AbstractThe 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

Bradburn, H.

Brison, D.r.

Bruce, K.

Collins, D.m.

Courtney, A.

Dand, P.

De Sousa, Paul

Downie, J.m.

Gardner, J.

Greenshields, A.

Mcdonald, K.

Russell, G.

Sneddon, S.

Tye, B.j.

Stem Cell Research

De Sousa, P.A.

Tye, B.J.

Collins, D.M.

Downie, J.M.

Bateman, M.

Elsevier - Publisher Connector 

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

Stem Cell Research 16 (2016) 537–540
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 RCe014-A (RC-10)P.A. De Sousa a,b,c,⁎, B.J. Tye a, K. Bruce a, P. Dand a, G. Russell a, D.M. Collins a, A. Greenshields a, H. Bradburn 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, UKA
In
P
C
D
Ty
Su
O
K
A
Li
In
E
⁎ Corresponding author at: Centres for Clinical Brain Sc
University of Edinburgh Chancellor's Building, 49 Little Fr
4SB, Scotland, UK.
E-mail addresses: paul.desousa@ed.ac.uk, paul.desousa
Sousa).
http://dx.doi.org/10.1016/j.scr.2016.02.034
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 16 February 2016
Accepted 19 February 2016
Available online 23 February 2016The 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. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).Resource tableName of stem cell
constructRCe014-Alternative name RC-10, RC10
stitution Roslin Cells Ltd.
erson who created
resourceB.J. Tye, K. Bruce, P. Dand, G. Russell, D.M. Collins,
A. Greenshields, H. Bradburnontact person and
emailPaul.desousa@roslincells.com; Paul.desousa@ed.ac.uk
Janet.downie@roslincells.com
Aidan.courtney@roslincells.com
Malcolm.bateman@roslinfoundation.comate archived/stock date 29 March 2010 (seed bank)
pe of resource Biological reagent: cell line
b-type hESC, research grade
rigin Zygote (oocyte/1PN)
ey transcription factors Oct4 (conﬁrmed by ﬂow cytometry)
uthentication See Quality Control test summary, Table 1
nk to related literature
(direct URL links and
full references)N/Aformation in public
databaseshttp://hpscreg.eu/cell-line/RCe014-Athics Informed consent obtained. Scotland A Research
Ethics committee approval obtained (07/MRE00/56).
Conducted under the UK Human Fertilisation and
Embryology Authority licence no R0136 to centre 0202.Resource detailsiences & Regenerative Medicine
ance Crescent, Edinburgh, EH16
@roslincells.com (P.A. De
. This is an open access article underRCe014-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
linewas derived bywhole embryo outgrowth onmitotically 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%, respective-
ly), whereas low expression of the differentiationmarker SSEA-1 (3.5%)
was observed (Fig. 1, Table 1). Differentiation to the three germ layers,
endoderm, ectoderm and mesoderm, was demonstrated using embry-
oid 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 AO1 (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 myco-
plasma contamination as determined by RC-qPCR. Microsatellite PCR
DNA proﬁling for cell identity is available (Table 2).
Materials and methods
Ethics
Derivation of hESC from surplus to requirement and failed to
fertilise/develop oocytes and embryos was approved by The Scotlandthe CC BY license (http://creativecommons.org/licenses/by/4.0/).
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.
538 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 537–540A 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.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% CO2, 5 ± 0.5% O2 in
drops under parafﬁn oil (Cook Medical) and transferred to fresh medi-
um at least every 2–3 days.
By day 8 of development, embryos were placed in derivation
conditions consisting of mitotically inactivated neonatal human dermal
ﬁbroblasts (HDFs) (Forticell Biosciences, NJ, USA) on tissue culture
plastic in XF KODMEM medium (Knockout-DMEM, 15% KOSR-XF,
2 mM L-glutamine, 1% MEM Non-essential amino acids, 2% XF Growth
Factor Cocktail, 0.1 mM β-mercaptoethanol, all ThermoFisher Scientiﬁc,
Paisley, UK) supplemented with 80 ng/ml human bFGF (ThermoFisher
Scientiﬁc).
HDF cells were cultured in DMEM (Lonza, Slough, UK), 10% Pharma
grade FCS (GE Healthcare (PAA), Buckinghamshire, UK) and 2 mM
L-glutamine (ThermoFisher Scientiﬁc). HDFs were mitotically
inactivated using gamma irradiation at 50GY using a Gammacell Elite
1000 machine. For use as a feeder layer, irradiated HDFs were platedTable 1
Summary of quality control testing and results for RC-10 (RCe014-A).
Classiﬁcation Test Purpose
Donor screening
HIV 1 + 2
Hepatitis B
Hepatitis C
Donor scree
Identity Microsatellite PCR (mPCR) DNA proﬁlin
Phenotype Flow cytometry Assess antig
commonly a
Genotype
(details provided in Table 2)
Blood group genotyping
(DNA analysis)
To establish
Karyology (G-banding) Conﬁrmation
HLA tissue typing To establish
Microbiology and virology
Mycoplasma Mycoplasma
Endotoxin Screening fo
Morphology Photography To capture a
Differentiation potential Embryoid body formation To show diffat 50,000 cells/cm2 in XF KODMEM medium supplemented with
80 ng/ml human bFGF (ThermoFisher Scientiﬁc). 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
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 samplewas 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%.Result
ning for adventitious agents Negative
g to give cell line its signature, gender/species Performed
en levels & cell surface markers
ssociated with hESC
Oct 3/4: 93.4%
Tra 1-60: 94.1%
SSEA-4: 98.9%
SSEA-1: 3.5%
blood group of the line AO1
of normal ploidy by G-banding
46XY (12 cells)
47 XY + 12 (3 cells)
full HLA type I and II genotype of the line HLA typed class I and class II
testing by RT-qPCR Negative
r endotoxin levels 1.2 EU/ml
visual record of the line Normal
erentiation to three germ layers
Expression of muscle actin,
β-tubulin and α-feto protein
Fig. 2. In vitro differentiation of RCe014-A (RC-10) to ectoderm (β-tubulin III), mesoderm (muscle actin), and endoderm (α-fetoprotein). Speciﬁc staining shown in green, cell nuclei are
counterstained with DAPI (blue).
Table 2
Microsatellite PCR, blood group and HLA tissue typing results for RCe014-A (RC-10).
Microsatellite PCR results
D3S1358 1 D3S1358 2 vWA 1 vWA 2 D16S539 1 D16S539 2 D2S1338 1 D2S1338 2
16 18 16 18 9 9 19 26
Amelogenin 1 Amelogenin 2 D8S1179 1 D8S1179 2 D21S11 1 D21S11 2 D18S51 1 D18S51 2
X Y 15 15 27 28 13 13
D19S433 1 D19S433 2 THO1 1 THO1 2 FGA 1 FGA 2 CSF1PO 1 CSF1PO 2
14 15 7 9.3 24 25 10 10
D5S818 1 D5S818 2 D7S820 1 D7S820 2 D13S317 1 D13S317 2 TPOX 1 TPOX 2
Blood group genotyping
11 11 8 12 10 13 10 11
RhD RhC Rhc RhE Rhe Fy a Fy b Fy GATA
pos pos pos neg pos neg pos neg
Jka Jkb K k M N S S
pos pos neg pos pos neg pos pos
Kp a Kp b Do a Do b ABO
ND ND pos pos AO1
ND = not determined
HLA tissue typing
HLA class I type HLA-A*01, A*02; B*40, B*57; Cw*03, Cw*06
HLA class II type HLA-DRB1*01, DRB1*04; DRB4*01; DQB1*03, DQB1*05
Comment B*40 is expressed serologically as B60, Cw*03 is expressed serologically as Cw10, DQB1*03 is expressed serologically as DQ8
539P.A. De Sousa et al. / Stem Cell Research 16 (2016) 537–540Flow 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 differentia-
tion marker. FITC conjugated Tra-1-60 (BD) was used as an additional
pluripotency marker. Fixed and permeabilised cells were analysedFig. 3. RCe014-A (RC-10) was analysed by Giesma staining and showedusing a FACS Aria ﬂow cytometer (BD). Percentage expression of each
marker was compared to isotype control or unstained cells.
Immunocytochemistry
hESCwasﬁxed inmethanol (ThermoFisher Scientiﬁc), blockedusing
10% goat serum (Sigma-Aldrich, Dorset, UK) in PBS (Lonza) containinga mixed 46XY (12 cells) and 47XY +12 (3 cells) male karyotype.
540 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 537–5400.01% Tween-20 (Sigma) and stained with AFP (1:500; Sigma),
β-tubulin III (1:1000; Sigma), muscle-speciﬁc actin (1:50; DAKO,
Glostrup, Denmark), and secondary antibody anti-goat IgG-AlexaFluor
488 (1:200; ThermoFisher Scientiﬁc). Images were acquired using a
Zeiss S100 Axiovert ﬂuorescence microscope or Nikon eC1 confocal
microscope.
In vitro differentiation
Conﬂuent hESC cells lifted using a cell scraper (Corning) and embry-
oid bodies EBs generated in ultra low attachment plates (Corning) in
EB medium (20% FBS (GE Healthcare (PAA)), 80% KO-DMEM 1 mM
L-glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids
(all ThermoFisher Scientiﬁc)). After 9 days in suspension culture, EBs
were being transferred onto glass slide tissue culture chambers
(Nunc, ThermoFisher Scientiﬁc) coated with 0.1% gelatin (Sigma) at
0.1 ml/cm2 and cultured for 14 days.
Genomic analysis
All outsourced assayswere carried out under a Quality and Technical
Agreement. DNA was extracted using the QIAamp DNA Mini kit
(Qiagen, Manchester, UK) according to manufacturer's recommenda-
tions 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 genotypingwas carried out by theMolecular 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 over-
night in warm containers, ﬁxed and analysed by standard G-banding
analysis. For research grade lines, 20 spreads were analysed.
Acknowledgements
Research culminating in the derivation of this line was funded by a
grant from Scottish Enterprise Economic Development Agency
(PM07321) to PDS, MB and AC.
Images of embroid body staining were kindly provided by
S. Greenhough and J. Gardner, Roslin Cellab Ltd.


