The KCL018 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the DMPK gene encoding the dystrophia myotonica protein kinase (2200 trinucleotide repeats; 14 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.
Resource table
Name of stem cell line
KCL018
Institution
King's College London, London UK
Derivation team
Neli Kadeva, Victoria Wood, Glenda Cornwell, Stefano Codognotto, Emma Stephenson
Link to related literature (direct URL links and full references)
Ilic, D., Stephenson, E., Wood, V., Jacquet, L., Stevenson, D., Petrova, A., Kadeva, N., Codognotto, S., Patel, H., Semple, M., Cornwell, G., Ogilvie, C., Braude, P., 2012. Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditions. Cytotherapy. 14 (1), 122–128.
KCL018 is a National Institutes of Health (NIH) registered hESC lineNIH Registration Number: 0218NIH Approval Number: NIHhESC-13-0218http://grants.nih.gov/stem_cells/registry/current.htm?id=658
Ethics
The hESC line KCL018 is derived under license from the UK Human Fertilisation and Embryology Authority (research license numbers: R0075 and R0133) and also has local ethical approval (UK National Health Service Research Ethics Committee Reference: 06/Q0702/90).Informed consent was obtained from all subjects and the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the NIH Belmont Report. No financial inducements are offered for donation.
Resource details
Consent signed
Aug 12, 2009
Embryo thawed
Aug 23, 2009
UK Stem Cell Bank Deposit Approval
Sep 23, 2010Reference: SCSC10-30
Sex
Female 46, XX
Grade
Research
Disease status(Fig. 1)
Mutation affecting one allele of the DMPK gene encoding dystrophia myotonica protein kinase (~ 2200 CTG repeats; 14 for the normal allele) associated with Myotonic dystrophy Type 1 (Ilic et al., 2012)
Karyotype (aCGH)
No copy number changes detected
DNA fingerprint
Allele sizes (in bp) of 17 microsatellite markers specific for chromosomes 13, 18 and 21 (Ilic et al., 2012)
Viability testing
Pass
Pluripotent markers(immunostaining)(Fig. 2)
NANOG, OCT4, TRA-1-60, TRA-1-81, AP activity (Ilic et al., 2012)
Three germ layers differentiation in vitro(immunostaining)(Fig. 3)
Endoderm: AFP (α-fetoprotein); Ectoderm: TUBB3 (tubulin, β3 class III); Mesoderm: ACTA2 (actin, α2, smooth muscle) (Ilic et al., 2012)
Sibling lines available
No
We generated KCL018 clinical grade hESC line following protocols, established previously (Ilic et al., 2012, Stephenson et al., 2012). The expression of the pluripotency markers was tested after freeze/thaw cycle (Ilic et al., 2012). Differentiation potential into three germ layers was verified in vitro (Ilic et al., 2012).
Materials and methodsConsenting process
We distribute Patient Information Sheet (PIS) and consent form to the in vitro fertilization (IVF) patients if they opted to donate to research embryos that were stored for 5 or 10 years. They mail signed consent back to us and that might be months after the PIS and consent were mailed to them. If in the meantime new versions of PIS/consent are implemented, we do not send these to the patients or ask them to resign; the whole process is done with the version that was given them initially. The PIS/consent documents (PGD-V.6) were created on Aug. 10, 2007. HFEA Code of Practice that was in effect at the time of document creation: Edition 7 — R.1 (http://www.hfea.gov.uk/2999.html). The donor couple signed the consent on Oct. 15, 2009. HFEA Code of Practice that was in effect at the time of donor signature: Edition 8 — R.1. HFEA Code of Practice Edition 7 — R.1 was in effect until Dec. 09, 2007 and Edition 8 — R.1 was in effect: Oct. 01, 2009–Apr. 06, 2010.
