Edinburgh Research Explorer Derivation of the human embryonic stem cell line RCe011-A (RC-7)

The human embryonic stem cell line RCe011-A (RC-7) was derived from a failed to fertilise 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 normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available. © 2016 The Authors


Resource details
RCe011-A (RC-7) was derived from a failed to fertilise oocyte/late developing embryo that had undergone activation using strontium chloride (SrCl 2 )-containing media (Bos-Mikich et al., 1997).As the oocyte resulting in RCe011-A (RC-7) divided shortly after activation, it is likely to be a late developing embryo rather than oocyte activation, although this has not been verified.The cell line was derived by whole embryo outgrowth on mitotically inactivated human fibroblast (HDF) feeder cells using xeno free medium and expanded under xeno free and feeder free conditions.
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 O 1 O 1 (Table 2).

Verification and authentication
The cell line was analysed for genome stability by G-banding (Fig. 3) and showed a normal 46XY male genotype.The cell line is free from mycoplasma contamination as determined by RC-qPCR.Microsatellite PCR DNA profiling for cell identity is shown in Table 2.

Ethics
Derivation of hESC from surplus to requirement and failed to fertilise/develop embryos and oocytes 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.
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).

Mycoplasma
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%.

Flow cytometry
Pluripotency was determined using the Human and Mouse Pluripotent Stem Cell Analysis kit (BD, Oxford, UK).Oct3/4 and SSEA-4 were included as pluripotency markers, and SSEA-1 as a differentiation marker.Fixed and permeabilised cells were analysed using a FACS Aria flow cytometer (BD).

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.
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Fig. 1 .
Fig. 1.RCe011-A (RC-7) 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 Table1.

Table 1
Summary of quality control testing and results for RCe011-A (RC-7).
Genotype(details provided in Table2)Bloodgroup genotyping (DNA analysis) To establish blood group of the line BO 1 Karyology (G-banding) Confirmation of normal ploidy by G-banding 46XY HLA tissue typing To establish full HLA type I and II genotype of the line HLA typed Class I and Class II Microbiology and virology Mycoplasma Mycoplasma testing by RT-qPCR Negative Endotoxin Screening for endotoxin levels 1.73 EU/ml Morphology Photography To capture a visual record of the line Normal Differentiation potential Embryoid body formation To show differentiation to three germ layers Expression of muscle actin, β-tubulin and α-feto protein

Table 2
Microsatellite PCR, blood group and HLA tissue typing results for RCe011-A (RC-7).