br Acknowledgments We would like to thank Dr Keisuke

Acknowledgments We would like to thank Dr. Keisuke Okita and Prof. Shinya Yamanaka for providing the plasmids for reprogramming and Dr. Feng Zhang for providing the plasmid for gene editing. We thank Ida Jørring and Bente Smith Thorup for excellent technical assistance in the cell culture. We thank Mihaela Mate for cytogenetic technical assistance. We thank the following agencies for financial support: The Danish Agency for Science, Technology and Innovation (6114-00003B-768138), the People Programme (Marie Curie Actions) of the European Union\'s Seventh Framework programme FP7 under REA grant agreement (STEMMAD, grant No. PIAPP-GA-2012-324451), Innovation Fund Denmark (BrainStem – Stem cell Centre of Excellence in Neurology, grant No. 4108-00008B).
Resource table
Resource details Fibroblasts from a male patient with PLX-4720 heterozygous variants in the tyrosine hydroxylase gene (TH, DYT14; OMIM: 191290; HGNC: 11782; c.[385-C>T]; [692-G>C]/p.[R129*]; [R231P]) were episomally reprogrammed using the reprogramming factors Oct3/4, Sox2, L-Myc, Klf4, Lin28 and an shRNA directed against p53 according to Okita et al. (2011). Karyotype was determined by fluorescence R-banding and provided a normal diploid 46,XY karyotype (Fig. 1A). Absence of episomal plasmids was analysed by RT-PCR using EBNA-and plasmid-specific primers (Fig. 1B; Table 1). Presence of the pathogenic variants was verified by PCR followed by Sanger Sequencing (Fig. 1C, Table 1). Expression of pluripotency markers was detected by immunofluorescence staining specific for Oct3/4, Sox2, Nanog, Lin28 and SSEA-4 (Fig. 1D) as well as by RT-PCR using gene-specific primers (Fig. 1E; Table 1). Differentiation potential was tested using embryoid body (EB) formation followed by RT-PCR (Haase et al., 2009) amplifying AFP (endoderm), MLC2A (mesoderm) and βIII-tubulin (ectoderm; Fig. 1F) and immunofluorescence staining for endodermal (AFP), mesodermal (SMA) and ectodermal (βIII-tubulin) markers (Fig. 1G).
Materials and methods
Acknowledgements pCXLE-hOCT3/4-shp53-F, pCXLE-hSK and pCXLE-hUL were kind gifts from Shinya Yamanaka (Addgene plasmids #27077, #27078, and #27080). This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy; EXC 62/3 to G.G.) and in part by the FP7-HEALTH-2012-INNOVATION-1 EU Grant No. 305444 (to N.B.).
Resource table. Resource details Spinocerebellar ataxia type3 (SCA3), also named as Machado Joseph disease (MJD), is the most common subtype of hereditary spinocerebellar ataxia, which is caused by an abnormal expansion of the CAG trinucleotide repeats in the 3′ end of ATXN3 gene and resulted in expanded polyglutamine stretches near the C-terminus of ATXN3 gene product, ataxin-3 (Kawaguchi et al., 1994). Human embryonic stem cell (hESC) line chHES-472 was derived from abnormal embryos diagnosis with Spinocerebellar ataxia type 3. In normal individuals, the number of CAG repeats in ATXN3 gene is 12 to 40 (Cummings and Zoghbi, 2000), as showed in the wild-type control (Fig. 1A). However, besides a normal band, chHES-472 cell line showed an expanded band with about 400bp, consistent with the positive control (Fig. 1A). This abnormal band indicates about 74 CAG repeats with pathogenicity. During long-term culture on the mitotically inactivated mouse embryonic fibroblasts (MEFs), those cells maintained a stable karyotype 46, XY (Fig. 1B), expressed the pluripotency related genes by RT-PCR (Fig. 1C) and were positive for OCT4, NANOG, TRA-1-60 and TRA-1-81 as well as alkaline phosphatase (Fig. 1D). The differentiation capacity of chHES-472 cells was confirmed through in vivo assays. Tissues of the three germ layers were detected in chHES-472 cells-derived teratomas by Hematoxylin and Eosin staining (Fig. 1E).
Materials and methods