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

A. Courtney

B. Tye

J. Gardner

J.M. Downie

K. Bruce

Ludwig

M. Bateman

P. Dand

P.A. De Sousa

Stem Cell Research

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

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AbstractThe 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

De Sousa, P.A.

Tye, B.

Bruce, K.

Dand, P.

Gardner, J.

Downie, J.M.

Bateman, M.

Courtney, A.

Elsevier - Publisher Connector 

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

Stem Cell Research 16 (2016) 607–610
Contents lists available at ScienceDirect
Stem Cell Research
j ourna l homepage: www.e lsev ie r .com/ locate /scrLab Resource: Stem Cell LineDerivation of the human embryonic stem cell line RCe008-A (RC-4)P.A. De Sousa a,b,c,⁎, B. Tye a, K. Bruce a, P. Dand a, J. Gardner a, J.M. Downie a, M. Bateman a, A. Courtney a
a Roslin Cells Limited, Nine Edinburgh Bio-Quarter, 9 Little France Road, Edinburgh EH16 4UX, UK
b Centre for Clinical Brain Sciences, University of Edinburgh, UK
c MRC Centre for Regenerative Medicine, University of Edinburgh, UKN
A
In
P
C
D
Ty
Su
O
K
A
Li
In
E
⁎ Corresponding author at: MSc Reader Centres f
Regenerative Medicine University of Edinburgh Chance
Crescent Edinburgh, EH16 4SB Scotland, UK.
http://dx.doi.org/10.1016/j.scr.2016.02.039
1873-5061/© 2016 The Authors. Published by Elsevier B.Va b s t r a c ta r t i c l e i n f oArticle history:
Received 25 February 2016
Accepted 29 February 2016
Available online 3 March 2016The 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 table.ame of stem cell construct RCe008-A
lternative name RC-4, RC4
stitution Roslin Cells Ltd.
erson who created resource B. Tye, K. Bruce, P. Dand, J. Gardner
ontact person and email Paul.desousa@roslincells.com;
Paul.desousa@ed.ac.uk
Janet.downie@roslincells.com
Aidan.courtney@roslincells.com
Malcolm.bateman@roslinfoundation.comate archived/stock date 11 June 2008
pe of resource Biological reagent: Cell line
b-type hESC, research grade
rigin Blastocyst with ICM and Trophoblast
ey transcription factors Oct4 and Nanog (conﬁrmed by
immunocytochemistry)
uthentication See Quality Control Test Summary, Table 1
nk to related literature (direct
URL links and full references)N/Aformation in public databases http://hpscreg.eu/cell-line/RCe008-A
thics Informed consent obtained. Scotland A Re-
search Ethics committee approval obtained
(07/MRE00/56). Conducted under the UK Hu-
man Fertilisation and Embryology Authority
licence no R0136 to centre 0202.Resource details
RCe008-A (RC-4) was derived from a fresh blastocyst that was sur-
plus to requirement or unsuitable for clinical use. The cell line wasor Clinical Brain Sciences &
llor's Building 49 Little France
. This is an open access article underderived 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) conditionedmediumand ex-
panded under feeder free conditions.
RCe008-A (RC-4)was shown to be pluripotent by expression of Oct-
4 and Nanog using immunocytochemistry (Table 1, Fig. 1). By ﬂow
cytometric analysis, the expression of pluripotency markers Tra-1-60,
Tra-1-81 and SSEA-4 was 74%, 58% and 75%, respectively, whereas low
expression of SSEA-1 (5%) was observed (Fig. 2). Germinal lineage
marker expression is not available due to poor EB formation.
A microsatellite PCR proﬁle has been obtained for the cell line and
blood group genotyping gave the blood group AO1 (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)
andmonosomy X (45X in 3 cells). The cell line is free frommycoplasma
