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Generation of KCL032 clinical grade human embryonic stem cell line

The KCL032 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.


Resource
We generated KCL032 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, containing a single gene, CDH18 (Cadherin-18), that was also present in one of two sibling cell lines, KCL033, 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 19,086,546 and ends at bp 21,585,311 as referred to Human Genome Build 38.
We also generated research grade of KCL032 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. For 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 (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 details 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 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.

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).

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 details .

SNP array
SNP array was performed as described in details (Canham et al., 2015).

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