Edinburgh Research Explorer Derivation of the human embryonic stem cell line RCe014-A (RC-10)

The human embryonic stem cell line RCe014-A (RC-10) was derived from a fresh oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XY and 47XY +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data is available. © 2016 The Authors

The human embryonic stem cell line RCe014-A (RC-10) was derived from a fresh oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XY and 47XY + 12 male karyotype and microsatellite PCR identity, HLA and blood group typing data is available. ©

Resource details
RCe014-A (RC-10) was received as a failed to fertilise oocyte/1PN (pro-nuclear) embryo that was surplus to requirement or unsuitable for clinical use. The embryo was grown to blastocyst stage and the cell line was derived by whole embryo outgrowth on mitotically inactivated human fibroblast (HDF) feeder cells using xeno-free medium (XF KODMEM) and expanded under xeno free and feeder free conditions. By flow cytometry, RCe014-A (RC-10) expressed the pluripotency makers Oct-4, Tra-1-60 and SSEA-4 (93.4%, 94.1% and 98.9%, respectively), whereas low expression of the differentiation marker SSEA-1 (3.5%) was observed (Fig. 1, Table 1). 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. 2).
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. 3) and showed a mixed 46XY and trisomy 12 (47XY +12) male genotype in 12 and 3 cells analysed, respectively. The cell line is free from mycoplasma contamination as determined by RC-qPCR. Microsatellite PCR DNA profiling for cell identity is available (Table 2).

Ethics
Derivation of hESC from surplus to requirement and failed to fertilise/develop oocytes and embryos was approved by The Scotland

Cell culture
Failed to activate oocytes and embryos were cultured in Sydney cleavage medium (Cook Medical, Hertfordshire, UK) until day 3 and Sydney blastocyst medium (Cook Medical) after day 3 of development. Embryos were cultured at 36.5-37.5°C, 5 ± 0.5% CO 2 , 5 ± 0.5% O 2 in drops under paraffin oil (Cook Medical) and transferred to fresh medium at least every 2-3 days.
The established cell line was expanded and banked using CellStart matrix and Stempro hESC Serum Free Medium (ThermoFisher Scientific). Passaging was performed mechanically using an EZ passage tool (ThermoFisher Scientific). hESC lines were expanded to 25-30 wells of a 6-well plate and cryopreserved in 0.5-1 ml Cryostor CS10 (Biolife Solution, Washington, USA).

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%. Fig. 1. RCe014-A (RC-10) was subjected to flow cytometry analysis for markers of pluripotency with specific antibody (top row) or isotype control (bottom row) as indicated above the histograms. Percentage staining is indicated in Table 1.

Flow cytometry
Pluripotency was determined using the Human and Mouse Pluripotent Stem Cell Analysis kit (BD, Oxford, UK). Oct 3/4 and SSEA-4 were included as pluripotency markers, and SSEA-1 as a differentiation marker. FITC conjugated Tra-1-60 (BD) was used as an additional pluripotency marker. Fixed and permeabilised cells were analysed using a FACS Aria flow cytometer (BD). Percentage expression of each marker was compared to isotype control or unstained cells.

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.