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Stem Cell Res. 2016 March; 16(2): 342–344.
PMCID: PMC4823665

Generation of KCL018 research grade human embryonic stem cell line carrying a mutation in the DMPK gene

Abstract

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.

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Resource details

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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 methods

Consenting 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).

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Author disclosure statement

There are no competing financial interests in this study.

Fig. 1
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.
Fig. 2
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 ...
Fig. 3
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 ...

Acknowledgments

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.

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. Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditions. Cytotherapy. 2012;14(1):122–128. [PubMed]
Ilic D., Caceres E., Lu S., Julian P., Foulk R., Krtolica A. Effect of karyotype on successful human embryonic stem cell derivation. Stem Cells Dev. 2010;19(1):39–46. [PubMed]
Ilic D., Genbacev O., Krtolica A. Derivation of hESC from intact blastocysts. Curr. Protoc. Stem Cell Biol. 2007 (Chapter 1: Unit 1 A.2) [PubMed]
Stephenson E., Jacquet L., Miere C., Wood V., Kadeva N., Cornwell G., Codognotto S., Dajani Y., Braude P., Ilic D. Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product-free environment. Nat. Protoc. 2012;7(7):1366–1381. [PubMed]