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Stem Cell Res. 2016 March; 16(2): 345–348.
PMCID: PMC4823766

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

Abstract

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.

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We generated KCL036 research grade hESC line following protocols established previously (Ilic et al., 2012, Stephenson et al., 2012, Jacquet et al., 2013). 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 (Fig. 3 and Fig. 5; Jacquet et al., 2015) and in vivo (Fig. 4; Jacquet et al., 2015).

Fig. 2
Expression of pluripotency markers. Pluripotency is confirmed by immunostaining (Oct4, Nanog, TRA-1-60, TRA-1-81) and alkaline phosphatase (AP) activity assay. Scale bar, 20 μm.
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 ...
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, ...
Fig. 5
TNNT2 (green) immunostaining on day 30 of cardiac differentiation. Nuclei are visualized with Hoechst 33342 (blue). Scale bar, 50 μm.

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).

Materials and methods

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. The PIS/consent documents (PGD-V.9) were created on Feb. 09, 2011. HFEA Code of Practice that was in effect at the time of document creation: Edition 8—R.2 (http://www.hfea.gov.uk/2999.html). The donor couple signed the consent on Jul. 21, 2011. HFEA Code of Practice that was in effect at the time of donor signature: Edition 8—R.3. HFEA Code of Practice Edition 8—R.2 was in effect: Apr. 07, 2010–Apr. 06, 2011. HFEA Code of Practice Edition 8—R.3 was in effect: Apr. 07, 2011–Oct. 01, 2011.

Embryo culture and micromanipulation

Embryo culture and laser-assisted dissection of inner cell mass (ICM) were carried out as previously described in details (Ilic et al., 2012, 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 (Ilic et al., 2012, 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 (Ilic et al., 2012, Stephenson et al., 2012).

Differentiation

Spontaneous differentiation into three germ layers was assessed in vitro and in vivo (Jacquet et al., 2015) as described (Ilic et al., 2012, Stephenson et al., 2012, Petrova et al., 2014). 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 (Ilic et al., 2012). 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 (Ilic et al., 2012).

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.

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.

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.

References

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