The human embryonic stem cell line RCe012-A (RC-8) was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was 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 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available

P.A. De Sousa

B.J. Tye

K. Bruce

P. Dand

G. Russell

D.M. Collins

A. Greenshields

H. Bradburn

J.M. Downie

M. Bateman

A. Courtney

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Derivation of the human embryonic stem cell line RCe012-A (RC-8)

S. Sneddon

K. McDonald

J. Gardner

D.R. Brison

Crossref

Derivation of the human embryonic stem cell line RCM1

The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available

McDonald, K.

Brison, D.R.

Enlighten

Stem Cell Research 16 (2016) 476–480Contents 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 RCM1P.A. De Sousa a,b,⁎, B.J. Tye a, S. Sneddon c, K. Bruce a, P. Dand a, G. Russell a, D.M. Collins a, A. Greenshields a,K. McDonald a, H. Bradburn a, J. Gardner a, J.M. Downie a, A. Courtney a, D.R. Brison ca Roslin Cells Limited, Nine Edinburgh Bio-Quarter, 9 Little France Road, Edinburgh EH16 4UX, UKb Centre for Clinical Brain Sciences, University of Edinburgh, UKc Department of Reproductive Medicine, Central Manchester University Hospitals, NHS Foundation Trust, St Mary's Hospital, Manchester M13 9WL, UKInPCDTSOKALi⁎ Corresponding author.http://dx.doi.org/10.1016/j.scr.2015.12.0201873-5061/© 2015 Published by Elsevier B.V. This is an oa b s t r a c ta r t i c l e i n f oArticle history:Received 23 December 2015Accepted 23 December 2015Available online 31 December 2015The human embryonic stem cell lineRCM-1wasderived from a failed to fertilise egg undergoing parthenogeneticstimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layersin vitro and in vivo. It has a normal 46XX female karyotype andmicrosatellite PCR identity, HLA and blood grouptyping data is available.© 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).(RResource tableName of stem cell construct RCM1stitution Roslin Cells Ltd.St Mary's Hospital, Manchestererson who created resource B. Tye, J. Gardner, C. Pope, S. Sneddon, K.Bruce, P. Dand, G. Russell, D.M. Collins, A.Greenshields, K. McDonald, H. BradburnInontact person and emailEPaul.desousa@roslincells.com;Paul.desousa@ed.ac.uk;Janet.downie@roslincells.comAidan.courtney@roslincells.comdaniel.brison@manchester.ac.ukate archived/stock date 21 December 2006 (passage 10 on feeders)07 February 2011 (seed bank at passage 24)ype of resource Biological reagent: Cell lineub-type hESC, research graderigin Blastocyst with ICM and trophoblast originatingfrom clinically failed egg receiving parthenoge-netic stimulus. Confirmed as biparental by wholegenome SNIP analysis.ey transcription factors Oct4 (confirmed by flow cytometry andimmunocytochemistry)uthentication See Quality Control Test Summary, Table 1nk to related literature (directURL links and full references)De Sousa PA*, Gardner J, Sneddon S, Pells S,Jorgenson Tye B, Dand P, Collins DM, Steward K,Shaw L, Przyborski S, Cooke M, McLauglin KJ,pen access article under the CC BY-NC-continued)esource table (continued)Name of stem cell constructND license (http://creativecommons.RCM1Kimber SJ, Lieberman BA, Wilmut I, Brison D.(2009) Clinically failed eggs as a source of normalhuman embryo stem cells. Stem Cell Research 2,188–197.http://www.sciencedirect.com/science/article/pii/S1873506109000026formation in public databases http://www.nibsc.org/science_and_research/advanced_therapies/uk_stem_cell_bank/cell_lines.aspxthics Informed consent obtained. Approved byparticipating institutional ethics advisoryboards and Central Manchester Local ResearchEthics Committee reference number03/CM/684. Conducted under the UK HumanFertilisation and Embryology Authority licenceno R0136.Resource details.RCM1was derived from a failed to fertilise egg subjected to partheno-genic activation. The cell linewas derived bywhole embryo outgrowth onmitotically inactivated human fibroblast (HDF) feeder cells using HDFconditioned medium and expanded under feeder free conditions.RCM1 was shown to be pluripotent by expression of Oct 4 usingimmunocytochemistry (Table 1, Fig. 1). By flow cytometric analysis, theexpression of pluripotency makers Oct4, Tra-1-60 and SSEA-4 was 97%,98% and 99%, respectively, whereas low expression of the differentiationmarker SSEA-1 (6%) was observed (Fig. 2).org/licenses/by-nc-nd/4.0/).Table 1Summary of quality control testing and results for RCM-1.Classification Test Purpose ResultDonor screening HIV 1 + 2Hepatitis BHepatitis CDonor screening for adventitious agents NegativeIdentity Microsatellite PCR (mPCR) DNA Profiling to give cell line its signature, gender/species PerformedPhenotype Immunocytochemisty To assess levels of staining for the pluripotency markers Oct4and NanogExpression of Oct4 and NanogFlow cytometry Assess antigen levels & cell surface markers commonlyassociated with hESCOct3/4: 97%Tra 1-60: 98%SSEA-4: 99%SSEA-1: 6%Genotype Blood group genotyping(DNA analysis)To establish blood group of the line AAKaryology (G-banding) Confirmation of normal ploidy by G-banding 46XXHLA tissue typing To establish full HLA Type I and Type II genotypes of the line HLA typed Class I and Class IIMicrobiology and virology Mycoplasma Mycoplasma testing by RT-qPCR NegativeMorphology Photography To capture a visual record of the line NormalDifferentiation potential Embryoid body formation To show differentiation to three germ layers Expression of muscle actin, β-tubulin and α-feto proteinTeratoma formation To show differentiation to three germ layers Teratoma formation observed, three germ layers detected477P.A. De Sousa et al. / Stem Cell Research 16 (2016) 476–480Differentiation to the three germ layers, endoderm, ectodermandme-soderm,was demonstrated using embryoid body formation and teratomaformation (Fig. 1).A microsatellite PCR profile has been obtained for the cell line, andHLA Class I and Class II typing is available (Table 1). Blood groupgenotyping gave the blood group AA (Table 1). The cell line wasanalysed for genome stability by G-banding and GCH (Fig. 3), as wellas a SNP array screen to determine whether RCM1 was derived from aparthenogenetic or fertilized embryo. This showed a normal, 46XXfemale genotype with no genetic deficiencies that had originated froma fertilized egg.The cell line is free from mycoplasma contamination as determinedby RC-qPCR.Materials and methodsEthicsDerivation of hESC from surplus to requirement and failed to fertilise/develop embryos was approved by The Scotland A Research Ethics Com-mittee and local ethics board at participating fertility clinics and conduct-ed under licence no R0136 from the UK HFEA with informed donorconsent.Cell cultureActivation and derivation of RCM1 is described in De Sousa et al.