Embryo culture and micromanipulation
Embryo culture and laser-assisted dissection of inner cell mass (ICM) were carried out as previously described in details (Ilic et al., 2012, Stephenson et al., 2012). The cellular area containing the ICM was then washed and transferred to plates containing mitotically inactivated human neonatal foreskin fibroblasts (HFF).
Cell culture
ICM plated on mitotically inactivated HFF were cultured as described (Ilic et al., 2012, Stephenson et al., 2012). TE cells were removed mechanically from outgrowth (Ilic et al., 2007, Ilic et al., 2010). hESC colonies were expanded and cryopreserved at the third passage.
Viability test
Straws with the earliest frozen passage (p. 2–3) are thawed and new colonies are counted three days later. These colonies are then expanded up to passage 8, at which point cells were part frozen and part subjected to standard battery of tests (pluripotency markers, in vitro and in vivo differentiation capability, genetics, sterility, mycoplasma).
Pluripotency markers
Pluripotency was assessed using two different techniques: enzymatic activity assay [alkaline phosphatase (AP) assay] and immunostaining as described (Ilic et al., 2012, Stephenson et al., 2012).
Genotyping
DNA was extracted from hES cell cultures using a Chemagen DNA extraction robot according to the manufacturer's instructions. Amplification of polymorphic microsatellite markers was carried out as described (Ilic et al., 2012). Allele sizes were recorded to give a unique fingerprint of each cell line.
Array comparative genomic hybridization (aCGH)
aCGH was performed as described in details (Ilic et al., 2012).
Chr
Marker
Allele 1
Allele 2
13
D13S252
299
299
D13S305
443
458
D13S325
289
297
D13S628
429
450
D13S634
397
417
18
D18S386
383
386
D18S390
372
372
D18S391
209
217
D18S535
482
486
D18S819
408
424
D18S976
479
479
D18S978
219
219
21
D21S11
244
251
D21S1409
216
224
D21S1411
308
308
D21S1435
184
188
D21S1437
331
335
Author disclosure statement
There are no competing financial interests in this study.
ReferencesIlicD.StephensonE.WoodV.JacquetL.StevensonD.PetrovaA.KadevaN.CodognottoS.PatelH.SempleM.CornwellG.OgilvieC.BraudeP.Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditionsCytotherapy141201212212822029654IlicD.CaceresE.LuS.JulianP.FoulkR.KrtolicaA.Effect of karyotype on successful human embryonic stem cell derivationStem Cells Dev.1912010394619485710IlicD.GenbacevO.KrtolicaA.Derivation of hESC from intact blastocystsCurr. Protoc. Stem Cell Biol.2007(Chapter 1: Unit 1 A.2)StephensonE.JacquetL.MiereC.WoodV.KadevaN.CornwellG.CodognottoS.DajaniY.BraudeP.IlicD.Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product-free environmentNat. Protoc.7720121366138122722371Acknowledgments
This work was supported by the UK Medical Research Council grants G0701172 and G0801061. We thank Dr. Yacoub Khalaf, Director of the Assisted Conception Unit of Guy's and St Thomas' NHS Foundation Trust and his staff for supporting the research program. We are especially indebted to Prof Peter Braude and to the patients who donated embryos.
Genetic pedigree tree. The couple undergoing IVF had only on embryo in this particular cycle. The embryo carried the mutation and was donated for research.
Expression of pluripotency markers. Pluripotency is confirmed by immunostaining (Oct4, Nanog, TRA-1-60, TRA-1-81) and alkaline phosphatase (AP) activity assay. Actin stress fibers, visualized with rhodamine-phalloidin (red), are present in both feeders and hES cell colonies, whereas AP activity (green) is detected only in hES cells. Scale bar, 50 μm.
Differentiation of three germ layers in vitro is confirmed by detection of markers: smooth muscle actin (red) for mesoderm, β-III tubulin (red) for ectoderm and α-fetoprotein (red) for endoderm. Nuclei are visualized with Hoechst 33,342 (blue). Scale bar, 50 μm.