br Materials and methods br Acknowledgments We would

Materials and methods
Acknowledgments We would like to thank Dr. Keisuke Okita and Prof. Shinya Yamanaka for providing the plasmids for reprogramming. We thank Bente Smith Thorup for excellent technical assistance in the cell culture. We thank Mehri Mirrajei 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. (See .) Resource details Lymphoblast glucose assay (Lymph4), derived from a 67-year-old AD patient with the TREM2 missense mutation, p.R47H, (Cuyvers et al., 2014) were reprogrammed employing oriP/EBNA-1-based episomal plasmids expressing OCT4, SOX2, KLF4, C-MYC, L-MYC, LIN28 and a p53 shRNA. Clone A and B of AD2-4 iPSC line were negative for EBNA-1 and oriP (Fig. 1A). Pluripotency was confirmed by (i) expression of OCT4, SOX2, c-MYC, NANOG, TRA-1-60 and TRA-1-81 (Fig. 1B) and (ii) embryoid body (EB)-based spontaneous differentiation into cell types representative of the three germ layers, namely ectoderm (Nestin, PAX6), mesoderm (SMA – smooth muscle actin) and endoderm (SOX17) (Fig. 1C). The DNA fingerprint of AD2-4 iPSC line was identical to the parental lymphoblast line Lymph4 (Fig.1D). Karyotype analysis was female (XX) and both lines exhibited a normal diploid chromosomal content (Fig. 1E). As depicted in the Cluster Dendrogram (Fig. 1F), the transcriptomes of the AD2-4 iPSCs and the embryonic stem cell line-H1, clustered together with a Pearson correlation of 0.961. The reprogramming process did not alter the TREM2 missense mutation as demonstrated in Fig. 1G.
Materials and methods
Acknowledgments JA acknowledges support from the Medical Faculty, Heinrich-Heine-University, Düsseldorf. Research at the Antwerp site is funded in part by the Belgian Science Policy Office Interuniversity Attraction Poles program (BELSPO, www.belspo.be), the Flanders Impulse Program on Networks for Dementia Research (VIND) and the University of Antwerp Research Fund (http://www.uantwerpen.be/). JA, FS, KS and CVB are members of the EU project- AgedBrainSYSBIO. The AgedBrainSYSBIO project received funding from the European Union\'s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 305299.
Resource table: Resource details Previously, we have generated an induced pluripotent stem cell (iPSC) line (H251 C3) from a symptomatic, 57-year-old woman carrying a heterozygous P301L mutation in the microtubule-associated protein tau (MAPT) gene (Rasmussen et al., 2016). Reprogramming was performed by electroporation of three episomal plasmids encoding hOCT4, hSOX2, hKLF4, hL-MYC, and hLIN28 (Okita et al., 2011; Takahashi et al., 2007). Here, we generated a gene-corrected clone of H251 C3 using the CRISPR/Cas9 technology, where the mutated triplet CTG (Leucine) was corrected to the wild-type triplet CCG (Proline) using a single stranded oligodeoxynucleotide (ssODN) as a homologous template (Fig. 1A). Successful gene-correction was validated by sequencing (Fig. 1B). Sequencing analysis of the region around the CRISPR cutting site confirmed that no further cutting occurred and that the DNA sequence remained intact without frameshift or other mutations. We finally ensured that the cells were still pluripotent after gene-correction (Fig. 1C, D and E) and showed a normal karyotype (Fig. 1F).
Materials and methods
Acknowledgments We would like to thank 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 Nevena Stoynova 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).