(2009), Stem Cell Research 2, 188–197. The established cell line wasexpanded and banked using CellStart matrix and Stempro hESC SerumFree Medium (ThermoFisher Scientific (Life Technologies), Paisley,UK). Passaging was performed mechanically using an EZ passage tool(ThermoFisher Scientific).MycoplasmaMycoplasma detection was performed using AppliedBiosystems PrepSEQ™ Mycoplasma Nucleic Acid Extraction Kitand MicroSEQ™ Mycoplasma Real-Time PCR Detection Kit(ThermoFisher Scientific (Applied Biosystems)) according to themanufacturer's instruction.Flow cytometryPluripotency was determined using the Human and Mouse Plu-ripotent Stem Cell Analysis kit (BD, Oxford, UK). Oct3/4 and SSEA-4were included as pluripotency markers, and SSEA-1 as a differenti-ation marker. FITC conjugated Tra-1-60 (BD) was used as an addi-tional pluripotency marker. Fixed and permeabilised cells wereanalysed using the Guava easyCyte flow cytometer (Millipore,Watford, UK).ImmunocytochemistryhESC were fixed in 4% paraformaldehyde (ThermoFisher Scientific(Alfa Aesar)), permeabilised using 100% ethanol (ThermoFisherScientific) and stained with AFP (1:500; Sigma Aldrich, Dorset, UK),β-tubulin III (1:1000; Sigma), muscle-specific actin (1:50; DAKO,Glostrup, Denmark), Oct4 (1:200; Santa Cruz Biotechnology, Texas,USA), Nanog (1:20; R&D Systems, Abingdon, UK) and secondary antibod-ies anti-mouse IgG-FITC (1:200; Sigma), anti mouse IgG — AlexaFluor488, anti-goat IgG — AlexaFluor 488 and anti-goat IgG— AlexaFluor-594(1:200 donkey polyclonal AlexaFluor-594) (all 1:200; ThermoFisher Sci-entific). Alkaline Phosphatase staining was as per manufacturer's recom-mended protocol (Vector labs, Peterborough, UK). Images were acquiredusing a Zeiss S100 Axiovert fluorescence microscope or Nikon eC1 confo-cal microscope.In vitro differentiationhESC cells were pre-treated for 1 h with 10 μMROCK inhibitor inStempro hESC SFM (ThermoFisher Scientific) and embryoid bodiesEBs generated in ultra low attachment plates (Corning) for 7 daysbefore being transferred into EB medium (20% FBS (GE Healthcare(PAA), Buckinghamshire, UK), 80% KO-DMEM 1 mM L-glutamine,0.1 mM β-mercaptoethanol, 1% nonessential amino acids (allThermoFisher Scientific)), on glass slide tissue culture chambersFig. 1. The characterization of RCM1 as a pluripotent hESC line. Undifferentiated colonies of RCM1 on human dermal fibroblast feeder cells are shown (A) in phase contrast, (B) stainedpositively for Oct4 and (C) stained positively for alkaline phosphatase activity. RCM1 cells in vitro differentiated via embryoid bodies and stained for markers of the three germ layers:(D) muscle-specific actin (mesoderm), (E) α-fetoprotein (endoderm), and (F) β-tubulin III (ectoderm). A histological assessment was made of teratoma tissue formed as a result oftransplantation of the hESC line, RCM1, into the testis of adult SCID mice. (G) Low-power image showing heterogeneity of tissue structure in tumor. Identifiable structures includebone (bn), cartilage (ct), connective tissue (cn), and small cysts (cy); higher power images show (H) neuroectoderm (ne) and neural ganglion (ng) and (I) bone (bn). (J) Cartilage;(K) wall of primitive intestine, including muscular layer (mu), submucosal tissue (sm), and goblet cells (gc) within epithelium releasing mucus secretion (mc); (L) primitive kidneyconsisting of renal corpuscle (rc) and associated tubules (tb). (G–J, L) Weigert's and (K) hematoxylin and eosin. Scale bars, 100 μm (A–J) and 50 μm (K and L).From De Sousa et al. (2009).Fig. 2. Flowcytometry analysis of undifferentiated RCM1. RCM-1was subjected to flowcytometry analysis formarkers of pluripotency. The specific antibody stained histograms are shownin the top row against the corresponding control histograms. Percentage staining is indicated in Table 1.478 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 476–480Fig. 3.Karyotype analysis of RCM-1. RCM-1was analysed byGiemsa staining of 20metaphase spreads and showed a normal 46XX female karyotype (top panel). No genomic copy numberimbalance was evaluated using a BlueGnome CytoChips array designed to improve detection of copy number variation associated with constitutional genetic disorders (bottom panel).Presented are SNP polymorphism copy number representation which did not exceed normal ranges (flanking green and red lines) depicted next to chromosome ideograms.From De Sousa et al. (2009).479P.A. De Sousa et al. / Stem Cell Research 16 (2016) 476–480(Nunc, ThermoFisher Scientific) coated with 0.5% gelatin (Sigma)at 0.1 ml/cm2 for 14 days.In vivo differentiationThe developmental potential of RCM-1 to form teratomas consistingof tissues representative of all three germ layers was evaluated480 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 476–480following transplantation under kidney capsule in adult SCID mice andteratoma formation assessed as described in De Sousa et al. (2009),Stem Cell Research 2, 188–197.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.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 theMolecular Diagnosticslaboratory 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 overnightin warm containers, fixed and analysed by standard G-banding analysis.For research grade lines, 20 spreads were analysed.Comparative genome hybridization (CGH)was carried out in collab-oration with ARK Genomics (Roslin Institute, UK), using BlueGnomeCytoChipTM arrayCGH v2. Genome-wide SNP analysis genomic DNAwas carried out with GeneChip Human Mapping 250 K Sty I and 250 KNsp arrays (Affymetrix), both as described in De Sousa et al. (2009),Stem Cell Research 2, 188–197.Verification and authenticationThe identity and phenotype of RCM1 was confirmed by qualitycontrol testing using the aforementioned methods the results of whichare summarised in Table 1, Figs. 1 & 2. Further information on this linecan be obtained from its original publication in De Sousa et al., 2009.AcknowledgementsResearch culminating in the derivation of this line was funded by agrant from the UK Medical Research Council (G0300484) to PDS andDRB, and Scottish Enterprise Economic Development Agency(PM07321) to PDS and AC.ReferencesDe Sousa, P.A., Gardner, J., Sneddon, S., Pells, S., Jorgenson Tye, B., Dand, P., DM, Collins,Steward, K., Shaw, L., Przyborski, S., Cooke, M., KJ, McLauglin, SJ, Kimber, BA,Lieberman, Wilmut, I., Brison, D., 2009. Clinically failed eggs as a source of normalhuman embryo stem cells. Stem Cell Res. 2, 188–197.

