Generation of KCL036 research grade human embryonic stem cell line carrying a mutation in the HTT gene

The KCL036 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomaldominantmutationaffectingonealleleofthe HTT geneencodinghuntingtin(38trinucleotiderepeats; 14 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 Authors. Elsevier B.V.

The KCL036 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 (38 trinucleotide repeats; 14 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.   (Ilic et al., 2015) Karyotype (aCGH) Decreased copy number at 2q37.3 (242,930,948,040) and at 3q25.1 (151,368,542,568). The imbalances are considered to be benign copy number variants. The chromosome 3 imbalance was not called by software.
Validation for sterility and specific and non-specific human pathogens confirmed that the cells in Master Bank were sterile, mycoplasma-free, and negative for human immunodeficiency virus 1 (HIV-1), Human T-lymphotropic virus type 1 (HTLV-1), hepatitis B and C (HBV and HCV), human herpes simplex virus HHV-4 (Epstein-Barr virus, EBV), and human cytomegalovirus (hCMV).
We also generated research grade of KCL036 line that is adapted to feeder-free conditions (Jacquet et al., 2015).

Consenting process
We distribute patient information sheets (PIS) and consent forms 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 3 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. Fig. 1. Genetic pedigree tree. The couple undergoing IVF had 16 embryos in this particular cycle. Three embryos were normal, whereas those that carried the mutation in HTT were donated for research. We derived hESC lines from two of them.
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 .

Special pathology
The Doctors Laboratory London (UK) tested the line for HIV1, HepB, HepC, CMV, and EBV by PCR.

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. Fig. 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 α-fetoprotein (AFP, red) for endoderm. Nuclei are visualized with Hoechst 33342 (blue). Scale bar, 25 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 AFP for endoderm. Scale bars are 100 μm.