contamination as determined by RT-qPCR.Materials and methods
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 in-
formed donor consent.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).
Classiﬁcation Test Purpose Result
Donor screening
HIV 1 + 2
Donor screening for adventitious agents NegativeHepatitis B
Hepatitis C
Identity
Microsatellite PCR (mPCR)
DNA proﬁling to give cell line its signature,
gender/species
Performed
Immunocytochemistry
To assess levels of staining for the
pluripotency markers.
Expression of Oct-4 and Nanog
Phenotype Flow cytometry
Assess antigen levels & cell surface markers
commonly associated with hESC
CD9: 84%
Tra 1-60: 74%
Tra 1-81: 58%
SSEA-4: 75%
SSEA-1: 5%
Genotype (details provided in
Table 2)
Blood group genotyping
To establish blood group of the line AO1(DNA analysis)
Karyology (G-banding) Conﬁrmation of normal ploidy by G-banding 46XX (16)/45X (3)
Microbiology and virology
Mycoplasma Mycoplasma testing by RT-qPCR Negative
Endotoxin Screening for endotoxin levels 1.87 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
Poor EB formation — germinal linage marker expression not
available
608 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 607–610Cell culture
Fresh embryos were cultured EmbryoAssist (Origio (Medicult),
Denmark) until Day3 or BlastAssist (Origio) after Day 3 of development.
Embryos were cultured at 36.5–37.5 °C, 5 ± 0.5% CO2, 5 ± 0.5% O2 in
drops under parafﬁn oil (Origio) and transferred to fresh medium at
least every 2–3 days.
By Day 8 of development, or when spontaneous hatching occurred,
embryos were placed in derivation conditions consisting of a mixture
of laminin (5 μg/cm2; Sigma Aldrich, Dorset, UK), ﬁbronectin
(5 μg/cm2; BD, Oxford, UK), vitronectin (0.2 μg/cm2; BD), and collagen
IV (10 μg/cm2; Sigma) (Ludwig et al., 2006) in human dermal ﬁbroblastFig. 1. RCe008-A (RC-4) expresses pluripotency markers Oct-4 a
Fig. 2. Flow cytometry staining of RCe008-A (RC-4) at Passage 13. Isotype/negative contro(HDF) conditioned medium (80% Knockout-DMEM, 20% Knockout
serum replacement (KOSR), 1 mM glutamine, 0.1 mM β-
mercaptoethanol, 1% nonessential amino acids, and 4 ng/ml human
bFGF (all ThermoFisher Scientiﬁc, Paisley, UK) over 24 h intervals over
7 days) supplemented with an additional 24 ng/ml human bFGF. If re-
quired, assisted hatching was performed by removing the zona pelluci-
dae mechanically using Swemed cutting tools (Vitrolife, Göteborg,
Sweden). Cells were cultured at 36.5–37.5 °C, 5 ± 0.5% CO2, 5 ± 0.5%
O2 and 50% medium exchanged 6 days a week.
The established cell line was expanded and banked using CellStart
matrix and Stempro hESC Serum Free Medium (ThermoFisher Scientif-
ic). Passaging was performed mechanically using an EZ passage toolnd Nanog. Cell nuclei are counterstained with DAPI (blue).
l is shown in red, speciﬁc staining in green. Percentage staining is indicated in Table 1.
Table 2
Microsatellite PCR, blood group and HLA tissue typing results for RCe008-A (RC-4).
Microsatellite PCR results
D3S1358 1 D3S1358 2 vWA 1 vWA 2 D16S539 1 D16S539 2 D2S1338 1 D2S1338 2
125 129 167 170 281 281 336 348
Amelogenin 1 Amelogenin 2 D8S1179 1 D8S1179 2 D21S11 1 D21S11 2 D18S51 1 D18S51 2
X X 145 150 209 215 ND ND
D19S433 1 D19S433 2 THO1 1 THO1 2 FGA 1 FGA 2 CSF1PO 1 CSF1PO 2
121 121 180 184 237 245 325 329
D5S818 1 D5S818 2 D7S820 1 D7S820 2 D13S317 1 D13S317 2 TPOX 1 TPOX 2
152 152 272 276 218 222 231 231
Blood group genotyping
RhD RhC Rhc RhE Rhe Fy a Fy b Fy GATA
pos neg pos pos pos pos pos neg