English

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

Edinburgh Research Explorer

     Edinburgh Research Explorer                                      Derivation of the human embryonic stem cell line RCe014-A (RC-10)Citation for published version:De Sousa, P, Tye, BJ, Bruce, K, Dand, P, Russell, G, Collins, DM, Greenshields, A, Bradburn, H, Downie,JM, Bateman, M & Courtney, A 2016, 'Derivation of the human embryonic stem cell line RCe014-A (RC-10)'Stem cell research, vol. 16, no. 2, pp. 537-540. DOI: 10.1016/j.scr.2016.02.034Digital Object Identifier (DOI):10.1016/j.scr.2016.02.034Link: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) 537–540Contents 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 RCe014-A (RC-10)P.A. De Sousa a,b,c,⁎, B.J. Tye a, K. Bruce a, P. Dand a, G. Russell a, D.M. Collins a, A. Greenshields a, H. Bradburn 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, UKAInPCDTySuOKALiInE⁎ Corresponding author at: Centres for Clinical Brain ScUniversity of Edinburgh Chancellor's Building, 49 Little Fr4SB, Scotland, UK.E-mail addresses: paul.desousa@ed.ac.uk, paul.desousaSousa).http://dx.doi.org/10.1016/j.scr.2016.02.0341873-5061/© 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 16 February 2016Accepted 19 February 2016Available online 23 February 2016The human embryonic stem cell line RCe014-A (RC-10) was derived from a fresh oocyte 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 the three germ layers in vitro. It has a mixed 46XY and 47XY +12male karyotype and microsatellite PCR identity, HLA 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 tableName of stem cellconstructRCe014-Alternative name RC-10, RC10stitution Roslin Cells Ltd.erson who createdresourceB.J. Tye, K. Bruce, P. Dand, G. Russell, D.M. Collins,A. Greenshields, H. Bradburnontact person andemailPaul.desousa@roslincells.com; Paul.desousa@ed.ac.ukJanet.downie@roslincells.comAidan.courtney@roslincells.comMalcolm.bateman@roslinfoundation.comate archived/stock date 29 March 2010 (seed bank)pe of resource Biological reagent: cell lineb-type hESC, research graderigin Zygote (oocyte/1PN)ey transcription factors Oct4 (conﬁrmed by ﬂow cytometry)uthentication See Quality Control test summary, Table 1nk to related literature(direct URL links andfull references)N/Aformation in publicdatabaseshttp://hpscreg.eu/cell-line/RCe014-Athics Informed consent obtained. Scotland A ResearchEthics committee approval obtained (07/MRE00/56).Conducted under the UK Human Fertilisation andEmbryology Authority licence no R0136 to centre 0202.Resource detailsiences & Regenerative Medicineance Crescent, Edinburgh, EH16@roslincells.com (P.A. De. This is an open access article underRCe014-A (RC-10) was received as a failed to fertilise oocyte/1PN(pro-nuclear) embryo that was surplus to requirement or unsuitablefor clinical use. The embryo was grown to blastocyst stage and the celllinewas derived bywhole embryo outgrowth onmitotically inactivatedhuman ﬁbroblast (HDF) feeder cells using xeno-free medium (XFKODMEM) and expanded under xeno free and feeder free conditions.By ﬂow cytometry, RCe014-A (RC-10) expressed the pluripotencymakers Oct-4, Tra-1-60 and SSEA-4 (93.4%, 94.1% and 98.9%, respective-ly), whereas low expression of the differentiationmarker SSEA-1 (3.5%)was observed (Fig. 1, Table 1). Differentiation to the three germ layers,endoderm, ectoderm and mesoderm, was demonstrated using embry-oid body formation and expression of the germ layer markersα-fetoprotein, β-tubulin and muscle actin (Fig. 2).A microsatellite PCR proﬁle has been obtained for the cell line, andHLA Class I and II typing is available (Table 2). Blood group genotypinggave the blood group AO1 (Table 2).Veriﬁcation and authenticationThe cell line was analysed for genome stability by G-banding (Fig. 3)and showed a mixed 46XY and trisomy 12 (47XY +12) male genotypein 12 and 3 cells analysed, respectively. The cell line is free from myco-plasma contamination as determined by RC-qPCR. Microsatellite PCRDNA proﬁling for cell identity is available (Table 2).Materials and methodsEthicsDerivation of hESC from surplus to requirement and failed tofertilise/develop oocytes and embryos was approved by The Scotlandthe CC BY license (http://creativecommons.org/licenses/by/4.0/).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 thehistograms. Percentage staining is indicated in Table 1.538 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 537–540A Research Ethics Committee and local ethics board at participatingfertility clinics and conducted under licence no R0136 from the UKHFEA with informed donor consent.Cell cultureFailed to activate oocytes and embryos were cultured in Sydneycleavage medium (Cook Medical, Hertfordshire, UK) until day 3 andSydney blastocyst medium (Cook Medical) 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 (Cook Medical) and transferred to fresh medi-um at least every 2–3 days.By day 8 of development, embryos were placed in derivationconditions consisting of mitotically inactivated neonatal human dermalﬁbroblasts (HDFs) (Forticell Biosciences, NJ, USA) on tissue cultureplastic in XF KODMEM medium (Knockout-DMEM, 15% KOSR-XF,2 mM L-glutamine, 1% MEM Non-essential amino acids, 2% XF GrowthFactor Cocktail, 0.1 mM β-mercaptoethanol, all ThermoFisher Scientiﬁc,Paisley, UK) supplemented with 80 ng/ml human bFGF (ThermoFisherScientiﬁc).HDF cells were cultured in DMEM (Lonza, Slough, UK), 10% Pharmagrade FCS (GE Healthcare (PAA), Buckinghamshire, UK) and 2 mML-glutamine (ThermoFisher Scientiﬁc). HDFs were mitoticallyinactivated using gamma irradiation at 50GY using a Gammacell Elite1000 machine. For use as a feeder layer, irradiated HDFs were platedTable 1Summary of quality control testing and