Edinburgh Research Explorer Derivation of the human embryonic stem cell line RCe009-A (RC-5)

ThehumanembryonicstemcelllineRCe009-A(RC-5)wasderivedfromafrozenandthawedDay2embryovolun- tarily donated asunsuitableandsurplus torequirement for fertility treatment following informedconsent under licence from the UK Human Fertilisation and Embryology Authority. RCe009-A carries the common DF508 mutation on the cystic ﬁ brosis trans-membrane regulator gene associated with the disease cystic ﬁ brosis. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro . It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

The human embryonic stem cell line RCe009-A (RC-5) was derived from a frozen and thawed Day 2 embryo voluntarily donated as unsuitable and surplus to requirement for fertility treatment following informed consent under licence from the UK Human Fertilisation and Embryology Authority. RCe009-A carries the common DF508 mutation on the cystic fibrosis trans-membrane regulator gene associated with the disease cystic fibrosis. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.
© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Resource Details
RCe009-A (RC-5) was derived from a frozen and thawed, pre-implantation genetic diagnosis embryo confirmed to have a heterozygous cystic fibrosis mutation (DF508 on the CTFR gene). The embryo was received as a Day 2 embryo and grown to blastocyst stage. The cell line was derived by whole embryo outgrowth on mitotically inactivated human fibroblast (HDF) feeder cells using HDF conditioned medium and expanded under feeder free conditions. RCe009-A (RC-5) was shown to be pluripotent by expression of the pluripotency markers Oct-4, Nanog, SSEA-4, Tra-1-60 and Tra-1-81, but not the differentiation marker SSEA-1, using immunocytochemistry (Table 1, Fig. 1). By flow cytometric analysis, the expression of pluripotency makers Tra-1-60, Tra-1-81 and SSEA-4 was 94.8, 93.6% and 94.8%, respectively, but some expression of the differentiation marker SSEA-1 (37.3%) was observed (Fig. 2). Differentiation to the three germ layers, endoderm, ectoderm and mesoderm, was demonstrated using embryoid body formation and expression of the germ layer markers α-fetoprotein, β-tubulin and muscle actin (Fig. 3).
A microsatellite PCR profile has been obtained for the cell line, and HLA Class I and II typing is available (Table 2). Blood group genotyping gave the blood group AO 1 (Table 2).

Verification and authentication
The cell line was analysed for genome stability by G-banding (Fig. 4) and showed a normal 46XX female genotype. The cell line is free from mycoplasma contamination as determined by RC-qPCR. Microsatellite PCR DNA profiling for cell identity is available.

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Ethics
Derivation of hESC from surplus to requirement and failed to fertilise/develop embryos was approved by The Scotland A Research Ethics Committee and local ethics board at participating fertility clinics and conducted under licence no. R0136 from the UK HFEA with informed donor consent.
By Day 8 of development, or when spontaneous hatching occurred, embryos were placed in derivation conditions consisting of mitotically inactivated neonatal human dermal fibroblasts (HDFs) (ThermoFisher Scientific (Cascade Biologics), Paisley, UK) on tissue culture plastic precoated with 2 μg/cm 2 human laminin (Sigma Aldrich, Dorset, UK) as per manufacturer's recommendation. If required, assisted hatching was performed by removing the zona pellucidae mechanically using Swemed Cutting tools (Vitrolife, Göteborg, Sweden).

Mycoplasma detection was performed using Applied Biosystems PrepSEQ™ Mycoplasma Nucleic Acid Extraction Kit and MicroSEQ™
Mycoplasma Real-Time PCR Detection Kit (ThermoFisher Scientific (Applied Biosystems)) according to manufacturer's instruction.

Endotoxin
Endotoxin levels were determined using the Kinetic-QCL assay (Lonza) and an incubating plate reader (BioTek ELx808) according to manufacturer's instructions. Briefly, an unknown sample was compared with a standard curve of known levels of control endotoxin. An assay was deemed valid if the coefficient of correlation, r ≥ 0.980 and the CV (%) for the standard curve was ≤10%.

Genomic analysis
All outsourced assays were carried out under a Quality and Technical Agreement. DNA was extracted using the QIAamp DNA Mini kit (Qiagen, Manchester, UK) according to manufacturer's recommendations and provided in recommended quantities to the service providers.
Microsatellite PCR, or Short Tandem Repeat analysis, was used to determine cell line identity and was carried out by Public Health England. A profile was obtained for the following core alleles: vWA, D16S539, Amelogenin, THO1, CSF1PO, D5S818, D75820, D135317 and TPOX.
Human Leukocyte Antigen (HLA) tissue typing was carried out by the Scottish National Blood Transfusion Service.  Blood group genotyping was carried out by the Molecular Diagnostics laboratory at NHSBT.
Karyotype analysis was carried out by The Doctors Laboratory (London, UK) or the Western General Cytogenetics Laboratory (Edinburgh, UK). Live cells at 60-70% confluency were shipped overnight in warm containers, fixed and analysed by standard G-banding analysis. For research grade lines, 20 spreads were analysed.