Jka Jkb K k M N S s
neg pos neg pos pos pos neg pos
Kp a Kp b Do a Do b ABO
neg pos neg pos AO1
ND = not determined
609P.A. De Sousa et al. / Stem Cell Research 16 (2016) 607–610(ThermoFisher Scientiﬁc). hESC lineswere expanded to 25–30wells of a
6-well plate and cryopreserved in 0.5–1 ml KOSR based cryopreserva-
tion solution (75% KO-DMEM, 15% Xeno-free KOSR (ThermoFisher Sci-
entiﬁc) 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 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 com-
pared 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. 3. RCe008-A (RC-4)was analysed by Giesma staining of 20metaphase spreads and showed
but a subpopulation (3 cells) showed 45X (monosomy X).Flow cytometry
Human embryonic stem cells were dissociated using Trypsin
(ThermoFisher Scientiﬁc). Non-speciﬁc staining was blocked using 5%
goat serum (Sigma) in PBS (Lonza) containing 0.01% Tween-20
(Sigma). Cells were stained with antibodies against SSEA-4, SSEA-1,
Tra-1-60 and Tra-1-81 (all BD, Oxford, UK), at 250 ng per reaction
followed by Goat F(ab)2 anti-mouse IgM-PE Goat F(ab)2 anti-mouse
IgG3-FITC (1:200; Santa Cruz Biotechnology, Texas, USA). Cells were
analysed using a FACS Aria ﬂow cytometer (BD).Immunocytochemistry
hESC were ﬁxed in 4% paraformaldehyde (ThermoFisher Scientiﬁc
(Alfa Aesar)), permeabilised using 100% ethanol (ThermoFisher Scien-
tiﬁc) and stained with AFP (1:500; Sigma), β-tubulin III (1:1000;
Sigma), muscle-speciﬁc actin (1:50; DAKO, Glostrup, Denmark), Oct-4
(1:200; Santa Cruz Biotechnology, Texas, USA), Nanog (1:20; R&D Sys-
tems, Abingdon, UK) and secondary antibodies anti-mouse IgG-FITC
(1:200; Sigma), anti mouse IgG-AlexaFluor 488, anti-goat IgG-
AlexaFluor 488, anti-goat IgG-AlexaFluor-594 and donkey polyclonal
AlexaFluor-594 (all 1:200; ThermoFisher Scientiﬁc). Images wereamixed karyotype. Themajority of cells analysed (16) showed a normal 46XX karyotype,
610 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 607–610acquired using a Zeiss S100 Axiovert ﬂuorescence microscope or Nikon
eC1 confocal microscope.
In vitro differentiation
hESCs were pre-treated for 1 h with 10 μM ROCK inhibitor in
Stempro hESC SFM (ThermoFisher Scientiﬁc) and embryoid bodies
EBs generated inultra low attachment plates (Corning) for 7 days before
being transferred into EBmedium (20% FBS (GE Healthcare (PAA)), 80%
KO-DMEM 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% nones-
sential amino acids (all ThermoFisher Scientiﬁc)), on glass slide tissue
culture chambers (Nunc, ThermoFisher Scientiﬁc) coated with 0.5% gel-
atin (Sigma) at 0.1 ml/cm2 for 14 days.
Genomic analysis
All outsourced assays were carried out under a Quality and Technical
Agreement. DNA was extracted using the QIAamp DNAMini kit (Qiagen,
Manchester, UK) according to the manufacturer's recommendations and
provided in recommended quantities to the service providers.
Microsatellite PCR, or Short TandemRepeat analysis, was used to de-
termine 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 Diagnos-
tics laboratory at NHSBT.
Karyotype analysis was carried out by The Doctors Laboratory
(London, UK) or the Western General Cytogenetics Laboratory (Edin-
burgh, 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.
Acknowledgments
Research culminating in the derivation of this line was funded by a
grant (PM07321) from Scottish Enterprise Economic Development
Agency to PDS, MB, and AC.
References
Ludwig, et al., 2006. Feeder-independent culture of human embryonic stem cells. Nat.
Methods 3 (8), 637–646.


