The human embryonic stem cell line RCe017-A (RC-13) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a frozen and thawed blastocyst stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe017-A (RC-13) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 47XY, +12/48XY, +1, +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data are available

A. Courtney

A. Greenshields

A. Laurie

Amps

B.J. Tye

Canham

D.M. Collins

Falls

G. Russell

H. Bradburn

J.M. Downie

K. Bruce

K. McDonald

Kearney

M. Bateman

M. Canham

P. Dand

P.A. De Sousa

Stephens

T. Kunath

Stem Cell Research

AbstractThe human embryonic stem cell line RCe018-A (RC-14) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a blastocyst stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe018-A (RC-14) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a male karyotype with an extra copy of chromosome 8 (47XY, +8). Microsatellite PCR identity, HLA and blood group typing data are available

De Sousa, P.A.

Tye, B.J.

Bruce, K.

Dand, P.

Russell, G.

Collins, D.M.

Greenshields, A.

McDonald, K.

Bradburn, H.

Laurie, A.

Downie, J.M.

Bateman, M.

Courtney, A.

Elsevier - Publisher Connector 

Stem Cell Research 16 (2016) 761–765
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 clinical grade human embryonic stem cell line
RCe018-A (RC-14)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, K. McDonald a,
H. Bradburn a, A. Laurie 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
⁎ Corresponding author at: Centres for Clinical Brain Sci
University of Edinburgh, Chancellor's Building, 49 Little Fr
4SB Scotland, UK and Roslin Cells Ltd, Scottish Centre for
France Drive, Edinburgh, EH16 4UU Scotland, UK. Tel.: +
791 759 4876.
E-mail addresses: paul.desousa@ed.ac.uk, paul.desousa@
URL's: http://www.crm.ed.ac.uk/research/associate/pl
http://www.roslincells.com (P.A. De Sousa).
http://dx.doi.org/10.1016/j.scr.2016.04.004
1873-5061/© 2016 Roslin Cells Ltd. Published by Elseviera b s t r a c ta r t i c l e i n f oArticle history:
Received 28 March 2016
Accepted 5 April 2016
Available online 13 April 2016The human embryonic stem cell line RCe018-A (RC-14) was derived under quality assured compliance with UK
regulation, EuropeanUnionDirectives and International guidance for tissue procurement, processing and storage
according toGoodManufacturing Practice (GMP) standards. The cell linewas derived from a blastocyst stage em-
bryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe018-A
(RC-14) showsnormal pluripotencymarker expression and differentiation to the three germ layers in vitro. It has
a male karyotype with an extra copy of chromosome 8 (47XY, +8). Microsatellite PCR identity, HLA and blood
group typing data are available.
© 2016 Roslin Cells Ltd. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).Li
(continued)Resource tableIn
Eame of stem cell construct RCe018-A
lternative name RC-14, RC14
stitution Roslin Cells Ltd.
erson who created resource B.J. Tye, K. Bruce, P. Dand, G. Russell, D.M.
Collins, A. Greenshields, K. McDonald, H.
Bradburn, A. Laurieontact 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 16 May 2011 (seed bank)
pe of resource Biological reagent: cell line
b-type hESC, clinical grade
rigin Blastocyst with ICM and trophoblast
ey transcription factors Oct4 (conﬁrmed by ﬂow cytometry),
uthentication See Quality Control Certiﬁcate of Analysis (Fig. 1)ences & RegenerativeMedicine,
ance Crescent, Edinburgh, EH16
Regenerative Medicine, 5 Little
44 (0) 131 242 6646, +44 (0)
roslincells.com (P.A.De Sousa).
uripotent-cell-translation,
B.V. This is an open access article under tnk to related literature (direct
URL links and full references)he CC BY license (http://creativecoN/Aformation in public databases http://hpscreg.eu/cell-line/RCe018-A
thics 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 and UK Human
Tissue Authority (HTA) licensing number 22631.Resource details
RCe018-A (RC-14)wasderived fromablastocyst stage embryo thatwas
surplus to requirement or unsuitable for clinical use. Human embryonic
stem cell (hESC) isolation, expansion and qualiﬁcation were performed
in facilities whose speciﬁcation, operation and monitoring complied with
GMP standards enabling; i) a fully traceable procurement procedure
with informed ethical consent which includes provision for commercial
use, ii) detailed medical history and blood borne virus (BBV) screening of
donors, and iii) compilation of a cell line history providing details on hESC
manufacturing process and quality control testing regime.
Human ESC culture and processing was performed in a grade A tissue
culture cabinet in a grade B clean room environment monitored for par-
ticulate and microbiological contamination during cell processing in ac-
cordance with Rules and Guidance for Pharmaceutical Manufacturers
and Distributors — The Orange Guide, compiled by the UK Medicines
Healthcare Products Regulatory Authority (go to: https://www.gov.uk/mmons.org/licenses/by/4.0/).
762 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 761–765guidance/good-manufacturing-practice-and-good-distribution-practice).
Accordingly, the facility was operating under a mature Quality Manage-
ment System, compliant with ISO9001:2008 standards. hESC derivation
was performed under licensure from the UK HFEA (R0136 to centre
0202) and HTA (Licensing Number 22631).
hESC derivation involved whole embryo outgrowth on mitotically
inactivated humandermalﬁbroblast (HDF) feeder cells. HDFswere derived
andmanufacturedaccording toGMPandhadbeenapproved for clinical use
by the Food and Drug Administration, USA. During derivation on HDFs,
hESCs were grown in a xeno-free cell therapy grade media (XF KODMEM)
supplemented with xeno-free human recombinant bFGF. hESCs were
subsequently expanded in a GMP grade serum-free medium (StemPro
hESC Serum Free Medium) on a xeno-free matrix (CellStart). The former
contained bovine serum albumin (BSA) from a Transmissible Spongiform
Encephalopathy (TSE)-free country of origin. The cell line was cryopre-
served in a GMP compliant cryopreservation solution (CryoStor CS10).
By ﬂow cytometry, RCe018-A (RC-14) expressed the pluripotency
makersOct-4, Tra-1-60 and SSEA-4 (87.7%, 55.4% and94.8%, respectively),
whereas low expression of the differentiation marker SSEA-1 (1.0%) was
observed (Figs. 1 and 2). Differentiation to the three germ layers,Fig. 1. Quality Control Certiﬁcate of Analysiendoderm, ectoderm and mesoderm, was demonstrated using embryoid
body formation in vitro, and expression of the germ layer markers α-
fetoprotein, β-tubulin and muscle actin was observed (Fig. 3).
A microsatellite PCR proﬁle has been obtained for the cell line, and
HLA Class I and II typing is available (Table 1). Blood group genotyping
gave the blood group O1O1 (Table 1).
