Abstract
The KCL038 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. 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 and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays.
1. Resource table
| Name of stem cell line | KCL038 |
| Institution | King's College London, London UK |
| Derivation team | Neli Kadeva, Victoria Wood, Glenda Cornwell, Stefano Codognotto, Emma Stephenson |
| Contact person and email | Dusko Ilic, email: dusko.ilic@kcl.ac.uk |
| Date archived/stock date | Dec 06, 2011 |
| Type of resource | Biological reagent: cell line |
| Sub-type | Human pluripotent stem cell line |
| Origin | Human embryo |
| Key marker expression | Pluripotent stem cell markers: NANOG, OCT4, TRA-1-60, TRA-1-81, alkaline phosphatase (AP) activity |
| Authentication | Identity and purity of line confirmed |
| Link to related literature (direct URL links and full references) |
http://www.ncbi.nlm.nih.gov/pubmed/22029654
http://www.ncbi.nlm.nih.gov/pubmed/22722371 |
| Information in public databases | KCL038 is a National Institutes of Health (NIH) registered hESC line NIH Registration Number: NIHhESC-14-0265 http://grants.nih.gov/stem_cells/registry/current.htm?id = 674 |
| Ethics | The hESC line KCL038 is derived under license from the UK Human Fertilisation and Embryology Authority (research licence 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. |
2. Resource details
| Consent signed | Jan 12, 2010 |
| Embryo thawed | Oct 24, 2011 |
| UK Stem Cell Bank Deposit Approval | Reference: SCSC12-37 |
| Sex | Male 46, XY |
| Grade | Clinical |
| Disease status | Healthy/Unaffected |
| Karyotype (aCGH) | Deletion of approximately 683 kb from band in the long arm of chromosome 5q13.2. The deletion represents benign copy number variant. |
| SNP Array | No abnormalities detected Canham et al. (2015) |
| DNA fingerprint | Allele sizes (in bp) of 16 microsatellite markers specific for chromosomes 13, 18 and 21 Jacquet et al. (2013) |
| HLA typing | HLA-A 03,11; B 07,15; Bw 06; C 03,07; DRB1 14,15; DRB3 02; DQB1 05,06 Jacquet et al. (2013), Canham et al. (2015) |
| Viability testing | Pass |
| Mycoplasma | Negative |
| Sterility | Pass |
| Pluripotent markers (immunostaining) (Fig. 1) |
NANOG, OCT4, TRA-1-60, TRA-1-81, AP activity |
| Three germ layers differentiation in vitro (immunostaining) (Fig. 2) |
Endoderm: AFP Ectoderm: TUBB3 (tubulin, beta 3 class III) Mesoderm: ACTA2 (actin, alpha 2, smooth muscle) |
| Sibling lines available | No |
We generated KCL038 clinical grade hESC line following protocols, established previously (Ilic et al., 2012, Stephenson et al., 2012), and now adapted to cGMP conditions. The expression of the pluripotency markers was tested after freeze/thaw cycle (Fig. 1). Differentiation potential into three germ layers was verified in vitro (Fig. 2).
Fig. 1.
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, 10 μm.
Fig. 2.
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 33342 (blue). Scale bar, 50 μm.
Molecular karyotyping identified deletion of approximately 683 kb from band q13.2 in the long arm of chromosome 5 (69,705,561 – 70,388,844). The deletion represents benign copy number variant.
The KCL038 line is confirmed by PCR to be negative for Human Immunodeficiency Virus 1 (HIV1), and Hepatitis C Virus (HepC, HCV). We did not retest the line.
We also generated research grade of KCL038 line that is adapted to feeder-free conditions.
3. Materials and methods
3.1. 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 meantime new versions of PIS/consent are implemented, we do not send these to the patients or ask them to re-sign; the whole process is done with the version that was given them initially. The PIS/consent documents (FRO-V.6) were created on Dec 18, 2008. HFEA Code of Practice that was in effect at the time of document creation: Edition 7 – R.4 (http://www.hfea.gov.uk/2999.html). The donor couple signed the consent on Jan 12, 2010. 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.4 was in effect: 02 Oct 2008–30 Sep 2009, whereas 8—R.1 was in effect: 01 Oct 2009–06 Apr 2010.
3.2. 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).
3.3. 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). hES colonies were expanded and cryopreserved at the third passage.
3.4. 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).
3.5. 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).
3.6. 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.
3.7. Array comparative genomic hybridization (aCGH)
aCGH was performed as described in details (Ilic et al., 2012).
3.8. SNP array
SNP array was performed as described in details (Canham et al., 2015).
3.9. HLA typing
HLA-A, -B and -DRB1 typing was performed with a PCR sequence-specific oligonucleotide probe (SSOP; Luminex, Austin, TX, USA) hybridization protocol at the certified Clinical Transplantation Laboratory, Guy's and St Thomas' NHS Foundation Trust and Serco Plc. (GSTS) Pathology (Guy's Hospital, London, UK) as described (Jacquet et al., 2013).
4. Author disclosure statement
There are no competing financial interests in this study.
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 patients who donated embryos.
References
- Canham A., Van Deusen A., Brison D.R., De Sousa P., Downie J., Devito L., Hewitt Z.A., Ilic D., Kimber S.J., Moore H.D., Murray H., Kunath T. The molecular karyotype of 25 clinical-grade human embryonic stem cells lines. Sci. Rep. 2015;5:17258. doi: 10.1038/srep17258. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ilic D., Genbacev O., Krtolica A. Derivation of hESC from intact blastocysts. Curr. Protoc. Stem Cell Biol. 2007 doi: 10.1002/9780470151808.sc01a02s1. (Chapter 1: Unit 1A.2) [DOI] [PubMed] [Google Scholar]
- 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. doi: 10.1089/scd.2009.0136. [DOI] [PubMed] [Google Scholar]
- 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. doi: 10.3109/14653249.2011.623692. [DOI] [PubMed] [Google Scholar]
- Jacquet L., Stephenson E., Collins R., Patel H., Trussler J., Al-Bedaery R., Renwick P., Ogilvie C., Vaughan R., Ilic D. Strategy for the creation of clinical grade hESC line banks that HLA-match a target population. EMBO Mol. Med. 2013;5(1):10–17. doi: 10.1002/emmm.201201973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. doi: 10.1038/nprot.2012.080. [DOI] [PubMed] [Google Scholar]