De Sousa, Paul

Downie, J.m.

Edinburgh Research Explorer

     Edinburgh Research Explorer                                      Derivation of the human embryonic stem cell line RCe008-A (RC-4)Citation for published version:De Sousa, P, Tye, B, Bruce, K, Dand, P, Gardner, J, Downie, JM, Bateman, M & Courtney, A 2016,'Derivation of the human embryonic stem cell line RCe008-A (RC-4)' Stem cell research, vol. 16, no. 3, pp.607-610. DOI: 10.1016/j.scr.2016.02.039Digital Object Identifier (DOI):10.1016/j.scr.2016.02.039Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Publisher's PDF, also known as Version of recordPublished In:Stem cell researchPublisher Rights Statement:Under a Creative Commons licenseGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 05. Apr. 2019Stem Cell Research 16 (2016) 607–610Contents lists available at ScienceDirectStem Cell Researchj ourna l homepage: www.e lsev ie r .com/ locate /scrLab Resource: Stem Cell LineDerivation of the human embryonic stem cell line RCe008-A (RC-4)P.A. De Sousa a,b,c,⁎, B. Tye a, K. Bruce a, P. Dand a, J. Gardner a, J.M. Downie a, M. Bateman a, A. Courtney aa Roslin Cells Limited, Nine Edinburgh Bio-Quarter, 9 Little France Road, Edinburgh EH16 4UX, UKb Centre for Clinical Brain Sciences, University of Edinburgh, UKc MRC Centre for Regenerative Medicine, University of Edinburgh, UKNAInPCDTySuOKALiInE⁎ Corresponding author at: MSc Reader Centres fRegenerative Medicine University of Edinburgh ChanceCrescent Edinburgh, EH16 4SB Scotland, UK.http://dx.doi.org/10.1016/j.scr.2016.02.0391873-5061/© 2016 The Authors. Published by Elsevier B.Va b s t r a c ta r t i c l e i n f oArticle history:Received 25 February 2016Accepted 29 February 2016Available online 3 March 2016The human embryonic stem cell line RCe008-A (RC-4) was derived from a blastocyst voluntarily donated asunsuitable and surplus to fertility requirements following ethics committee approved informed consent underlicence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotencymarker expression and differentiation to ectoderm and mesoderm in vitro. It has a mixed 46XX/45X femalekaryotype 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 table.ame of stem cell construct RCe008-Alternative name RC-4, RC4stitution Roslin Cells Ltd.erson who created resource B. Tye, K. Bruce, P. Dand, J. Gardnerontact person and email Paul.desousa@roslincells.com;Paul.desousa@ed.ac.ukJanet.downie@roslincells.comAidan.courtney@roslincells.comMalcolm.bateman@roslinfoundation.comate archived/stock date 11 June 2008pe of resource Biological reagent: Cell lineb-type hESC, research graderigin Blastocyst with ICM and Trophoblastey transcription factors Oct4 and Nanog (conﬁrmed byimmunocytochemistry)uthentication See Quality Control Test Summary, Table 1nk to related literature (directURL links and full references)N/Aformation in public databases http://hpscreg.eu/cell-line/RCe008-Athics Informed consent obtained. Scotland A Re-search Ethics committee approval obtained(07/MRE00/56). Conducted under the UK Hu-man Fertilisation and Embryology Authoritylicence no R0136 to centre 0202.Resource detailsRCe008-A (RC-4) was derived from a fresh blastocyst that was sur-plus to requirement or unsuitable for clinical use. The cell line wasor Clinical Brain Sciences &llor's Building 49 Little France. This is an open access article underderived by whole embryo outgrowth on a chemically deﬁned matrixconsisting of laminin, ﬁbronectin, Collagen IV and vitronectin (Ludwiget al., 2006) using human ﬁbroblast (HDF) conditionedmediumand ex-panded under feeder free conditions.RCe008-A (RC-4)was shown to be pluripotent by expression of Oct-4 and Nanog using immunocytochemistry (Table 1, Fig. 1). By ﬂowcytometric analysis, the expression of pluripotency markers Tra-1-60,Tra-1-81 and SSEA-4 was 74%, 58% and 75%, respectively, whereas lowexpression of SSEA-1 (5%) was observed (Fig. 2). Germinal lineagemarker expression is not available due to poor EB formation.A microsatellite PCR proﬁle has been obtained for the cell line andblood group genotyping gave the blood group AO1 (Table 2).Veriﬁcation and authenticationThe 