results for RC-10 (RCe014-A).Classiﬁcation Test PurposeDonor screeningHIV 1 + 2Hepatitis BHepatitis CDonor screeIdentity Microsatellite PCR (mPCR) DNA proﬁlinPhenotype Flow cytometry Assess antigcommonly aGenotype(details provided in Table 2)Blood group genotyping(DNA analysis)To establishKaryology (G-banding) ConﬁrmationHLA tissue typing To establishMicrobiology and virologyMycoplasma MycoplasmaEndotoxin Screening foMorphology Photography To capture aDifferentiation potential Embryoid body formation To show diffat 50,000 cells/cm2 in XF KODMEM medium supplemented with80 ng/ml human bFGF (ThermoFisher Scientiﬁc). Cells were culturedat 36.5–37.5 °C, 5 ± 0.5% CO2, 5 ± 0.5% O2 and 50%medium exchanged6 days a week.The established cell line was expanded and banked using CellStartmatrix and Stempro hESC Serum Free Medium (ThermoFisherScientiﬁc). Passaging was performed mechanically using an EZ passagetool (ThermoFisher Scientiﬁc). hESC lines were expanded to 25–30wells of a 6-well plate and cryopreserved in 0.5–1 ml Cryostor CS10(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 manufacturer's instruction.EndotoxinEndotoxin levels were determined using the Kinetic-QCL assay(Lonza) and an incubating plate reader (BioTek ELx808) according tomanufacturer's instructions. Brieﬂy, an unknown samplewas comparedwith a standard curve of known levels of control endotoxin. An assaywas deemed valid if the coefﬁcient of correlation, r ≥ 0.980 and the CV(%) for the standard curve was ≤10%.Resultning for adventitious agents Negativeg to give cell line its signature, gender/species Performeden levels & cell surface markersssociated with hESCOct 3/4: 93.4%Tra 1-60: 94.1%SSEA-4: 98.9%SSEA-1: 3.5%blood group of the line AO1of normal ploidy by G-banding46XY (12 cells)47 XY + 12 (3 cells)full HLA type I and II genotype of the line HLA typed class I and class IItesting by RT-qPCR Negativer endotoxin levels 1.2 EU/mlvisual record of the line Normalerentiation to three germ layersExpression of muscle actin,β-tubulin and α-feto proteinFig. 2. In vitro differentiation of RCe014-A (RC-10) to ectoderm (β-tubulin III), mesoderm (muscle actin), and endoderm (α-fetoprotein). Speciﬁc staining shown in green, cell nuclei arecounterstained with DAPI (blue).Table 2Microsatellite PCR, blood group and HLA tissue typing results for RCe014-A (RC-10).Microsatellite PCR resultsD3S1358 1 D3S1358 2 vWA 1 vWA 2 D16S539 1 D16S539 2 D2S1338 1 D2S1338 216 18 16 18 9 9 19 26Amelogenin 1 Amelogenin 2 D8S1179 1 D8S1179 2 D21S11 1 D21S11 2 D18S51 1 D18S51 2X Y 15 15 27 28 13 13D19S433 1 D19S433 2 THO1 1 THO1 2 FGA 1 FGA 2 CSF1PO 1 CSF1PO 214 15 7 9.3 24 25 10 10D5S818 1 D5S818 2 D7S820 1 D7S820 2 D13S317 1 D13S317 2 TPOX 1 TPOX 2Blood group genotyping11 11 8 12 10 13 10 11RhD RhC Rhc RhE Rhe Fy a Fy b Fy GATApos pos pos neg pos neg pos negJka Jkb K k M N S Spos pos neg pos pos neg pos posKp a Kp b Do a Do b ABOND ND pos pos AO1ND = not determinedHLA tissue typingHLA class I type HLA-A*01, A*02; B*40, B*57; Cw*03, Cw*06HLA class II type HLA-DRB1*01, DRB1*04; DRB4*01; DQB1*03, DQB1*05Comment B*40 is expressed serologically as B60, Cw*03 is expressed serologically as Cw10, DQB1*03 is expressed serologically as DQ8539P.A. De Sousa et al. / Stem Cell Research 16 (2016) 537–540Flow cytometryPluripotency was determined using the Human and MousePluripotent Stem Cell Analysis kit (BD, Oxford, UK). Oct 3/4 and SSEA-4 were included as pluripotency markers, and SSEA-1 as a differentia-tion marker. FITC conjugated Tra-1-60 (BD) was used as an additionalpluripotency marker. Fixed and permeabilised cells were analysedFig. 3. RCe014-A (RC-10) was analysed by Giesma staining and showedusing a FACS Aria ﬂow cytometer (BD). Percentage expression of eachmarker was compared to isotype control or unstained cells.ImmunocytochemistryhESCwasﬁxed inmethanol (ThermoFisher Scientiﬁc), blockedusing10% goat serum (Sigma-Aldrich, Dorset, UK) in PBS (Lonza) containinga mixed 46XY (12 cells) and 47XY +12 (3 cells) male karyotype.540 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 537–5400.01% Tween-20 (Sigma) and stained with AFP (1:500; Sigma),β-tubulin III (1:1000; Sigma), muscle-speciﬁc actin (1:50; DAKO,Glostrup, Denmark), and secondary antibody anti-goat IgG-AlexaFluor488 (1:200; ThermoFisher Scientiﬁc). Images were acquired using aZeiss S100 Axiovert ﬂuorescence microscope or Nikon eC1 confocalmicroscope.In vitro differentiationConﬂuent hESC cells lifted using a cell scraper (Corning) and embry-oid bodies EBs generated in ultra low attachment plates (Corning) inEB medium (20% FBS (GE Healthcare (PAA)), 80% KO-DMEM 1 mML-glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids(all ThermoFisher Scientiﬁc)). After 9 days in suspension culture, EBswere being transferred onto glass slide tissue culture chambers(Nunc, ThermoFisher Scientiﬁc) coated with 0.1% gelatin (Sigma) at0.1 ml/cm2 and cultured for 14 days.Genomic analysisAll outsourced assayswere carried out under a Quality and TechnicalAgreement. DNA was extracted using the QIAamp DNA Mini kit(Qiagen, Manchester, UK) according to manufacturer's recommenda-tions 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 bythe Scottish National Blood Transfusion Service.Blood group genotypingwas carried out by theMolecular Diagnosticslaboratory 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 over-night in warm containers, ﬁxed and analysed by standard G-bandinganalysis. For research grade lines, 20 spreads were analysed.AcknowledgementsResearch culminating in the derivation of this line was funded by agrant from Scottish Enterprise Economic Development Agency(PM07321) to PDS, MB and AC.Images of embroid body staining were kindly provided byS. Greenhough and J. Gardner, Roslin Cellab Ltd.

