Generation of KCL033 clinical grade human embryonic stem cell line

The KCL033 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. The line was also validated for sterility and specific and non-specific human pathogens.


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Stem Cell Research j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / s c r

Resource details
We generated KCL033 clinical grade hESC line following protocols, established previously 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).
Molecular karyotyping identified a novel 2.4 Mb gain on chromosome 5p14.3 and a gain on chromosome 12p11.21, which was also found in KCL040.
The gain on chromosome 5p14.3 containing a single gene, CDH18 (Cadherin-18), was also present in one of two sibling cell lines, KCL032, but not in KCL034, a third sibling. A duplication of this size has not been reported to date, but its presence in two sibling hESC lines strongly suggests that it was inherited from one of the parents rather than by acquisition during hESC derivation and culture (Canham et al., 2015). The 2498.8 bp gain starts at bp 19086546 and ends at bp 21585311 as referred to Human Genome Build 38.
The gain on chromosome 12p11.21 contains no genes and it has been also reported in at least 14 submissions at Database of Genomic Variants (DGV; http://dgv.tcag.ca), which has collected structural variations in more than 14,000 healthy individuals from worldwide population (MacDonald et al., 2014). Estimated frequency in the human population is 4.70% (Canham et al., 2015).
We also generated research grade of KCL033 line that is adapted to feeder-free conditions.

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 resign; the whole process is done with the version that was given them initially. The PIS/consent documents (FRO-V.6) were created on Dec.

Embryo culture and micromanipulation
Embryo culture and laser-assisted dissection of inner cell mass (ICM) were carried out as previously described in detail 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).

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 with immunostaining for pluripotency markers as described Stephenson et al., 2012).

Differentiation
Spontaneous differentiation into three germ layers was assessed in vitro and in vivo as described 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 . Allele sizes were recorded to give a unique fingerprint of each cell line.
Array comparative genomic hybridization (aCGH) aCGH was performed as described in detail .
Whole-genome single nucleotide polymorphism (SNP) array SNP array was performed as described in detail (Canham et al., 2015).

HLA typing
HLA-A, -B and -DRB1 typing was performed with a PCR sequencespecific 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). HLA typing was also performed independently by other group (Canham et al., 2015).

Validation for sterility and specific and non-specific human pathogens
Validation for sterility and specific and non-specific human pathogens was performed as described . All validation studies were conducted by SGS Vitrology (Glasgow, U.K., http:// www.sgs.com), in compliance with the principles of GMP as set out in Directive 2003/94/EC for medicinal products for human use (Directive 2003/94/EC, 2003 and 91/412/EEC for veterinary medicinal products (Directive 91/412/EEC, 1991).
Sterility testing was performed in accordance with the current requirements of the European Pharmacopoeia, Section 2.6.1 Sterility, U.S. Pharmacopeia, 71. Sterility Tests, and International Conference on Harmonisation Topic Q5D guidelines.
Mycoplasma testing was performed in accordance with the current requirements of the European Pharmacopoeia, Section 2.6.7, Mycoplasmas.
All PCR-based assays used were compliant with the current edition of the European Pharmacopoeia, 2.6.21, Nucleic Acid Amplification Techniques.

Author disclosure statement
There are no competing financial interests in this study.