Generation of KCL013 research grade human embryonic stem cell line carrying a mutation in the HTT gene

The KCL013 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomaldominantmutationaffectingonealleleofthe HTT geneencodinghuntingtin(42trinucleotiderepeats; 17 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ -irradiated human fore- skin ﬁ broblasts.Boththederivationandcelllinepropagationwereperformedinananimalproduct-freeenviron-ment. Pluripotent state and differentiation potential were con ﬁ rmed by in vitro assays.


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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 KCL013 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. 3; Ilic et al., 2012). Differentiation potential into three germ layers was verified in vitro ( Fig. 4; Ilic et al., 2012).

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 form 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 was cultured as described Stephenson et al., 2012). Trophectoderm cells were removed mechanically from outgrowth (Ilic et al., 2007;Ilic et al., 2010). hESC colonies were expanded and cryopreserved at the third passage. Fig. 1. Genetic pedigree tree. The couple undergoing IVF had 12 embryos in this particular cycle. Three embryos were normal, whereas nine carried the mutation in HTT and were donated for research. We derived hESC lines from two of them. Fig. 2. A modal karyotype (in 19 cells) showed a normal male chromosome complement and banding pattern. In addition, one anomalous cellwas seen (45,XY,−19), believed to be the result of harvesting artifact.

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).
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.

Differentiation
Spontaneous differentiation into three germ layers was assessed in vitro and in vivo as described (Petrova et al., 2014;Stephenson et al., 2012).
Array comparative genomic hybridization (aCGH) aCGH was performed as described in detail .