Generation of KCL034 clinical grade human embryonic stem cell line

research. 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 and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were con ﬁ rmed by in vitro assays. The line was also validated for sterility, speci ﬁ c and non-speci ﬁ c human pathogens. The the CC


Stem Cell Research
Molecular karyotyping identified a gain on chromosome 6p22.1. The gain on chromosome 5p14.3 containing the following genes: HIST1H2BL, HIST1H2AI, HIST1H3H, HIST1H2AJ, HIST1H2BM, HIST1H4J, HIST1H4K, HIST1H2AK, HIST1H2BN, HIST1H2AL, HIST1H1B, HIST1H3I, HIST1H4L, HIST1H3J, HIST1H2AM, HIST1H2BO, OR2B2 and OR2B6 (Canham et al., 2015). The 330.8 kb gain starts at bp 27627265 and ends at bp 27958049 as referred to Human Genome Build 38. This duplication that contained part of the Histone 1 gene cluster was not fully present on the database of genomic variants (DGV; http://dgv.tcag.ca), which has collected structural variations in more than 14,000 healthy individuals from worldwide population (MacDonald et al., 2014). It is probable that this gain represents a benign event as other histone clusters have been shown to be preferentially duplicated during evolution (Canham et al., 2015;Braastad et al., 2004).
We also generated research grade of KCL034 line that is adapted to feeder-free conditions. (continued) 3. Materials and methods

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 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. hES 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;Stephenson Fig. 2. 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, 50 μm. Fig. 3. 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 (adjacent section of the one stained for desmin). Scale bars are 100 μm. et al., 2012). Targeted differentiation in cardiomyocytes (Jacquet et al., 2015;Laflamme et al., 2007) and definitive endoderm (Cvoro et al., 2015;Cheng et al., 2012), keratinocytes (Petrova et al., 2014), followed the protocols described earlier. Nuclei are visualized with Hoechst 33342.

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) 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). HLA typing was also performed independently by other group (Canham et al., 2015).

Validation for sterility and specific and non-specific human pathogens
Validation for sterility and specific and non-specific human pathogens was performed as described (Devito et al., 2014). All validation studies were conducted by SGS Vitrology (Glasgow, U.K., http://www. sgs.com), in compliance with the principles of GMP as set out in Directive 2003/94/EC for medicinal products for human use (Directive 2003/94/EC, 2003 and 91/412/EEC for veterinary medicinal products (Directive 91/412/EEC, 1991).
Sterility testing was performed in accordance with the current requirements of the European Pharmacopoeia, Section 2.6.1 Sterility, U.S. Pharmacopeia, 71. Sterility Tests, and International Conference on Harmonisation Topic Q5D guidelines.
Mycoplasma testing was performed in accordance with the current requirements of the European Pharmacopoeia, Section 2.6.7, Mycoplasmas.
All PCR-based assays used were compliant with the current edition of the European Pharmacopoeia, 2.6.21, Nucleic Acid Amplification Techniques.

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