Veriﬁcation and authentication
The cell line was analysed for genome stability by G-banding and
showed amale genotypewith trisomy 8 (47XY,+8) in all cells analysed
(Fig. 4). The cell line is free from mycoplasma contamination as deter-
mined 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 Researchs for RCe018-A (RC-14) P28A seed lot.
Fig. 1 (continued).
Fig. 2. RCe018-A (RC-14) 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 the Certiﬁcate of Analysis (Fig. 1).
763P.A. De Sousa et al. / Stem Cell Research 16 (2016) 761–765
Fig. 3. In vitro differentiation of RCe018-A (RC-14) to ectoderm (β-tubulin III), mesoderm (muscle Actin), and endoderm (α-fetoprotein). Speciﬁc staining shown in green, cell nuclei are
counterstained with DAPI (blue).
764 P.A. De Sousa et al. / Stem Cell Research 16 (2016) 761–765Ethics 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 processing and storage of hESC for human
application were conducted under licence number 22631 from the UK
Human Tissue Authority.Cell culture
Fresh embryoswere cultured in Sydneyblastocystmedium(CookMed-
ical, Hertfordshire, UK) after day 3 of development. Embryoswere cultured
at 36.5–37.5 °C, 5.0 ± 0.5% CO2, 5.0 ± 0.5% O2 in drops under parafﬁn oil
(Cook 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 of mitotically inactivated GMP grade neonatal human
dermal ﬁbroblasts (HDFs) (Forticell Biosciences, NJ, USA) on tissue cul-
ture plastic in XF KODMEM medium (Knockout-DMEM, 15% KOSR-XF,
2 mM L-glutamine, 1% MEM nonessential amino acids, 2% XF Growth
Factor Cocktail, 0.1 mM β-mercaptoethanol (ThermoFisher Scientiﬁc,
Paisley, UK) supplemented with 80 ng/ml human bFGF (ThermoFisher
Scientiﬁc). When available, cell therapy system quality reagents were
used. Assisted hatchingwas performed by removing the zonapellucidae
mechanically using Swemed cutting tools (Vitrolife, Göteborg, Sweden).
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). HDF were mitotically inactivated
using gamma irradiation at 50 Gy using a Gammacell Elite 1000 ma-
chine. For use as a feeder layer, irradiated HDFs were plated at
50,000 cells/cm2 in XF KODMEM medium supplemented withTable 1
Microsatellite PCR, blood group and HLA tissue typing results for RCe018-A (RC-14).
Microsatellite PCR results
D3S1358 1 D3S1358 2 vWA 1 vWA 2
16 16 16 16
Amelogenin 1 Amelogenin 2 D8S1179 1 D8S1179 2
X Y 12 12
D19S433 1 D19S433 2 THO1 1 THO1 2
13 16 9.3 9.3
D5S818 1 D5S818 2 D7S820 1 D7S820 2
9 11 8.2 12
Blood group genotyping
RhD RhC Rhc RhE
pos pos neg neg
Jka Jkb K k
pos neg neg pos
Do a Do b ABO
neg pos O1O1
HLA tissue typing
HLA Class I Type
HLA Class II Type
Comment
*Peak falls below threshold to conﬁdently score.80 ng/ml human bFGF (ThermoFisher Scientiﬁc). Cells were cultured
at 36.5–37.5 °C, 5.0 ± 0.5% CO2, 5.0 ± 0.5% O2 and 50% medium ex-
changed 6 days a week.
The established cell linewas expandedandbankedusingCellStartma-
trix and Stempro hESC Serum Free Medium (cell therapy system quality
reagents, ThermoFisher Scientiﬁc). This contained BSA from a TSE-free
country of origin. Passagingwas performedmechanically using an EZ pas-
sage 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) using an EF600-107 controlled rate
freezer (Grant Instruments, Cambridge, UK) before being stored in a
−150 °C freezer (Panasonic Biomedical, Loughborough, UK).
Mycoplasma
In process mycoplasma detection was performed using Applied
Biosystems PrepSEQ™ Mycoplasma Nucleic Acid Extraction Kit and
MicroSEQ™MycoplasmaReal-TimePCRDetectionKit (ThermoFisher Sci-
entiﬁc (Applied Biosystems)) according to themanufacturer'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%, and the reaction time ofD16S539 1 D16S539 2 D2S1338 1 D2S1338 2
10 12 17 24
D21S11 1 D21S11 2 D18S51 1 D18S51 2
30 31.2 16 16
FGA 1 FGA 2 CSF1PO 1 CSF1PO 2
20 24 10 10
D13S317 1 D13S317 2 TPOX 1 TPOX 2
11 14 8 12
Rhe Fy a Fy b Fy GATA
pos pos neg neg
M N S s
pos pos neg pos
HLA-A*01, A*02; B*07, B*08; C*07
HLA-DRB1*03; DRB3*01/02; DQB1*02
DRB1*03 is expressed serologically as DR17.
Fig. 4. RCe018-A (RC-14) was analysed by Giemsa staining of 30 metaphase spreads and showed a 47XY, +8 male karyotype in all spreads analysed.
765P.A. De Sousa et al. / Stem Cell Research 16 (2016) 761–765the negative control was greater than the reaction time of the lowest
standard on the standard curve.
Flow cytometry
Pluripotency was determined using the Human and Mouse Pluripo-
tent Stem Cell Analysis kit (BD, Oxford, UK). Oct 3/4 and SSEA-4 were in-
cluded as pluripotency markers, and SSEA-1 as a differentiation marker.
FITC conjugated Tra-1-60 (BD) was used as an additional pluripotency
marker. Fixed and permeabilised cells were analysed using a FACS Aria
ﬂowcytometer (BD) or aGuava easyCyteﬂowcytometer (Millipore,Wat-
ford, UK). Percentage expression of eachmarkerwas compared to isotype
control or unstained cells.
Viability
Viability was determined using the Guava ViaCount assay. Brieﬂy, the
Guava ViaCount reagent (Millipore) containing a nuclear and a viability
dye, was mixed with a single cell suspension, incubated for 5 min and
analysed using the Guava easyCyte ﬂow cytometer (Millipore). Total
cell count, viable cell count and percentage viable cells were obtained.
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 in ultra low attachment plates (Corning) for 7 days be-
fore being transferred into EBmedium (20% FBS (GE Healthcare (PAA)),
80% KO-DMEM 1 mM L-glutamine, 3.5 μM β-mercaptoethanol, 1% non-
essential amino acids (all ThermoFisher Scientiﬁc)), on glass slide tissue
culture chambers (Nunc, ThermoFisher Scientiﬁc) coated with 0.2% gel-
atin (Sigma Aldrich, Dorset, UK) at 0.1 ml/cm2 for 14 days.
Immunocytochemistry
Cells were ﬁxed in 4% paraformaldehyde (ThermoFisher Scientiﬁc
(Alfa Aesar)), permeabilised using 100% ethanol (ThermoFisherScientiﬁc) and stained with AFP (1:500; Sigma), β-tubulin III (1:1000;
Sigma) and muscle-speciﬁc actin (1:50; DAKO, Glostrup, Denmark)
and secondary antibody anti mouse IgG-AlexaFluor 488 (1:200;
Sigma). Images were acquired using a Zeiss S100 Axiovert ﬂuorescence
microscope or Nikon eC1 confocal microscope.
Genomic analysis and outsourced assays
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
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 Western General Cytoge-
netics Laboratory (Edinburgh, UK). Live cells at 60–70% conﬂuencywere
shipped in warm containers, ﬁxed and analysed by standard G-banding
analysis. For clinical grade lines, 30 spreads were analysed.
Viral screening for cytomegalovirus (CMV), Human T-cell
lymphotropic virus (HTLV)-1, Human immunodeﬁciency virus
(HIV)-1, Hepatitis C virus (HCV), Hepatitis B virus (HBV) and
Epstein–Barr virus (EBV) was carried out by The Doctors Laboratory
(London, UK).
Acknowledgements
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.


