Generation of KCL024 research grade human embryonic stem cell line carrying a mutation in NF1 gene

The KCL024 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation in the NF1 gene encoding neuro ﬁ bromin (c.3739 – 3742 Δ TTTG). Mutations in this gene have been linked to neuro ﬁ bromatosis type 1, juvenile myelomonocytic leukemia and Watson syn- drome. The ICM was isolated using laser microsurgery and plated on γ -irradiated human foreskin ﬁ broblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripo- tent state and differentiation potential were con ﬁ rmed by in vitro assays.

We generated KCL024 clinical grade hESC line following protocols, established previously Stephenson et al., 2012). The expression of the pluripotency markers was tested after freeze/thaw cycle. Differentiation potential into three germ layers was verified in vitro.

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 re-sign; the whole process is done with the version that was given them initially. The PIS/ consent documents (PGD-V.8) were created on Jul. 01, 2010. HFEA Code of Practice that was in effect at the time of document creation: Edition 8 -R.2 (http://www.hfea.gov.uk/2999.html). The donor couple signed the consent on Oct. 28, 2010. HFEA Code of Practice that was in effect at the time of donor signature: Edition 8 -R.2. HFEA Code of Practice Edition 8 -R.2 was in effect 07 Apr. 2010-Apr. 06, 2011.

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). 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 Stephenson et al., 2012).

Differentiation
Spontaneous differentiation into three germ layers was assessed in vitro as described Stephenson et al., 2012;Petrova et al., 2014).

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