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

The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.</p

De Sousa, P. A.

Tye, B. J.

Collins, D. M.

Downie, J. M.

Brison, D. R.

The University of Manchester - Institutional Repository

AbstractThe human embryonic stem cell line RCe012-A (RC-8) was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was 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 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available

Stem Cell Research 16 (2016) 489–492
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 RCe012-A (RC-8)P.A. De Sousa a,b,c,⁎, B.J. Tye a, K. Bruce a, P. Dand a, G. Russell a, D.M. Collins a, A. Greenshields a, H. Bradburn 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, UK⁎ Corresponding author at: Roslin Cells Limited, Nine
France Road, Edinburgh, EH16 4UX, UK.
Name of stem cell construct RCe012-A
Alternative name RC-8, RC8
Institution Roslin Cells Ltd.
Person who created resource B. J. Tye, K. Bruce,
Collins, A. Greens
Contact person and email Paul.desousa@ros
Paul.desousa@ed.
Janet.downie@ros
Aidan.courtney@r
Malcolm.bateman
Date archived/stock date 06 September 201
Type of resource Biological reagent
Sub-type hESC, research gr
Origin Blastocyst with IC
Key transcription factors Oct4 (conﬁrmed b
Authentication See Quality Contr
Link to related literature (direct
URL links and full references)
N/A
Information in public databases http://hpscreg.eu
Ethics Informed consent
Ethics committee a
(07/MRE00/56). Co
Fertilisation and Em
R0136 to centre 02
http://dx.doi.org/10.1016/j.scr.2016.02.033
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 16 February 2016
Accepted 19 February 2016
Available online 23 February 2016The human embryonic stem cell line RCe012-A (RC-8) was derived from a frozen and thawed day 5 embryo cul-
tivated to the blastocyst stage. The embryowas voluntarily donated as unsuitable and surplus to fertility require-
ments 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 46XX female karyotype and microsatellite PCR identity, HLA 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 tableP. Dand, G. Russell, D.M.
hields, H. Bradburn
lincells.com;
ac.uk
lincells.com
oslincells.com
@roslinfoundation.com
0 (seed bank)
: cell line
ade
M and trophoblast
y ﬂow cytometry)
ol test summary, Table 1
/cell-line/RCe012-A
obtained. Scotland A Research
pproval obtained
nducted under the UK Human
bryology Authority licence no
02.Resource details
RCe012-A (RC-8) was derived from a thawed day 5 blastocyst that
was surplus to requirement or unsuitable for clinical use. The cell lineEdinburgh Bio-Quarter, 9 Little
. This is an open access article underwas 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, RCe012-A (RC-8) expressed the pluripotency
makers Oct-4, Tra-1-60 and SSEA-4 (94.7%, 89.4% and 99.8%, respective-
ly), whereas low expression of the differentiationmarker SSEA-1 (2.2%)
was observed (Fig. 1, Table 1). Differentiation to the three germ layers,
endoderm, ectoderm and mesoderm, was demonstrated using embry-
oid 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 AO1 (Table 2).
Veriﬁcation and authentication
The cell line was analysed for genome stability by G-banding (Fig. 3)
and showed a normal 46XX female genotype. The cell line is free from
mycoplasma contamination as determined by RC-qPCR. Microsatellite
PCR DNA proﬁling for cell identity is available (Table 2).
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/).
Fig. 1. RCe012-A (RC-8) 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.
490 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 489–492Cell culture
Vitriﬁed embryos were thawed using Vitriﬁed Embryo Safety
Thawing Pack (Kitazato/Dibimed, Valencia, Spain) according toman-
ufacturers' instruction and were cultured in system SAGE Quinn's
Advanced Blastocyst medium (Rochford Medical, Coventry) 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 (Rochford Medical)
and transferred to fresh medium at least every 2–3 days.
By day 8 of development, embryos were placed in derivation condi-
tions consisting ofmitotically inactivated neonatal human dermal ﬁbro-
blasts (HDFs) (ThermoFisher Scientiﬁc (Cascade Biologics), Paisley, UK)
on tissue culture plastic in XF KODMEM medium (Knockout-DMEM,
15% KOSR-XF, 2 mM L-glutamine, 1% MEM Non-essential amino acids,
2% XF Growth Factor Cocktail, 0.1 mM β-mercaptoethanol, all
ThermoFisher Scientiﬁc, Paisley, UK) supplemented with 80 ng/ml
human bFGF (ThermoFisher Scientiﬁc). Assisted hatching was per-
formed by removing the zona pellucidae mechanically using Swemed
Cutting tools (Vitrolife, Göteborg, Sweden).
HDF cells were cultured in DMEM (Lonza, Slough, UK), 10% FCS (GE
Healthcare (PAA), Buckinghamshire, UK) and 2 mM L-glutamine
(ThermoFisher Scientiﬁc). HDFs were mitotically inactivated using
gamma irradiation at 50GY using a Gammacell Elite 1000 machine.
For use as a feeder layer, irradiated HDFs were plated at 50,000 cells/
cm2 in XF KODMEM medium supplemented with 80 ng/ml humanTable 1
Summary of quality control testing and results for RCe012-A (RC-8).
Classiﬁcation
Test Purpose
Donor screening HIV 1 + 2
Hepatitis B
Hepatitis C
Donor screenin
Identity Microsatellite PCR (mPCR) DNA proﬁling
gender/species
Phenotype Flow cytometry Assess antigen
commonly ass
Genotype
(details provided in Table 2)
Blood group genotyping
(DNA analysis)
To establish bl
Karyology (G-banding) Conﬁrmation o
HLA tissue typing To establish fu
Microbiology and virology Mycoplasma Mycoplasma te
Endotoxin Screening for e
Morphology Photography To capture a vi
Differentiation potential Embryoid body formation To show differbFGF (ThermoFisher Scientiﬁc). 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 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 So-
lution, Washington, USA).
Mycoplasma
Mycoplasma detection was performed using Applied Biosystems
PrepSEQ™ Mycoplasma Nucleic Acid Extraction Kit and MicroSEQ™
MycoplasmaReal-Time PCRDetection Kit (ThermoFisher Scientiﬁc (Ap-
plied 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 samplewas 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%.Result
g for adventitious agents Negative
to give cell line its signature, Performed
levels & cell surface markers
ociated with hESC
Oct 3/4: 94.7%
Tra 1-60: 89.4%
SSEA-4: 99.8%
SSEA-1: 2.2%
ood group of the line O1O1
f normal ploidy by G-banding 46XX
ll HLA type I and II genotype of the line HLA typed class I and class II
sting by RT-qPCR Negative
ndotoxin levels 0.54 EU/ml
sual record of the line Normal
entiation to three germ layers Expression of muscle actin, β-tubulin
and α-feto protein
Fig. 2. In vitro differentiation of RCe012-A (RC-8) to ectoderm (β-tubulin III), mesoderm (muscle actin), and endoderm (α-fetoprotein). Speciﬁc staining shown in green, cell nuclei are
counterstained with DAPI (blue).
491P.A. De Sousa et al. / Stem Cell Research 16 (2016) 489–492Flow cytometry
Pluripotency was determined using the Human andMouse Pluripo-
tent StemCell Analysis kit (BD,Oxford, UK). Oct 3/4 and SSEA-4were in-
cluded as pluripotencymarkers, and SSEA-1 as a differentiationmarker.
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.
Immunocytochemistry
hESC were ﬁxed in methanol (ThermoFisher Scientiﬁc), blocked using
10% goat serum (Sigma-Aldrich, Dorset, UK) in PBS (Lonza) containing
0.01% Tween-20 (Sigma) and stained with AFP (1:500; Sigma), β-
tubulin III (1:1000; Sigma), muscle-speciﬁc actin (1:50; DAKO, Glostrup,
Denmark), and secondary antibody anti-goat IgG-AlexaFluor 488 (1:200;
ThermoFisher Scientiﬁc). Images were acquired using a Zeiss S100
Axiovert ﬂuorescence microscope or Nikon eC1 confocal microscope.
In vitro differentiation
Conﬂuent hESC cells lifted using a cell scraper (Corning) and embry-
oid bodies EBs generated in ultra low attachment plates (Corning) in EB
medium (20% FBS (GE Healthcare (PAA)), 80% KO-DMEM 1mM L-gluta-
mine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids (all
ThermoFisher Scientiﬁc)). After 9 days in suspension culture, EBs were
being transferred onto glass slide tissue culture chambers (Nunc,
ThermoFisher Scientiﬁc) coated with 0.1% gelatin (Sigma) at 0.1 ml/cm2
and cultured for 14 days.Table 2
Microsatellite PCR, blood group and HLA tissue typing results for RCe012-A (RC-8).
Microsatellite PCR results
D3S1358 1 D3S1358 2 vWA 1 vWA 2
14 16 17 19
Amelogenin 1 Amelogenin 2 D8S1179 1 D8S1179 2
X X 14 16
D19S433 1 D19S433 2 THO1 1 THO1 2
14 14 7 9.3
D5S818 1 D5S818 2 D7S820 1 D7S820 2
9 11 8 10
Blood group genotyping
RhD RhC Rhc RhE
pos pos neg neg
Jka Jkb K k
neg pos neg pos
Kp a Kp b Do a Do b
neg pos pos pos
HLA tissue typing
HLA class I type HLA-A*02, A*32; B*15, B*38; C*03, C*1
HLA class II type HLA-DRB1*04, DRB1*15; DRB4*01; DR
Comment C*03 is expressed serologically as C10,
serologically as B62, DQB1*03 is expreGenomic analysis
All outsourced assays were carried out under a Quality and Tech-
nical Agreement. DNA was extracted using the QIAamp DNAMini kit
(Qiagen, Manchester, UK) according to manufacturer's recommen-
dations 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 En-
gland. 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.Acknowledgements
Research culminating in the derivation of this line was funded by a
grant from Scottish Enterprise Economic Development Agency
(PM07321) to PDS, MB and AC.
Images of embroid body staining were kindly provided by S.
Greenhough and J. Gardner, Roslin Cellab Ltd.D16S539 1 D16S539 2 D2S1338 1 D2S1338 2
12 13 17 20
D21S11 1 D21S11 2 D18S51 1 D18S51 2
28 30 12 16
FGA 1 FGA 2 CSF1PO 1 CSF1PO 2
23 26 12 13
D13S317 1 D13S317 2 TPOX 1 TPOX 2
11 12 8 8
Rhe Fy a Fy b Fy GATA
pos pos pos neg
M N S S
pos pos pos pos
ABO
O1O1
2
B5*01; DQB1*06, DQB1*03
B*15 is expressed
ssed serologically as a DQ8.
Fig. 3. RCe012-A (RC-8) was analysed by Giesma staining of 20 metaphase spreads at passage 23 and showed a normal 46XX female karyotype in 17 of 20 spreads. Abnormalities in the
three remaining cells are believed to be results of harvesting artefacts. An independent assessment of karyology on the same cell line at passage 54 indicated normal 46XX karyology all 20
cells examined.
492 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 489–492