Derivation of the clinical grade human embryonic stem cell line RCe018-A (RC-14) 

Crossref

Derivation of the clinical grade human embryonic stem cell line RCe018-A (RC-14)

Derivation of the clinical grade human embryonic stem cell line RCe016-A (RC-12)

Edinburgh Research Explorer

English

Derivation of the clinical grade human embryonic stem cell line RCe015-A (RC-11)

AbstractThe human embryonic stem cell line RCe016-A (RC-12) was derived under quality assured compliance with UK regulations, EU Directives and International guidance for tissue procurement, processing and storage according to good manufacturing practice (GMP) standards. The cell line was derived from a cryopreserved blastocyst stage embryo voluntarily donated as surplus to fertility requirements following informed consent. RCe016-A (RC-12) shows normal pluripotency marker expression and differentiation to three germ layers in vitro. Karyology revealed a mixed male karyotype at early passage (P15), which resolved as normal 46XY by passage 33. Microsatellite PCR identity, HLA and blood group typing data is available

Derivation of the clinical grade human embryonic stem cell line RCe016-A (RC-12) 

The human embryonic stem cell line RCe018-A (RC-14) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a blastocyst stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe018-A (RC-14) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a male karyotype with an extra copy of chromosome 8 (47XY, +8). Microsatellite PCR identity, HLA and blood group typing data are available

Directory of Open Access Journals

De Sousa, Paul

Tye, B.j.

Collins, D.m.

Mcdonald, K.

Downie, J.m.

