Human ESC culture and processing were performed

Human ESC culture and processing were performed in a grade A tissue culture cabinet in a grade B clean room environment monitored for particulate and microbiological contamination during cell processing in accordance with Rules and Guidance for Pharmaceutical Manufacturers and Distributors — The Orange Guide, compiled by the UK Medicines Healthcare Products Regulatory Authority (go to: https://www.gov.uk/guidance/good-manufacturing-practice-and-good-distribution-practice). Accordingly, the facility was operating under a mature Quality Management System, compliant with ISO9001:2008 standards. HESC derivation was performed under licensure from the UK HFEA (R0136 to centre 0202) and HTA (Licensing Number 22631). By flow cytometry, RCe021-A (RC-17) expressed the pluripotency makers Oct-4, Tra-1-60 and SSEA-4 (89.8%, 70.8% and 94.7%, respectively), whereas lower expression of the differentiation marker SSEA-1 (10.2%) was observed (Figs. 1, 2). Differentiation to the three germ layers, endoderm, ectoderm and mesoderm, was demonstrated using embryoid body formation in vitro, and expression of the germ layer markers α-fetoprotein, β-tubulin and muscle buy Z-YVAD-FMK was observed (Fig. 3). A microsatellite PCR profile has been obtained for the cell line, and HLA Class I and II typing is available (Table 1). Blood group genotyping gave the blood group O1O1, expected to give rise to blood group O+ (Table 1). The cell line is free from mycoplasma contamination as determined by RT-qPCR.
Materials and methods
SNP genotyping and analysis DNA samples were assayed using the Illumina HumanCytoSNP-12 v2.1 BeadChip. Genotyping data was initially assessed using GenomeStudio genotyping module (v1.94, Illumina). Karyostudio (v1.4, Illumina) was employed to perform automatic normalisation and to identify genomic aberrations utilising default settings of the built-in cnvPartition algorithm (3.07, Illumina) to generate B-allele frequency and smoothened Log R ratio plots for detected regions. These parameters are designed to detect CNVs greater than 75kb and CN-LOH regions larger than 1MB with a confidence value greater than 35. All identified regions were first cross-matched to the Database of Genomic Variants (DGV; http://dgv.tcag.ca) to identify naturally-occurring structural variations in the human. CNVs that were not identified on the DGV were then checked against a list of ES cell-associated culture adaptation genomic variants published by the International Stem Cell Initiative (Amps et al. 2011). See also Canham et al. 2015 for further details.
Genomic analysis and outsourced assays
Acknowledgments
Resource table
Resource details Expanded T cells isolated from a 38year-old Japanese healthy male 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, WT-iPSC2, was confirmed by PCR analysis (Fig. 1A). DNA fingerprint analysis of WT-iPSC2 and expanded T cells provided the origin of cell source (Fig. 1B). The authenticity of WT-iPSC2 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, WT-iPSC2 maintains normal karyotype (46, XY) (Fig. 1F), and Bisulfite sequencing revealed that the NANOG promoter region in WT-iPSC2 was unmethylated (Fig. 1G).
Materials and methods
Acknowledgements 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 following financial supports; JSPS KAKENHI Grant-in-Aid for Research Activity Start-up (Grant Number 24890250) and Grant-in-Aid for Scientific Research (C) (Grant Number 15K09789), Lydia O\'Leary Memorial Foundation, and Takeda Science Foundation.