Derivation of the human embryonic stem cell line RCe012-A (RC-8) 

AbstractThe human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available

Derivation of the human embryonic stem cell line RCM1 

     Edinburgh Research Explorer                                      Derivation of the human embryonic stem cell line RCM1Citation for published version:De Sousa, P, Tye, BJ, Sneddon, S, Bruce, K, Dand, P, Russell, G, Collins, DM, Greenshields, A, Mcdonald,K, Bradburn, H, Gardner, J, Downie, JM, Courtney, A & Brison, DR 2016, 'Derivation of the humanembryonic stem cell line RCM1' Stem cell research, vol. 16, no. 2, pp. 476-480. DOI:10.1016/j.scr.2015.12.020Digital Object Identifier (DOI):10.1016/j.scr.2015.12.020Link: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) 476–480Contents 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 RCM1P.A. De Sousa a,b,⁎, B.J. Tye a, S. Sneddon c, K. Bruce a, P. Dand a, G. Russell a, D.M. Collins a, A. Greenshields a,K. McDonald a, H. Bradburn a, J. Gardner a, J.M. Downie a, A. Courtney a, D.R. Brison ca Roslin Cells Limited, Nine Edinburgh Bio-Quarter, 9 Little France Road, Edinburgh EH16 4UX, UKb Centre for Clinical Brain Sciences, University of Edinburgh, UKc Department of Reproductive Medicine, Central Manchester University Hospitals, NHS Foundation Trust, St Mary's Hospital, Manchester M13 9WL, UKInPCDTSOKALi⁎ Corresponding author.http://dx.doi.org/10.1016/j.scr.2015.12.0201873-5061/© 2015 Published by Elsevier B.V. This is an oa b s t r a c ta r t i c l e i n f oArticle history:Received 23 December 2015Accepted 23 December 2015Available online 31 December 2015The human embryonic stem cell lineRCM-1wasderived from a failed to fertilise egg undergoing parthenogeneticstimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layersin vitro and in vivo. It has a normal 46XX female karyotype andmicrosatellite PCR identity, HLA and blood grouptyping data is available.© 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).(RResource tableName of stem cell construct RCM1stitution Roslin Cells Ltd.St Mary's Hospital, Manchestererson who created resource B. Tye, J. Gardner, C. Pope, S. Sneddon, K.Bruce, P. Dand, G. Russell, D.M. Collins, A.Greenshields, K. McDonald, H. BradburnInontact person and emailEPaul.desousa@roslincells.com;Paul.desousa@ed.ac.uk;Janet.downie@roslincells.comAidan.courtney@roslincells.comdaniel.brison@manchester.ac.ukate archived/stock date 21 December 2006 (passage 10 on feeders)07 February 2011 (seed bank at passage 24)ype of resource Biological reagent: Cell lineub-type hESC, research graderigin Blastocyst with ICM and trophoblast originatingfrom clinically failed egg receiving parthenoge-netic stimulus. Conﬁrmed as biparental by wholegenome SNIP analysis.ey transcription factors Oct4 (conﬁrmed by ﬂow cytometry andimmunocytochemistry)uthentication See Quality Control Test Summary, Table 1nk to related literature (directURL links and full references)De Sousa PA*, Gardner J, Sneddon S, Pells S,Jorgenson Tye B, Dand P, Collins DM, Steward K,Shaw L, Przyborski S, Cooke M, McLauglin KJ,pen access article under the CC BY-NC-continued)esource table (continued)Name of stem cell constructND license (http://creativecommons.RCM1Kimber SJ, Lieberman BA, Wilmut I, Brison D.(2009) Clinically failed eggs as a source of normalhuman embryo stem cells. Stem Cell Research 2,188–197.http://www.sciencedirect.com/science/article/pii/S1873506109000026formation in public databases http://www.nibsc.org/science_and_research/advanced_therapies/uk_stem_cell_bank/cell_lines.aspxthics Informed consent obtained. Approved byparticipating institutional ethics advisoryboards and Central Manchester Local ResearchEthics Committee reference number03/CM/684. Conducted under the UK HumanFertilisation and Embryology Authority licenceno R0136.Resource details.RCM1was derived from a failed to fertilise egg subjected to partheno-genic activation. The cell linewas derived bywhole embryo outgrowth onmitotically inactivated human ﬁbroblast (HDF) feeder cells using HDFconditioned medium and expanded under feeder free conditions.RCM1 was shown to be pluripotent by expression of Oct 4 usingimmunocytochemistry (Table 1, Fig. 1). By ﬂow cytometric analysis, theexpression of pluripotency makers Oct4, Tra-1-60 and SSEA-4 was 97%,98% and 99%, respectively, whereas low expression of the differentiationmarker SSEA-1 (6%) was observed (Fig. 2).org/licenses/by-nc-nd/4.0/).Table 1Summary of quality control testing and results for RCM-1.Classiﬁcation Test Purpose ResultDonor screening HIV 1 + 2Hepatitis BHepatitis CDonor screening for adventitious agents NegativeIdentity Microsatellite PCR (mPCR) DNA Proﬁling to give cell line its signature, gender/species PerformedPhenotype Immunocytochemisty To assess levels of staining for the pluripotency markers Oct4and NanogExpression of Oct4 and NanogFlow cytometry Assess antigen levels & cell surface markers commonlyassociated with hESCOct3/4: 97%Tra 1-60: 98%SSEA-4: 99%SSEA-1: 6%Genotype Blood group genotyping(DNA analysis)To establish blood group of the line AAKaryology (G-banding) Conﬁrmation of normal ploidy by G-banding 46XXHLA tissue typing To establish full HLA Type I and Type II genotypes of the line HLA typed Class I and Class IIMicrobiology and virology Mycoplasma Mycoplasma testing by RT-qPCR NegativeMorphology Photography To capture a visual record of the line NormalDifferentiation potential Embryoid body formation To show differentiation to three germ layers Expression of muscle actin, β-tubulin and α-feto proteinTeratoma formation To show differentiation to three germ layers Teratoma formation observed, three germ layers detected477P.A. De Sousa et al. / Stem Cell Research 16 (2016) 476–480Differentiation to the three germ layers, endoderm, ectodermandme-soderm,was demonstrated using embryoid body formation and teratomaformation (Fig. 1).A microsatellite PCR proﬁle has been obtained for the cell line, andHLA Class I and Class II typing is available (Table 1). Blood groupgenotyping gave the blood group AA (Table 1). The cell line wasanalysed for genome stability by G-banding and GCH (Fig. 3), as wellas a SNP array screen to determine whether RCM1 was derived from aparthenogenetic or fertilized embryo. This showed a normal, 46XXfemale genotype with no genetic deﬁciencies that had originated froma fertilized egg.The cell line is free from mycoplasma contamination as determinedby RC-qPCR.Materials and methodsEthicsDerivation of hESC from surplus to requirement and failed to fertilise/develop embryos was approved by The Scotland A Research Ethics Com-mittee and local ethics board at participating fertility clinics and conduct-ed under licence no R0136 from the UK HFEA with informed donorconsent.Cell cultureActivation and derivation of RCM1 is described in De Sousa et al.