     Edinburgh Research Explorer                                      Derivation of the clinical grade human embryonic stem cell lineRCe016-A (RC-12)Citation for published version:De Sousa, P, Tye, BJ, Bruce, K, Dand, P, Russell, G, Collins, DM, Greenshields, A, Mcdonald, K, Bradburn,H, Laurie, A, Downie, JM, Bateman, M & Courtney, A 2016, 'Derivation of the clinical grade humanembryonic stem cell line RCe016-A (RC-12)', Stem cell research. https://doi.org/10.1016/j.scr.2016.04.001Digital Object Identifier (DOI):10.1016/j.scr.2016.04.001Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Peer reviewed versionPublished 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: 11. May. 2020  	Derivation of the clinical grade human embryonic stem cell line RCe016-A(RC-12)P.A. De Sousa, B.J. Tye, K. Bruce, P. Dand, G. Russell, D.M. Collins,A. Greenshields, K. McDonald, H. Bradburn, A. Laurie, J.M. Downie, M.Bateman, A. CourtneyPII: S1873-5061(16)30012-5DOI: doi: 10.1016/j.scr.2016.04.001Reference: SCR 742To appear in: Stem Cell ResearchReceived date: 28 March 2016Accepted date: 5 April 2016Please cite this article as: De Sousa, P.A., Tye, B.J., Bruce, K., Dand, P., Russell, G.,Collins, D.M., Greenshields, A., McDonald, K., Bradburn, H., Laurie, A., Downie, J.M.,Bateman, M., Courtney, A., Derivation of the clinical grade human embryonic stem cellline RCe016-A (RC-12), Stem Cell Research (2016), doi: 10.1016/j.scr.2016.04.001This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTTitle: Derivation of the clinical grade human embryonic stem cell line RCe016-A (RC-12) Authors:  P.A. De Sousaa,b,c, B.J. Tyea, K. Brucea, P. Danda, G. Russella, D.M. Collinsa, A. Greenshieldsa, K. McDonalda, H. Bradburna, A. Lauriea, J.M. Downiea, M. Batemana, A. Courtneya Affiliations  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  Abstract The human embryonic stem cell line RCe016-A (RC-12) was derived under quality assured compliance with UK regulation, EU Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards.  The cell line was derived from a cryopreserved blastocyst stage embryo voluntarily donated as surplus to fertility requirements following informed consent.  RCe016-A (RC-12) shows normal pluripotency marker expression and differentiation to three germ layers in vitro.  Karyology revealed a mixed male karyotype at early passage (P15), which resolved as normal 46XY by passage 33.  Microsatellite PCR identity, HLA and blood group typing data is available.   Resource table  Name of stem cell construct     RCe016-A   Alternative name R -12, RC12   Institution   Roslin Cells Ltd.   Person who created resource   B.J. Tye, K. Bruce, P. Dand, G. Russell, D.M.  Collins, A. Greenshields, K. McDonald, H. Bradburn, A. Laurie   Contact person and email   Paul.desousa@roslincells.com; Paul.desousa@ed.ac.uk Janet.downie@roslincells.com Aidan.courtney@roslincells.com Malcolm.bateman@roslinfoundation.com   Date archived/stock 07 September 2010 (seed bank) ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTdate     Type of resource   Biological reagent: cell line   Sub-type   hESC, clinical grade   Origin  Blastocyst with ICM and Trophoblast   Key transcription factors   Oct4 (confirmed by flow cytometry),   Authentication   See Quality Control Certificate of Analysis (Fig. 1)   Link to related literature (direct URL links and full references)   N/A   Information in public databases   http://hpscreg.eu/cell-line/RCe016-A Ethics   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 and UK Human Tissue Authority (HTA) licensing number 22631.    Resource details RCe016-A (RC-12) was derived from a frozen and thawed blastocyst stage embryo that was surplus to fertility treatment requirement.   Human embryonic stem cell (hESC) isolation, expansion and qualification was performed in a facilities whose specification, operation and monitoring complied with GMP standards enabling; i) a fully traceable procurement procedure with informed ethical consent which includes provision for commercial use, ii) detailed medical history and blood borne virus (BBV) screening of donors, and iii) compilation of a cell line history providing details on hESC manufacturing process and quality control testing regime.  Human ESC culture and processing was performed in a grade A tissue culture cabinet in a grade B clean room environment monitored for particulate and microbiological contamination during cell processing in accordance with Rules and Guidance for Pharmaceutical Manufacturers and Distributors - The Orange Guide, compiled by the UK Medicines Healthcare Products Regulatory Authority (Go to: https://www.gov.uk/guidance/good-manufacturing-practice-and-good-ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTdistribution-practice). Accordingly, the facility was operating under a mature Quality Management System, compliant with ISO9001:2008 standards. HESC derivation was performed under licensure from the UK HFEA (R0136 to centre 0202) and HTA (Licensing Number 22631).   HESC derivation involved whole embryo outgrowth on mitotically inactivated human dermal fibroblast (HDF) feeder cells.  HDFs were derived and manufactured according to GMP and had been approved for clinical use by the Food and Drug Administration, USA.  During derivation on HDFs, hESCs were grown in a xeno-free cell therapy grade media (XF KODMEM) supplemented with xeno-free human recombinant bFGF. HESCs were subsequently expanded in a GMP grade serum-free medium (StemPro hESC Serum Free Medium,) on a xeno-free matrix (CellStart). The former contained bovine serum albumin (BSA) from a Transmissible Spongioform Encephalopathy (TSE)-free country of origin.  The cell line was cryopreserved in a GMP compliant cryopreservation solution (CryoStor CS10,). By flow cytometry, RCe016-A (RC-12) expressed the pluripotency makers Oct-4, Tra-1-60 and SSEA-4 (88.8%, 85.8% and 95.0%, respectively), whereas low expression of the differentiation marker SSEA-1 (0.1%) was observed (Fig. 1, Fig. 2).  Differentiation to the three germ layers, endoderm, ectoderm and mesoderm, was demonstrated using embryoid body formation in vitro, and expression of the germ layer markers α-fetoprotein, β-tubulin and muscle actin was observed (Fig. 3).  A microsatellite PCR profile has been obtained for the cell line, and HLA Class I and II typing is available (Table 1).  Blood group genotyping gave the blood group AO1 (Table 1).   Verification and authentication  The cell line was analysed for genome stability by G-banding and showed a mixed male genotype at passage 15 (Certificate of Analysis, Fig. 1).  A normal 46XY genotype was present in 18 of 30 cells analysed, but a subpopulation (12 of 30 cells) exhibited trisomy 12 (47XY, +12).  By passage 33, the cell line had stabilised on a normal male 46XY karyotype (Fig. 4).  The cell line is free from mycoplasma 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 informed donor consent.  The processing and storage of hESC cells for human application was conducted under licence number 22631 from the UK Human Tissue Authority.  