Generation of KCL027 research grade human embryonic stem cell line carrying a mutation in the HTT gene

The KCL027 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomaldominantmutationaffectingonealleleofthe HTT geneencodinghuntingtin(43trinucleotiderepeats; 21 for the normal allele). 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. Pluripotent state and differentiation potential were con ﬁ rmed by in vitro and in vivo 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/).

The KCL027 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele). 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. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.  We generated KCL027 research 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;Jacquet et al., 2015). Differentiation potential into three germ layers was verified in vitro (Figs. 3 and 5; Jacquet et al., 2015) and in vivo (Fig. 4;Jacquet et al., 2015).

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). 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 (Jacquet et al., 2015). Targeted differentiation in cardiomyocytes followed the protocols described earlier (Jacquet et al., 2015;Laflamme et al., 2007).
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 .

HLA typing
HLA-A, -B and -DRB1 typing was performed with a PCR sequencespecific oligonucleotide probe (SSOP; Luminex, Austin, TX, USA) 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. (continued) hybridization protocol at the certified Clinical Transplantation Laboratory, Guy's and St Thomas' NHS Foundation Trust and Serco Plc. (GSTS) Pathology (Guy's Hospital, London, UK) as described (Jacquet et al., 2013).

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

Acknowledgments
This work was supported by the UK Medical Research Council grants G0701172 and G0801061. We thank Dr. Yacoub Khalaf, Director of the Assisted Conception Unit of Guy's and St Thomas' NHS Foundation Trust and his staff for supporting the research program. We are especially indebted to Prof Peter Braude and to the patients who donated embryos.   3. Differentiation of three germ layers in vitro is confirmed by detection of markers: smooth muscle actin (ACTA2, red) for mesoderm, β-III tubulin (TUBB3, red) for ectoderm and αfetoprotein (AFP, red) for endoderm. Nuclei are visualized with Hoechst 33342 (blue). Scale bar, 100 μm. Fig. 4. 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). Germ layer marker: DES for mesoderm, TUBB3 for ectoderm, and GATA4 for endoderm. Scale bars are 100 μm.