(2009), Stem Cell Research 2, 188–197. The established cell line wasexpanded and banked using CellStart matrix and Stempro hESC SerumFree Medium (ThermoFisher Scientiﬁc (Life Technologies), Paisley,UK). Passaging was performed mechanically using an EZ passage tool(ThermoFisher Scientiﬁc).MycoplasmaMycoplasma detection was performed using AppliedBiosystems PrepSEQ™ Mycoplasma Nucleic Acid Extraction Kitand MicroSEQ™ Mycoplasma Real-Time PCR Detection Kit(ThermoFisher Scientiﬁc (Applied Biosystems)) according to themanufacturer's instruction.Flow cytometryPluripotency was determined using the Human and Mouse Plu-ripotent Stem Cell Analysis kit (BD, Oxford, UK). Oct3/4 and SSEA-4were included as pluripotency markers, and SSEA-1 as a differenti-ation marker. FITC conjugated Tra-1-60 (BD) was used as an addi-tional pluripotency marker. Fixed and permeabilised cells wereanalysed using the Guava easyCyte ﬂow cytometer (Millipore,Watford, UK).ImmunocytochemistryhESC were ﬁxed in 4% paraformaldehyde (ThermoFisher Scientiﬁc(Alfa Aesar)), permeabilised using 100% ethanol (ThermoFisherScientiﬁc) and stained with AFP (1:500; Sigma Aldrich, Dorset, UK),β-tubulin III (1:1000; Sigma), muscle-speciﬁc actin (1:50; DAKO,Glostrup, Denmark), Oct4 (1:200; Santa Cruz Biotechnology, Texas,USA), Nanog (1:20; R&D Systems, Abingdon, UK) and secondary antibod-ies anti-mouse IgG-FITC (1:200; Sigma), anti mouse IgG — AlexaFluor488, anti-goat IgG — AlexaFluor 488 and anti-goat IgG— AlexaFluor-594(1:200 donkey polyclonal AlexaFluor-594) (all 1:200; ThermoFisher Sci-entiﬁc). Alkaline Phosphatase staining was as per manufacturer's recom-mended protocol (Vector labs, Peterborough, UK). Images were acquiredusing a Zeiss S100 Axiovert ﬂuorescence microscope or Nikon eC1 confo-cal microscope.In vitro differentiationhESC cells were pre-treated for 1 h with 10 μMROCK inhibitor inStempro hESC SFM (ThermoFisher Scientiﬁc) and embryoid bodiesEBs generated in ultra low attachment plates (Corning) for 7 daysbefore being transferred into EB medium (20% FBS (GE Healthcare(PAA), Buckinghamshire, UK), 80% KO-DMEM 1 mM L-glutamine,0.1 mM β-mercaptoethanol, 1% nonessential amino acids (allThermoFisher Scientiﬁc)), on glass slide tissue culture chambersFig. 1. The characterization of RCM1 as a pluripotent hESC line. Undifferentiated colonies of RCM1 on human dermal ﬁbroblast feeder cells are shown (A) in phase contrast, (B) stainedpositively for Oct4 and (C) stained positively for alkaline phosphatase activity. RCM1 cells in vitro differentiated via embryoid bodies and stained for markers of the three germ layers:(D) muscle-speciﬁc actin (mesoderm), (E) α-fetoprotein (endoderm), and (F) β-tubulin III (ectoderm). A histological assessment was made of teratoma tissue formed as a result oftransplantation of the hESC line, RCM1, into the testis of adult SCID mice. (G) Low-power image showing heterogeneity of tissue structure in tumor. Identiﬁable structures includebone (bn), cartilage (ct), connective tissue (cn), and small cysts (cy); higher power images show (H) neuroectoderm (ne) and neural ganglion (ng) and (I) bone (bn). (J) Cartilage;(K) wall of primitive intestine, including muscular layer (mu), submucosal tissue (sm), and goblet cells (gc) within epithelium releasing mucus secretion (mc); (L) primitive kidneyconsisting of renal corpuscle (rc) and associated tubules (tb). (G–J, L) Weigert's and (K) hematoxylin and eosin. Scale bars, 100 μm (A–J) and 50 μm (K and L).From De Sousa et al. (2009).Fig. 2. Flowcytometry analysis of undifferentiated RCM1. RCM-1was subjected to ﬂowcytometry analysis formarkers of pluripotency. The speciﬁc antibody stained histograms are shownin the top row against the corresponding control histograms. Percentage staining is indicated in Table 1.478 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 476–480Fig. 3.Karyotype analysis of RCM-1. RCM-1was analysed byGiemsa staining of 20metaphase spreads and showed a normal 46XX female karyotype (top panel). No genomic copy numberimbalance was evaluated using a BlueGnome CytoChips array designed to improve detection of copy number variation associated with constitutional genetic disorders (bottom panel).Presented are SNP polymorphism copy number representation which did not exceed normal ranges (ﬂanking green and red lines) depicted next to chromosome ideograms. (Forinterpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)From De Sousa et al. (2009).479P.A. De Sousa et al. / Stem Cell Research 16 (2016) 476–480(Nunc, ThermoFisher Scientiﬁc) coated with 0.5% gelatin (Sigma)at 0.1 ml/cm2 for 14 days.In vivo differentiationThe developmental potential of RCM-1 to form teratomas consistingof tissues representative of all three germ layers was evaluated480 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 476–480following transplantation under kidney capsule in adult SCID mice andteratoma formation assessed as described in De Sousa et al. (2009),Stem Cell Research 2, 188–197.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.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 theMolecular Diagnosticslaboratory 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 overnightin warm containers, ﬁxed and analysed by standard G-banding analysis.For research grade lines, 20 spreads were analysed.Comparative genome hybridization (CGH)was carried out in collab-oration with ARK Genomics (Roslin Institute, UK), using BlueGnomeCytoChipTM arrayCGH v2. Genome-wide SNP analysis genomic DNAwas carried out with GeneChip Human Mapping 250 K Sty I and 250 KNsp arrays (Affymetrix), both as described in De Sousa et al. (2009),Stem Cell Research 2, 188–197.Veriﬁcation and authenticationThe identity and phenotype of RCM1 was conﬁrmed by qualitycontrol testing using the aforementioned methods the results of whichare summarised in Table 1, Figs. 1 & 2. Further information on this linecan be obtained from its original publication in De Sousa et al., 2009.AcknowledgementsResearch culminating in the derivation of this line was funded by agrant from the UK Medical Research Council (G0300484) to PDS andDRB, and Scottish Enterprise Economic Development Agency(PM07321) to PDS and AC.ReferencesDe Sousa, P.A., Gardner, J., Sneddon, S., Pells, S., Jorgenson Tye, B., Dand, P., DM, Collins,Steward, K., Shaw, L., Przyborski, S., Cooke, M., KJ, McLauglin, SJ, Kimber, BA,Lieberman, Wilmut, I., Brison, D., 2009. Clinically failed eggs as a source of normalhuman embryo stem cells. Stem Cell Res. 2, 188–197.

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

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