Cell culture Frozen embryos were thawed using Vitrified Embryo Safety Thawing Pack (Kitazato/Dibimed, Valencia, Spain) according to manufacturers 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 ± 0.5% CO2, 5.0 ± 0.5% O2 in drops under paraffin oil (Rochford Medical) and transferred to fresh medium at least every 2-3 days.   ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTBy Day 8 of development, embryos were placed in derivation conditions consisting of mitotically inactivated GMP grade 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 (ThermoFisher Scientific, Paisley, UK) supplemented with 80 ng/ml human bFGF (ThermoFisher Scientific).      When available, cell therapy system quality reagents were used.  Assisted hatching was performed by removing the zona pellucidae mechanically using Swemed cutting tools (Vitrolife, Göteborg, Sweden).   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).  HDF 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 50000 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 ± 0.5% CO2, 5.0 ± 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 (cell therapy system quality reagents, 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) using an EF600-107  controlled rate freezer (Grant Instruments, Cambridge, UK) before being stored in a -150°C freezer (Panasonic Biomedical, Loughborough, UK).   Mycoplasma In process 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. Eurpoean pharmacopoeia (EP) mycoplasma testing was carried out by Moredun Scientific Ltd. (Edinburgh, UK), under a quality and technical agreement.   Endotoxin Endotoxin levels were determined using the Kinetic-QCL assay (Lonza) and an incubating plate reader (BioTek ELx808) according to the manufacturer’s instructions. Briefly, an unknown sample was compared with a standard curve of known levels of control endotoxin.  An assay was deemed valid if the coefficient of correlation, r ≥ 0.980 and the CV (%) for the standard curve was ≤10%, and the reaction time of the negative control was greater than the reaction time of the lowest standard on the standard curve.   Flow cytometry  Pluripotency was determined using the Human and Mouse Pluripotent Stem Cell Analysis kit (BD, Oxford, UK).   Oct 3/4 and SSEA-4 were included as pluripotency markers, and SSEA-1 as a differentiation marker.   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) or a Guava easyCyte flow cytometer (Millipore, Watford, UK).  Percentage expression of each marker was compared to isotype control or unstained cells.   Viability ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTViability was determined using the Guava ViaCount assay.  Briefly, the Guava Viacount reagent (Millipore) containing a nuclear and a viability dye, was mixed with a single cell suspension, incubated for 5 minutes and analysed using the Guava easyCyte flow cytometer (Millipore).  Total cell count, viable cell count and percentage viable cells was obtained.    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 in ultra low attachment plates (Corning) for 7 days before being transferred into EB medium (20% FBS (GE Healthcare (PAA)), 80% KO-DMEM 1 mM L-glutamine, 3.5 µM -mercaptoethanol, 1 % nonessential amino acids (all ThermoFisher Scientific)), on glass slide tissue culture chambers (Nunc, ThermoFisher Scientific) coated with 0.2% gelatin (Sigma Aldrich, Dorset, UK) at 0.1 ml/cm2 for 14 days. Immunocytochemistry Cells were fixed in 4% paraformaldehyde (ThermoFisher Scientific (Alfa Aesar)), permeabilised using 100% ethanol (ThermoFisher Scientific) and stained with AFP (1:500; Sigma), β-tubulin III (1:1000; Sigma) and muscle-specific actin (1:50; DAKO, Glostrup, Denmark) and secondary antibody anti mouse IgG-AlexaFluor 488 (1:200; Sigma).  Images were acquired using a Zeiss S100 Axiovert fluorescence microscope or Nikon eC1 confocal microscope Genomic analysis and outsourced assays All outsourced assays were carried out under a Quality and Technical Agreement.   DNA was extracted using the QIAamp DNA Mini kit (Qiagen, Manchester, UK) according to manufacturer’s recommendations and provided in recommended quantities to the service providers.     Microsatellite PCR, or Short Tandem Repeat analysis, was used to determine cell line identity and was carried out by Public Health England.   A profile was obtained for the following core alleles: vWA, D16S539, Amelogenin, THO1, CSF1PO, D5S818, D75820, D135317 and TPOX.   Human Leukocyte Antigen (HLA) tissue typing was carried out by the Scottish National Blood Transfusion Service. Blood group genotyping was carried out by the Molecular Diagnostics laboratory at NHSBT. Karyotype analysis was carried out by The Doctors Laboratory (London, UK) or the Western General Cytogenetics Laboratory (Edinburgh, UK).  Live cells at 60-70% confluency were shipped in warm containers, fixed and analysed by standard G-banding analysis.  For clinical grade lines, 30 spreads were analysed.    Viral screening for cytomegalovirus (CMV), Human T-cell lymphotropic virus (HTLV), Human immunodeficiency virus (HIV)-1, Hepatitis C virus (HCV), Hepatitis B virus (HBV), Epstein-Barr virus (EBV) and syphilis was carried out by The Doctors Laboratory.   Figures and tables  ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT Fig. 1.  Quality Control Certificate of Analysis for RCe016-A (RC-12) P8B seed lot.    ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT Fig. 2.  RCe016-A (RC-12) 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 the Certificate of Analysis (Fig. 1).    Fig. 3.  In vitro differentiation of RCe016-A (RC-12) to ectoderm (β-tubulin III), mesoderm (muscle Actin), and endoderm (α-fetoprotein).  Specific staining shown in green, cell nuclei are counterstained with DAPI (blue).    ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTFig. 4.  RCe016-A (RC-12) was analysed by Giesma staining of 20 metaphase spreads at passage 33 and showed a normal male karyotype of 46XY.   Table 1.  Microsatellite PCR, blood group and HLA tissue typing results for RCe016-A (RC-12).   Microsatellite PCR results D3S1358 1 D3S1358 2 vWA 1 vWA 2 D16S539 1 D16S539 2 D2S1338 1 D2S1338 2 16 17 17 18 9 11 24 25         Amelogenin 1 Amelogenin 2 D8S1179 1 D8S1179 2 D21S11 1 D21S11 2 D18S51 1 D18S51 2 X Y 13 13 27 29 11 14         D19S433 1 D19S433 2 THO1 1 THO1 2 FGA 1 FGA 2 CSF1PO 1 CSF1PO 2 14 14 7 8 23 26 12 12         D5S818 1 D5S818 2 D7S820 1 D7S820 2 D13S317 1 D13S317 2 TPOX 1 TPOX 2 12 13 11 11 11 13 8 11         Blood group genotyping RhD RhC Rhc RhE Rhe Fy a Fy b Fy GATA pos neg pos neg pos pos pos neg         Jka Jkb K k  M N S  S  neg pos neg pos pos neg neg pos         ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTDo a Do b ABO      neg pos AO1              HLA tissue typing HLA Class I Type HLA-A*01, A*11; B*08, B*35; Cw*04, Cw*07 HLA Class II Type HLA-DRB1*03, DRB1*04; DRB3*01; DRB4*01; DQB1*02, DQB1*03; DPB1*04 Comment DRB1*03 is expressed serologically as DR17, DQB1*03 is expressed serologically as DQ8, Unable to exclude DPB1*2302N/2402/9901.  Acknowledgements  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.  