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)andmonosomy X (45X in 3 cells). The cell line is free frommycoplasmacontamination as determined by RT-qPCR.Materials and methodsEthicsDerivation of hESC from surplus to requirement and failed tofertilise/develop embryos was approved by The Scotland A ResearchEthics Committee and local ethics board at participating fertility clinicsand conducted under licence no. R0136 from the UK HFEA with in-formed donor consent.the CC BY license (http://creativecommons.org/licenses/by/4.0/).Table 1Summary of quality control testing and results for RCe008-A (RC-4).Classiﬁcation Test Purpose ResultDonor screeningHIV 1 + 2Donor screening for adventitious agents NegativeHepatitis BHepatitis CIdentityMicrosatellite PCR (mPCR)DNA proﬁling to give cell line its signature,gender/speciesPerformedImmunocytochemistryTo assess levels of staining for thepluripotency markers.Expression of Oct-4 and NanogPhenotype Flow cytometryAssess antigen levels & cell surface markerscommonly associated with hESCCD9: 84%Tra 1-60: 74%Tra 1-81: 58%SSEA-4: 75%SSEA-1: 5%Genotype (details provided inTable 2)Blood group genotypingTo establish blood group of the line AO1(DNA analysis)Karyology (G-banding) Conﬁrmation of normal ploidy by G-banding 46XX (16)/45X (3)Microbiology and virologyMycoplasma Mycoplasma testing by RT-qPCR NegativeEndotoxin Screening for endotoxin levels 1.87 EU/mlMorphology Photography To capture a visual record of the line NormalDifferentiation potential Embryoid body formation To show differentiation to three germ layersPoor EB formation — germinal linage marker expression notavailable608 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 607–610Cell cultureFresh embryos were cultured EmbryoAssist (Origio (Medicult),Denmark) until Day3 or BlastAssist (Origio) after Day 3 of development.Embryos were cultured at 36.5–37.5 °C, 5 ± 0.5% CO2, 5 ± 0.5% O2 indrops under parafﬁn oil (Origio) and transferred to fresh medium atleast every 2–3 days.By Day 8 of development, or when spontaneous hatching occurred,embryos were placed in derivation conditions consisting of a mixtureof laminin (5 μg/cm2; Sigma Aldrich, Dorset, UK), ﬁbronectin(5 μg/cm2; BD, Oxford, UK), vitronectin (0.2 μg/cm2; BD), and collagenIV (10 μg/cm2; Sigma) (Ludwig et al., 2006) in human dermal ﬁbroblastFig. 1. RCe008-A (RC-4) expresses pluripotency markers Oct-4 aFig. 2. Flow cytometry staining of RCe008-A (RC-4) at Passage 13. Isotype/negative contro(HDF) conditioned medium (80% Knockout-DMEM, 20% Knockoutserum replacement (KOSR), 1 mM glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, and 4 ng/ml humanbFGF (all ThermoFisher Scientiﬁc, Paisley, UK) over 24 h intervals over7 days) supplemented with an additional 24 ng/ml human bFGF. If re-quired, assisted hatching was performed by removing the zona pelluci-dae mechanically using Swemed cutting tools (Vitrolife, Göteborg,Sweden). Cells were cultured at 36.5–37.5 °C, 5 ± 0.5% CO2, 5 ± 0.5%O2 and 50% medium exchanged 6 days a week.The established cell line was expanded and banked using CellStartmatrix and Stempro hESC Serum Free Medium (ThermoFisher Scientif-ic). Passaging was performed mechanically using an EZ passage toolnd Nanog. Cell nuclei are counterstained with DAPI (blue).l is shown in red, speciﬁc staining in green. Percentage staining is indicated in Table 1.Table 2Microsatellite PCR, blood group and HLA tissue typing results for RCe008-A (RC-4).Microsatellite PCR resultsD3S1358 1 D3S1358 2 vWA 1 vWA 2 D16S539 1 D16S539 2 D2S1338 1 D2S1338 2125 129 167 170 281 281 336 348Amelogenin 1 Amelogenin 2 D8S1179 1 D8S1179 2 D21S11 1 D21S11 2 D18S51 1 D18S51 2X X 145 150 209 215 ND NDD19S433 1 D19S433 2 THO1 1 THO1 2 FGA 1 FGA 2 CSF1PO 1 CSF1PO 2121 121 180 184 237 245 325 329D5S818 1 D5S818 2 D7S820 1 D7S820 2 D13S317 1 D13S317 2 TPOX 1 TPOX 2152 152 272 276 218 222 231 231Blood group genotypingRhD RhC Rhc RhE Rhe Fy a Fy b Fy GATApos neg pos pos pos pos pos negJka Jkb K k M N S sneg pos neg pos pos pos neg posKp a Kp b Do a Do b ABOneg pos neg pos AO1ND = not determined609P.A. De Sousa et al. / Stem Cell Research 16 (2016) 607–610(ThermoFisher Scientiﬁc). hESC lineswere