Edinburgh Research Explorer Derivation of the human embryonic stem cell line RCe010-A (RC-6)

The human embryonic stem cell line RCe010-A (RC-6) was derived from a frozen and thawed blastocyst volun-tarilydonatedas unsuitableand surplus to fertility requirementsfollowing ethicscommitteeapprovedinformed 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. 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
RCe010-A (RC-6) was derived from a frozen and thawed blastocyst that was surplus to requirement or unsuitable for clinical use.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.
A microsatellite PCR profile has been obtained for the cell line, and HLA Class I and II typing is available (

Verification and authentication
The cell line was analysed for genome stability by G-banding (Fig. 4) 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 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 KOSR based Fig. 2. RCe010-A (RC-6) 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.cryopreservation solution (75% KO-DMEM, 15% Xeno-free KOSR (ThermoFisher Scientific ) and 10% DMSO (Origen Biomedical, Texas, USA)) or 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).Oct 3/4 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.RCe010-A (RC-6) expresses pluripotency markers Oct-4, Nanog, SSEA-4 and Tra-1-60, but no significant expression of the differentiation maker SSEA-1.Specific staining shown in green, cell nuclei are counterstained with DAPI (blue).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3 .
Fig. 3.In vitro differentiation of RCe010-A (RC-6) to ectoderm (β-tubulin III), mesoderm (muscle Actin), and endoderm (α-fetoprotein).Specific staining shown in green, cell nuclei are counterstained with DAPI (blue).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1
Summary of quality control testing and results for RC-6 (RCe010-A).

Table 2
Microsatellite PCR, blood group and HLA tissue typing results for RCe010-A (RC-6).