The DNA fingerprint of Lymph AD iPS cell line was

The DNA fingerprint of Lymph2-AD2-iPS cell line was identical to the parental lymphoblast line AD2-2 (Fig. 1D). Karyotype analysis was male (XY) and both lines exhibited a normal diploid chromosomal content (Fig. 1E). As depicted in the Dendrogram (Fig. 1F), the transcriptome of the parental lymphoblast SBI-0206965 differs from the pluripotent cell lines. The transcriptomes of the Lymph2-AD2-iPS cells and the embryonic stem cell line-H1, clustered together with a Pearson correlation of 0.966. 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: PH1-PBMCs-hiPSC4F1 and PH1-Fib-hiPSC4F1
Resource details We have generated human induced pluripotent stem cell (hiPSC) lines from peripheral blood mononuclear cells (PBMCs) and human dermal fibroblasts of a Primary Hyperoxaluria Type I (PH1)-diagnosed patient with c.731T>C mutation (p.I244T) in AGXT gene, which is highly prevalent in Canary Islands due to founder effect (Santana et al. 2003). PH1-PBMCs-hiPSC4F1 and PH1-Fib-hiPSC4F1 lines were generated using the CytoTune®-iPS 2.0 Reprogramming System (Life Technologies, Invitrogen), which includes the reprogramming factors SOX2, POU5F1, cMYC and KLF4. This reprogramming system is based on a modified and non-transmissible form of Sendai virus (SeV) (Ban et al. 2011). Both PH1-hiPSC lines displayed a typical round shape ESC-like morphology with small and tightly packed cells, with a high nucleus/cytoplasm ratio and prominent nucleoli (Fig. 1A). The presence of the c.731T>C mutation in AGXT gene was confirmed in both PH1-hiPSC lines (Fig. 1B) and the expression of several pluripotency-associated markers was corroborated by qPCR (Fig. 1C), immunofluorescence (Fig. 2A) and FACS analyses (Fig. 2B). Moreover, the absence of exogenous reprogramming transgenes was observed by RT-PCR after 8–10 passages (Fig. 1D). Differentiation capacity into three germ layers was demonstrated by in vivo teratoma formation (Fig. 3A). Finally, PH1-hiPSC lines showed normal karyotype (46, XY) (Fig. 3B) and cell line identity was corroborated by DNA fingerprinting.
Material and methods
Author disclosure statement
Acknowledgments We thank the Genomics Core Facility of the CIMA and the CIMA LAB Diagnostics. This work was supported by the “Torres Quevedo” Subprogram, Ministry of Economy and Competitiveness (PTQ-11-04777) to JRRM; the Institute of Health Carlos III (PI13/00862) to JRRM and RETIC (RD12/0019/0031) to FP; and the Fundación Bancaria Caja Navarra (70270) to JRRM.
Resource table
Resource details The generation of the human iPSC line, PG64SV.2, 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 a defect of intergenomic communication (Hirano et al., 2001) were provided by EuroBiobank. The patient\'s fibroblasts carried a homozygous mutation in the gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma (POLG) (c.2243G>C; p.Trp748Ser). The presence of this mutation in the iPSC line was confirmed by Sanger sequencing (Fig. 1A). PG64SV.2 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, Crypto 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 cells (Fig. 1F). Promoters of the pluripotency associated genes, OCT4 and NANOG, heavily methylated in the original fibroblasts were almost demethylated in the PG64SV.2 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, XX) after more than twenty culture passages (Fig. 1H). We also confirmed by DNA fingerprinting analysis that the line PG64SV.2 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).