Derivation of the clinical grade human embryonic stem cell line RCe017-A (RC-13)

     Edinburgh Research Explorer                                      Derivation of the clinical grade human embryonic stem cell lineRCe017-A (RC-13)Citation for published version:De Sousa, P, Tye, BJ, Bruce, K, Dand, P, Russell, G, Collins, DM, Greenshields, A, Mcdonald, K, Bradburn,H, Laurie, A, Downie, JM, Bateman, M & Courtney, A 2016, 'Derivation of the clinical grade humanembryonic stem cell line RCe017-A (RC-13)' Stem cell research. DOI: 10.1016/j.scr.2016.04.006Digital Object Identifier (DOI):10.1016/j.scr.2016.04.006Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Peer reviewed versionPublished 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. 2019  	Derivation of the clinical grade human embryonic stem cell line RCe017-A(RC-13)P.A. De Sousa, B.J. Tye, K. Bruce, P. Dand, G. Russell, D.M. Collins,A. Greenshields, K. McDonald, H. Bradburn, A. Laurie, J.M. Downie, M.Bateman, A. CourtneyPII: S1873-5061(16)30017-4DOI: doi: 10.1016/j.scr.2016.04.006Reference: SCR 747To appear in: Stem Cell ResearchReceived date: 28 March 2016Accepted date: 5 April 2016Please cite this article as: De Sousa, P.A., Tye, B.J., Bruce, K., Dand, P., Russell, G.,Collins, D.M., Greenshields, A., McDonald, K., Bradburn, H., Laurie, A., Downie, J.M.,Bateman, M., Courtney, A., Derivation of the clinical grade human embryonic stem cellline RCe017-A (RC-13), Stem Cell Research (2016), doi: 10.1016/j.scr.2016.04.006This is a PDF ﬁle of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its ﬁnal form. Please note that during the production processerrors may be discovered which could aﬀect the content, and all legal disclaimers thatapply to the journal pertain.ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTTitle: Derivation of the clinical grade human embryonic stem cell line RCe017-A (RC-13) Authors:  P.A. De Sousaa,b,c, B.J. Tyea, K. Brucea, P. Danda, G. Russella, D.M. Collinsa, A. Greenshieldsa, K. McDonalda, H. Bradburna, A. Lauriea, J.M. Downiea, M. Batemana, A. Courtneya Affiliations  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  Abstract The human embryonic stem cell line RCe017-A (RC-13) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards.  The cell line was derived from a frozen and thawed blastocyst stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent.   RCe017-A (RC-13) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro.  It has a mixed 47XY, +12/48XY, +1, +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data is available.    Resource Table  Name of stem cell construct     RCe017-A   Alternative name RC-13, RC13   Institution   Roslin Cells Ltd.   Person who created resource   B.J.Tye, K. Bruce, P. Dand, G. Russell, D.M.  Collins, A. Greenshields, K. McDonald, H. Bradburn, A. Laurie   Contact person and email   Paul.desousa@roslincells.com; Paul.desousa@ed.ac.uk Janet.downie@roslincells.com Aidan.courtney@roslincells.com Malcolm.bateman@roslinfoundation.com   ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTDate archived/stock date   11 March 2011 (seed bank)   Type of resource   Biological reagent: cell line   Sub-type   hESC, clinical grade   Origin  Blastocyst with ICM and Trophoblast   Key transcription factors   Oct4 (confirmed by flow cytometry),   Authentication   See Quality Control Certificate of Analysis (Fig. 1)   Link to related literature (direct URL links and full references)   N/A   Information in public databases   http://hpscreg.eu/cell-line/RCe017-A   Ethics   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 and UK Human Tissue Authority (HTA) licensing number 22631.    Resource details RCe017-A (RC-13) was derived from a frozen and thawed blastocyst stage embryo that was surplus to requirement or unsuitable for clinical use.   Human embryonic stem cell (hESC) isolation, expansion and qualification was performed in a facilities whose specification, operation and monitoring complied with GMP standards enabling; i) a fully traceable procurement procedure with informed ethical consent which includes provision for commercial use, ii) detailed medical history and blood borne virus (BBV) screening of donors, and iii) compilation of a cell line history providing details on hESC manufacturing process and quality control testing regime.  Human ESC culture and processing was performed in a grade A tissue culture cabinet in a grade B clean room environment monitored for particulate and microbiological contamination during cell processing in accordance with Rules and Guidance for Pharmaceutical Manufacturers and ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTDistributors - The Orange Guide, compiled by the UK Medicines Healthcare Products Regulatory Authority (Go to: https://www.gov.uk/guidance/good-manufacturing-practice-and-good-distribution-practice). Accordingly, the facility was operating under a mature Quality Management System, compliant with ISO9001:2008 standards. HESC derivation was performed under licensure from the UK HFEA (R0136 to centre 0202) and HTA (Licensing Number 22631).   