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Resource table. Resource details RCe008-A (RC-4) was derived from a fresh order RGFP966 that was surplus to requirement or unsuitable for clinical use. The cell line was derived by whole embryo outgrowth on a chemically defined matrix consisting of laminin, fibronectin, Collagen IV and vitronectin (Ludwig et al., 2006) using human fibroblast (HDF) conditioned medium and expanded under feeder free conditions. RCe008-A (RC-4) was shown to be pluripotent by expression of Oct-4 and Nanog using immunocytochemistry (Table 1, Fig. 1). By flow cytometric analysis, the expression of pluripotency markers Tra-1-60, Tra-1-81 and SSEA-4 was 74%, 58% and 75%, respectively, whereas low expression of SSEA-1 (5%) was observed (Fig. 2). Germinal lineage marker expression is not available due to poor EB formation. A microsatellite PCR profile has been obtained for the cell line and blood group genotyping gave the blood group AO1 (Table 2).
Verification and authentication The cell line was analysed for genome stability by G-banding (Fig. 3) and showed a mixture of a normal female karyotype (46XX in 16 cells) and monosomy X (45X in 3 cells). The cell line is free from mycoplasma contamination as determined by RT-qPCR.
Materials and methods
Acknowledgments Research culminating in the derivation of this line was funded by a grant (PM07321) from Scottish Enterprise Economic Development Agency to PDS, MB, and AC.
Resource table. Resource details Expanded T cells isolated from a patient with lymphedema-distichiasis syndrome (LDS) were reprogrammed employing Sendai virus vectors (SeVdp) expressing four reprogramming factors, OCT3/4, SOX2, cMYC, KLF4. SeVdp is integration-free vector, and the absence of reprogramming genes in established iPSC line, LDS-iPSC8, was confirmed by PCR analysis (Fig. 1A). The same pathogenic mutation in the FOXC2 gene was also detected in genomic DNA isolated from LDS-iPSC8 (Fig. 1B), proving the origin of cell source. The authenticity of LDS-iPSC8 was confirmed by the followings; (1) the expression of stem cell markers by immunostaining (Fig. 1C) and RT-PCR (Fig. 1D), (2) the differentiation capability into three germ layers using in vitro differentiation through embryoid bodies (EBs) and teratoma formation (Fig. 1E). In addition, LDS-iPSC8 maintains normal karyotype (46, XY) (Fig. 1F), and bisulfite sequencing revealed that the NANOG promoter region in LDS-iPSC8 was unmethylated (Fig. 1G).
Materials and methods
Acknowledgments We would like to thank Dr. Mahito Nakanishi, Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology, for providing SeVdp. We would also like to thank the financial support by Lydia O\'Leary Memorial Foundation.
Resource Table Resource Details Tail-tip fibroblasts (TTF) and cardiac fibroblasts (CF) derived from Mef2c-AHF-Cre (Verzi et al., 2005) x Ai6(RCLZsGreen) (Madisen et al., 2010) mice were transduced with Oct4, Sox2, Klf4 and c-Myc (OSKM) with the pMXs retrovirus vectors. Mouse ES-like colonies appeared 15–20days after transduction and were picked and seeded on irradiated mouse embryonic fibroblasts (ɣMEFs) and maintained with LIF. Established iPSC, AHF-iPSC, derived from TTF (AHFiPS7) and CF (AHFiPS19) encoded the expected genomic insertions (Fig. 1a). The karyotypes of AHF-iPSC were normal (Fig. 1b) and the transgenes were silenced in established AHF-iPSC lines as shown by qRT-PCR (Fig. 1c). Endogenous pluripotency-associated markers such Oct4, Sox2, Nanog and Zfp42 were expressed in AHF-iPSC, analyzed by qRT-PCR (Fig. 1d), and Nanog (Fig. 1e) and alkaline phosphatase (AP, Fig. 1f) expression was verified by cell staining. To demonstrate the capacity of AHF-iPSC lines to differentiate into the three germ layers we performed in vivo teratoma and in vitro embryoid body (EB) differentiation assays. Teratomas contained tissues derived from the three germ layers (Fig. 2a). We collected RNA from undifferentiated AHF-iPSC and from EB on day 7 and 14 of differentiation. Increased expression of Cxcl12/Mash1 (ectoderm), Acta2/Myh6 (mesoderm) and Hnf4a/Afp (endoderm), (Fig. 2b) was observed. Mef2c and ZsGreen were not detected in undifferentiated AHF-iPSC, however, the expression of Mef2c and ZsGreen was confirmed by qRT-PCR on day 7 and 14 of differentiation (Fig. 2b), and ZsGreen was observed under fluorescence microscopy (Fig. 2c).