluripotency of MPSII line

The pluripotency of MPSII-4.1 line was confirmed by alkaline phosphatase staining (ALP) and by immunocytochemistry staining (ICC) for endogenous NANOG and -CADHERIN (B) after silencing of the exogenous hOKSM construct. The spontaneous differentiation potential of the iPSC line towards the three germ layers was demonstrated by the expression of ectodermal (βIII-TUBULIN), mesodermal (BRACHYURY) and endodermal (GATA4) markers (B) using ICC. The karyotype of the MPSII-4.1 iPSC line was determined by Giemsa-banding, proving normal diploid 46, XY karyotype, without any detectable abnormalities (B). Materials and methods
Acknowledgement We would like to thank for Department of Pediatrics, University of Pécs (Hungary) for collecting the patient blood samples and providing clinical data. This work was supported by grants from EU FP7 projects (IDPBYNMR, PITN-GA-2010-264257; Anistem, PIAPP-GA-2011-286264; EpiHealth, HEALTH-2012-F2-278418); and Research Centre of Excellence – 11476-3/2016/FEKUT.
Resource details A human induced pluripotent stem cell (hiPSC) line was previously generated from a skin biopsy obtained from a 58-year-old male Alzheimer\'s disease (AD) patient carrying a heterozygous point mutation (c.449C>T) in exon 6 of the presenilin 1 (PSEN1) gene (Tubsuwan et al., 2016). The single base pair mutation results in the substitution of leucine (L) to proline (P) at position 150 of the amino FRAX597 manufacturer sequence (i.e., L150P). To generate a patient-specific and integration-free hiPSC line, 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 patient\'s fibroblasts into hiPSCs (Tubsuwan et al., 2016). Here, we used this hiPSC line to further generate an isogenic control line, L150P-GC-hiPSC, by employing the CRISPR/Cas9 system (Ran et al., 2013) to correct the c.449C>T mutation. Genomic sequences manipulated for gene editing are shown in Fig. 1A. Correction of the point mutation (C>T) was validated by sequencing (Fig. 1B). The L150P–GC-hiPSC was characterized to be karyotype normal with 46, XY (Fig. 1C) and expressed common pluripotency markers: OCT4, NANOG, SSEA3, SSEA4, TRA-1-60 and TRA-1-81 (Fig. 1D). This study was approved by the “De Videnskabsetiske Komiteer for Region Hovedstaden” (protocol number H-4-2011-157), Copenhagen, Denmark, and informed consent was obtained from the patient and his family. As part of privacy protection, no personal information of the patient is provided here.
Materials and methods
Acknowledgements We would like to thank Dr. Keisuke Okita and Prof. Shinya Yamanaka for providing the plasmids for reprogramming, Dr. Feng Zhang for providing the plasmid for gene editing and Addgene for their distribution of the plasmids. Furthermore, we would like to thank Ulla Bekker Poulsen for her excellent technical assistance. Our work is supported by the European Union 7th Framework Program (PIAP-GA-2012-324451-STEMMAD), the Innovation Fund Denmark for BrainStem (4108-00008B), and the Programme of Excellence 2016 (Copenhagen as the next leader in precise genetic engineering CDO2016: 2016CDO04210) from the University of Copenhagen.
Resource table.
Resource details A human induced pluripotent stem cell (hiPSC) line was generated from skin fibroblasts collected from a familial 64-year-old male Alzheimer\'s disease (AD) patient carrying a heterozygous L282F mutation in the presenilin 1 (PSEN1) gene. The underlying C→T transversion at the first position of codon 282 (Chr14:73198105, Ensembl genome assembly: GRCh38.p7) results in the substitution of leucine (L) to phenylalanine (F) as previously described (Hamaguchi et al., 2009). We confirmed the presence of this L282F mutation in patient fibroblasts through sequencing (Fig. 1A). In addition, we detected a heterozygous variation (Chr14: 73198104 G>A) in our patient (Fig. 1A). Genetic variation at this position does not lead to any changes at the amino acid level and has been previously reported as a silent single nucleotide polymorphism (The 1000 Genomes Project Consortium, 2015).