Generation of KCL029 research grade human embryonic stem cell line carrying a mutation in WAS gene

The KCL029 human embryonic stem cell line was derived from an embryo donated for research that carried a c.814 T N C mutation in the WAS gene, which is linked to the Wiskott-Aldrich syndrome, a rare, inherited, X-linked, recessive disease characterized by immune dysregulation and microthrombocytopenia. The line is also carrier for a mutation p.N1152H in the gene encoding the cystic ﬁ brosis transmembrane conductance regulator CFTR. The ICMwas isolatedusing laser microsurgeryandplatedon γ -irradiatedhumanforeskin ﬁ broblasts.Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were con ﬁ rmed by in vitro assays. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).


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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 We generated KCL029 clinical grade hESC line following protocols, established previously Stephenson et al., 2012). The expression of the pluripotency markers was tested after freeze/thaw cycle (Fig. 2). Differentiation potential into three germ layers was verified in vitro (Fig. 3).

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.

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 and in vivo as described (Petrova et al., 2014;Ilic et al., 2012;Stephenson et al., 2012).  Fig. 1. Genetic pedigree tree. Both parents were carrying mutation in CFTR gene. Maternal CFTR mutation was p.N1152H and paternal ΔF508 and exon 2 deletion. In addition, the mother carried c.814 T N C mutation in WAS gene. The embryos were first genotyped for mutation in CFTR gene and then only normal and career embryos were assessed further for a mutation in WAS gene. Ab, abnormal; Af, affected; C, carrier; NA, non-applicable; ND, not determined.

Genotyping
DNA was extracted from hESC 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 .

Author disclosure statement
There are no competing financial interests in this study. 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 and hES cell colonies, whereas AP activity (green) is detected only in hES cells. Scale bar, 100 μm. 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 (blue). Scale bar, 100 μm.