Generation of KCL012 research grade human embryonic stem cell line carrying a mutation in the HTT gene

The KCL012 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomaldominantmutationaffectingonealleleoftheHTTgeneencodinghuntingtin(46trinucleotiderepeats; 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 and in vivo assays. The


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 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.

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).    5. Differentiation of three germ layers in vivo. Teratomas were encapsulated and did not invade surrounding tissue. Sections are counterstained with hematoxylin and eosin and specific stains are brown (immunohistochemistry) or light blue (Alcian blue). Germ layer markers: Alcian blue-PAS-stained cartilage and DES for mesoderm, TUBB3 and GFAP for ectoderm, GATA4 and AFP for endoderm. Positive immunostaining for complex IV type II marker confirms the human origin of the tumor. Scale bars are 100 μm.
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 details .

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