HESC derivation involved whole embryo outgrowth on mitotically inactivated human dermal fibroblast (HDF) feeder cells.  HDFs were derived and manufactured according to GMP and had been approved for clinical use by the Food and Drug Administration, USA.  During derivation on HDFs, hESCs were grown in a xeno-free cell therapy grade media (XF KODMEM) supplemented with xeno-free human recombinant bFGF. HESCs were subsequently expanded in a GMP grade serum-free medium (StemPro hESC Serum Free Medium) on a xeno-free matrix (CellStart). The former contained bovine serum albumin (BSA) from a Transmissible Spongioform Encephalopathy (TSE)-free country of origin.  The cell line was cryopreserved in a GMP compliant cryopreservation solution (CryoStor CS10). By flow cytometry, RCe017-A (RC-13) expressed the pluripotency makers Oct-4, Tra-1-60 and SSEA-4 (80.8%, 88.3% and 79.6%, respectively), whereas low expression of the differentiation marker SSEA-1 (0.7%) was observed (Fig. 1, Fig. 2).  Differentiation to the three germ layers, endoderm, ectoderm and mesoderm, was demonstrated using embryoid body formation in vitro, and expression of the germ layer markers α-fetoprotein, β-tubulin and muscle actin was observed (Fig. 3).  A microsatellite PCR profile has been obtained for the cell line, and HLA Class I and II typing is available (Table 1).  Blood group genotyping gave the blood group AA (Table 1).   Verification and authentication  The cell line was analysed for genome stability by G-banding and showed a mixed male genotype.  Trisomy 12 (47XY, +12) was present in 26 of 30 cells analysed, but a subpopulation (4 of 30 cells) has an additional copy of chromosome 1 (48XY, +1, +12) (Fig. 4).  The cell line is free from mycoplasma 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 informed donor consent.  The processing and storage of hESC cells for human application was conducted under licence number 22631 from the UK Human Tissue Authority.  Cell culture Frozen embryos were thawed using Vitrified Embryo Safety Thawing Pack (Kitazato/Dibimed, Valencia, Spain) according to manufacturers 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 ± 0.5% CO2, 5.0 ± 0.5% O2 in drops under paraffin oil (Rochford Medical) and transferred to fresh medium at least every 2-3 days.   ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTBy Day 8 of development, embryos were placed in derivation conditions consisting of mitotically inactivated GMP grade 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 (ThermoFisher Scientific, Paisley, UK) supplemented with 80 ng/ml human bFGF (ThermoFisher Scientific).      When available, cell therapy system quality reagents were used.  Assisted hatching was performed by removing the zona pellucidae mechanically using Swemed cutting tools (Vitrolife, Göteborg, Sweden).   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).  HDF were mitotically inactivated using gamma irradiation at 50 Gy using a Gammacell Elite 1000 machine.  For use as a feeder layer, irradiated HDFs were plated at 50000 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 ± 0.5% CO2, 5.0 ± 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 (cell therapy system quality reagents, ThermoFisher Scientific).  This contained BSA from a TSE-free country of origin. 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) using an EF600-107  controlled rate freezer (Grant Instruments, Cambridge, UK) before being stored in a -150°C freezer (Panasonic Biomedical, Loughborough, UK).   Mycoplasma In process mycoplasma detection was performed using Applied Biosystems PrepSEQ™ Mycoplasma Nucleic Acid Extraction Kit and MicroSEQ™ Mycoplasma Real-Time PCR Detection Kit (ThermoFisher Scientific (Applied Biosystems)) according to the manufacturer’s instruction.  Endotoxin Endotoxin levels were determined using the Kinetic-QCL assay (Lonza) and an incubating plate reader (BioTek ELx808) according to the manufacturer’s instructions. Briefly, an unknown sample was compared with a standard curve of known levels of control endotoxin.  An assay was deemed valid if the coefficient of correlation, r ≥ 0.980 and the CV (%) for the standard curve was ≤10%, and the reaction time of the negative control was greater than the reaction time of the lowest standard on the standard curve.   Flow cytometry  Pluripotency was determined using the Human and Mouse Pluripotent Stem Cell Analysis kit (BD, Oxford, UK).   Oct 3/4 and SSEA-4 were included as pluripotency markers, and SSEA-1 as a differentiation marker.   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) or a Guava easyCyte flow cytometer (Millipore, Watford, UK).  Percentage expression of each marker was compared to isotype control or unstained cells.   Viability ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTViability was determined using the Guava ViaCount assay.  