Edinburgh Research Explorer Derivation of the human embryonic stem cell line RCe008-A (RC-4)

The human embryonic stem cell line RCe008-A (RC-4) was derived from a blastocyst 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 ectoderm and mesoderm in vitro . It has a mixed 46XX/45X female karyotype and microsatellite PCR identity and blood group typing data is 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
RCe008-A (RC-4) was derived from a fresh blastocyst that was surplus to requirement or unsuitable for clinical use.The cell line was derived by whole embryo outgrowth on a chemically defined matrix consisting of laminin, fibronectin, Collagen IV and vitronectin (Ludwig et al., 2006) using human fibroblast (HDF) conditioned medium and expanded under feeder free conditions.
A microsatellite PCR profile has been obtained for the cell line and 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 mixture of a normal female karyotype (46XX in 16 cells) and monosomy X (45X in 3 cells).The cell line is free from mycoplasma contamination as determined by RT-qPCR.

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.Embryos were cultured at 36.5-37.5 °C, 5 ± 0.5% CO 2 , 5 ± 0.5% O 2 in drops under paraffin oil (Origio) 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  Fig. 2. Flow cytometry staining of RCe008-A (RC-4) at Passage 13.Isotype/negative control is shown in red, specific staining in green.Percentage staining is indicated in Table 1.

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 the manufacturer's instruction.

Endotoxin
Endotoxin levels were determined using the Kinetic-QCL assay (Lonza) and an incubating plate reader (BioTek ELx808) according to the 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 the 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.
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.
Contents lists available at ScienceDirectStem Cell Researchj o u r n a l h o m e p a g e : w w w .e l s e v i e r .c o m / l o c a t e / s c r Cell culture Fresh embryos were cultured EmbryoAssist (Origio (Medicult), Denmark) until Day 3 or BlastAssist (Origio) after Day 3 of development.
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/).

Table 1
Summary of quality control testing and results for RCe008-A (RC-4).

Table 2
Microsatellite PCR, blood group and HLA tissue typing results for RCe008-A (RC-4).