Generation of KCL031 clinical grade human embryonic stem cell line

TheKCL031 human embryonicstemcelllinewasderivedfromanormalhealthyblastocystdonatedfor 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 and in vivo assays. © 2016 The Authors. Elsevier B.V. This is an the CC license (http://creativecommons.org/licenses/by/4.0/).

Molecular karyotyping using array comparative genomic hybridization aCGH identified deletion at 7q22.3 (105,465,516,305). Whole-genome single nucleotide polymorphism (SNP) array analysis detected loss at 8q24.23 (136,718,837,768) (Canham et al., 2015). The gain contains no genes and it has been also reported previously to occur in healthy individuals from worldwide population (Macdonald et al., 2014). Estimated frequency in the human population is 3.85% (Canham et al., 2015).
Donors were tested negative for Human Immunodeficiency Virus 1 (HIV1), Hepatitis B (HepB, HCB) and C Virus (HepC, HCV). We did not retest the line.
We also generated research grade of KCL031 line that is adapted to feeder-free conditions.

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. The PIS/consent documents (FRO-V.5) were created on Aug. 10, 2007. HFEA Code of Practice that was in effect at the time of document creation: Edition 7 -R.1 (http://www.hfea.gov.uk/2999.html). The donor

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 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
Pluripotency in vitro 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). Targeted differentiation in cardiomyocytes followed the protocols described earlier (Laflamme et al., 2007;Jacquet et al., 2015). Fig. 2. Differentiation of three germ layers in vitro is confirmed by detection of markers: smooth muscle actin (red) for mesoderm, β-III tubulin (red) for ectoderm and α-fetoprotein (red) for endoderm. Nuclei are visualized with Hoechst 33,342 (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.

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).

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