Briefly, the Guava Viacount reagent (Millipore) containing a nuclear and a viability dye, was mixed with a single cell suspension, incubated for 5 minutes and analysed using the Guava easyCyte flow cytometer (Millipore).  Total cell count, viable cell count and percentage viable cells was obtained.    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 in ultra low attachment plates (Corning) for 7 days before being transferred into EB medium (20% FBS (GE Healthcare (PAA)), 80% KO-DMEM 1 mM L-glutamine, 3.5 µM -mercaptoethanol, 1 % nonessential amino acids (all ThermoFisher Scientific)), on glass slide tissue culture chambers (Nunc, ThermoFisher Scientific) coated with 0.2% gelatin (Sigma Aldrich, Dorset, UK) at 0.1 ml/cm2 for 14 days. Immunocytochemistry Cells were fixed in 4% paraformaldehyde (ThermoFisher Scientific (Alfa Aesar)), permeabilised using 100% ethanol (ThermoFisher Scientific) and stained with AFP (1:500; Sigma), β-tubulin III (1:1000; Sigma) and muscle-specific actin (1:50; DAKO, Glostrup, Denmark) and secondary antibody anti mouse IgG-AlexaFluor 488 (1:200; Sigma).  Images were acquired using a Zeiss S100 Axiovert fluorescence microscope or Nikon eC1 confocal microscope Genomic analysis and outsourced assays All outsourced assays were carried out under a Quality and Technical Agreement.   DNA was extracted using the QIAamp DNA Mini kit (Qiagen, Manchester, UK) according to manufacturer’s recommendations and provided in recommended quantities to the service providers.     Microsatellite PCR, or Short Tandem Repeat analysis, was used to determine cell line identity and was carried out by Public Health England.   A profile was obtained for the following core alleles: vWA, D16S539, Amelogenin, THO1, CSF1PO, D5S818, D75820, D135317 and TPOX.   Human Leukocyte Antigen (HLA) tissue typing was carried out by the Scottish National Blood Transfusion Service. Blood group genotyping was carried out by the Molecular Diagnostics laboratory at NHSBT. Karyotype analysis was carried out by the Western General Cytogenetics Laboratory (Edinburgh, UK).  Live cells at 60-70% confluency were shipped in warm containers, fixed and analysed by standard G-banding analysis.  For clinical grade lines, 30 spreads were analysed.    Viral screening for cytomegalovirus (CMV), Human T-cell lymphotropic virus (HTLV)-1, Human immunodeficiency virus (HIV)-1, Hepatitis C virus (HCV), Hepatitis B virus (HBV) and Epstein-Barr virus (EBV) was carried out by The Doctors Laboratory (London, UK).   Figures and tables  ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT Fig. 1.  Quality Control Certificate of Analysis for RCe017-A (RC-13) P19A seed lot.    ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPT Fig. 2.  RCe017-A (RC-13) 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 the Certificate of Analysis (Fig. 1).    Fig. 3.  In vitro differentiation of RCe017-A (RC-13) to ectoderm (β-tubulin III), mesoderm (muscle Actin), and endoderm (α-fetoprotein).  Specific staining shown in green, cell nuclei are counterstained with DAPI (blue).     Fig. 4.  RCe017-A (RC-13) was analysed by Giesma staining of 30 metaphase spreads and showed a mixed male karyotype of 47XY, +12 (26 of 30 cells) and  48XY, +1, +12 (4 of 30 cells).   Table 1.  Microsatellite PCR, blood group and HLA tissue typing results for RCe017-A (RC-13).   Microsatellite PCR results D3S1358 1 D3S1358 2 vWA 1 vWA 2 D16S539 1 D16S539 2 D2S1338 1 D2S1338 2 14 17 17 18 9 12 24 24         ACCEPTED MANUSCRIPTACCEPTED MANUSCRIPTAmelogenin 1 Amelogenin 2 D8S1179 1 D8S1179 2 D21S11 1 D21S11 2 D18S51 1 D18S51 2 X Y 13 13 27 29 12 14         D19S433 1 D19S433 2 THO1 1 THO1 2 FGA 1 FGA 2 CSF1PO 1 CSF1PO 2 13 14 6 8 23 26 12* 12*         D5S818 1 D5S818 2 D7S820 1 D7S820 2 D13S317 1 D13S317 2 TPOX 1 TPOX 2 11 12 12* 12* 13 13 8 11 * Peak falls below threshold to confidently score in 1 triplicate.          Blood group genotyping RhD RhC Rhc RhE Rhe Fy a Fy b Fy GATA pos neg pos neg pos pos pos neg         Jka Jkb K k  M N S  s neg pos neg pos pos pos neg pos         Do a Do b ABO      neg pos AA              HLA tissue typing HLA Class I Type HLA-A*02, A*31; B*13, B*40; C*03, C*06 HLA Class II Type HLA-DRB1*04, DRB1*07; DRB4*01; DQB1*02, DQB1*03 Comment B*40 is expressed serologically as B60, C*03 is expressed serologically as Cw10, DQB1*03 is expressed serologically as DQ8.  Acknowledgements  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.  

file:///data/core-remote/dit/data/elsevier/pdf/71f/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMTg3MzUwNjExNjMwMDE1MA==.pdf

Derivation of the clinical grade human embryonic stem cell line RCe017-A (RC-13)

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector

Crossref

Crossref

Edinburgh Research Explorer

Crossref

Elsevier - Publisher Connector

Directory of Open Access Journals

Edinburgh Research Explorer

Directory of Open Access Journals

Edinburgh Research Explorer