expanded to 25–30wells of a6-well plate and cryopreserved in 0.5–1 ml KOSR based cryopreserva-tion solution (75% KO-DMEM, 15% Xeno-free KOSR (ThermoFisher Sci-entiﬁc) and 10% DMSO (Origen Biomedical, Texas, USA)) or CryostorCS10 (Biolife Solution, Washington, USA).MycoplasmaMycoplasma detection was performed using Applied BiosystemsPrepSEQ™ Mycoplasma Nucleic Acid Extraction Kit and MicroSEQ™Mycoplasma Real-Time PCR Detection Kit (ThermoFisher Scientiﬁc(Applied Biosystems)) according to the manufacturer's instruction.EndotoxinEndotoxin levels were determined using the Kinetic-QCL assay(Lonza) and an incubating plate reader (BioTek ELx808) according tothe manufacturer's instructions. Brieﬂy, an unknown sample was com-pared with a standard curve of known levels of control endotoxin. Anassay was deemed valid if the coefﬁcient of correlation, r ≥ 0.980 andthe CV (%) for the standard curve was ≤10%.Fig. 3. RCe008-A (RC-4)was analysed by Giesma staining of 20metaphase spreads and showedbut a subpopulation (3 cells) showed 45X (monosomy X).Flow cytometryHuman embryonic stem cells were dissociated using Trypsin(ThermoFisher Scientiﬁc). Non-speciﬁc staining was blocked using 5%goat serum (Sigma) in PBS (Lonza) containing 0.01% Tween-20(Sigma). Cells were stained with antibodies against SSEA-4, SSEA-1,Tra-1-60 and Tra-1-81 (all BD, Oxford, UK), at 250 ng per reactionfollowed by Goat F(ab)2 anti-mouse IgM-PE Goat F(ab)2 anti-mouseIgG3-FITC (1:200; Santa Cruz Biotechnology, Texas, USA). Cells wereanalysed using a FACS Aria ﬂow cytometer (BD).ImmunocytochemistryhESC were ﬁxed in 4% paraformaldehyde (ThermoFisher Scientiﬁc(Alfa Aesar)), permeabilised using 100% ethanol (ThermoFisher Scien-tiﬁc) and stained with AFP (1:500; Sigma), β-tubulin III (1:1000;Sigma), muscle-speciﬁc actin (1:50; DAKO, Glostrup, Denmark), Oct-4(1:200; Santa Cruz Biotechnology, Texas, USA), Nanog (1:20; R&D Sys-tems, Abingdon, UK) and secondary antibodies anti-mouse IgG-FITC(1:200; Sigma), anti mouse IgG-AlexaFluor 488, anti-goat IgG-AlexaFluor 488, anti-goat IgG-AlexaFluor-594 and donkey polyclonalAlexaFluor-594 (all 1:200; ThermoFisher Scientiﬁc). Images wereamixed karyotype. Themajority of cells analysed (16) showed a normal 46XX karyotype,610 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 607–610acquired using a Zeiss S100 Axiovert ﬂuorescence microscope or NikoneC1 confocal microscope.In vitro differentiationhESCs were pre-treated for 1 h with 10 μM ROCK inhibitor inStempro hESC SFM (ThermoFisher Scientiﬁc) and embryoid bodiesEBs generated inultra low attachment plates (Corning) for 7 days beforebeing transferred into EBmedium (20% FBS (GE Healthcare (PAA)), 80%KO-DMEM 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% nones-sential amino acids (all ThermoFisher Scientiﬁc)), on glass slide tissueculture chambers (Nunc, ThermoFisher Scientiﬁc) coated with 0.5% gel-atin (Sigma) at 0.1 ml/cm2 for 14 days.Genomic analysisAll outsourced assays were carried out under a Quality and TechnicalAgreement. DNA was extracted using the QIAamp DNAMini kit (Qiagen,Manchester, UK) according to the manufacturer's recommendations andprovided in recommended quantities to the service providers.Microsatellite PCR, or Short TandemRepeat analysis, was used to de-termine 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 bythe Scottish National Blood Transfusion Service.Blood group genotyping was carried out by the Molecular Diagnos-tics laboratory at NHSBT.Karyotype analysis was carried out by The Doctors Laboratory(London, UK) or the Western General Cytogenetics Laboratory (Edin-burgh, UK) Live cells at 60–70% conﬂuency were shipped overnight inwarm containers, ﬁxed and analysed by standard G-banding analysis.For research grade lines, 20 spreads were analysed.AcknowledgmentsResearch culminating in the derivation of this line was funded by agrant (PM07321) from Scottish Enterprise Economic DevelopmentAgency to PDS, MB, and AC.ReferencesLudwig, et al., 2006. Feeder-independent culture of human embryonic stem cells. Nat.Methods 3 (8), 637–646.

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

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