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Materials and methods
Verification and authentication
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Materials and methods
Verification and authentication
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Resource details: Materials and methods
Verification and authentication Ethics/consents: Ethics approval for the project (‘Derivation of human embryonic stem purchase PD173074 from embryos identified through pre-implantation genetic diagnosis to be affected by known genetic conditions’) was obtained from the Genea Ethics Committee on 13 September 2005. Excess ART embryos were fully consented for stem cell derivation by all responsible people through an informed consent process (signed de-identified consent form can be provided upon request). Donors have received no payment or financial benefits for their donation. Genea080 has been derived from a donated, fully commercially consented embryo, originally created by assisted reproduction technology (ART) for the purpose of procreation. The embryo was identified through pre-implantation genetic diagnosis to be affected by a genetic mutation and was declared excess to reproductive needs. Derivation was performed under Australian National Health and Medical Research Council (NHMRC) licence 309710. This licence was issued to GENEA on 7 May 2007. More information about the licence can be obtained from the NHMRC webpage at http://www.nhmrc.gov.au/health-ethics/human-embryos-and-cloning/database-licences-authorising-use-excess-art-embryos. PGD analysis conclusion: Mutation; Compound heterozygous exon 55 deletion & c.15110dupA in NEB gene. Family tree; both parents are carriers. Nemaline Myopathy 2 (NEM2) affected. Morphology: The derived stem cell line, Genea080, morphologically displays adherent monolayer of compact cells in well-defined colonies with high nuclear to cytoplasmic ratio and prominent nucleoli. Genetic analysis: The cell line has been karyotype tested by CGH (Table 1, Supplementary Fig. 1), which demonstrated 46, XY karyotype, consistent with original derivation and pre-implantation genetic diagnosis (PGD). Analysis of STR markers showed allele pattern consistent with male genotype (Table 2, Supplementary Fig. 2). Pluripotency: GENEA080 is pluripotent by; Sterility: The cell line is tested and found negative for Mycoplasma and any visible contamination (Supplementary Fig. 3). The following are the supplementary data related to this article.
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Resource details RCe006-A (RC-2) was shown to be pluripotent by expression of the pluripotency markers Oct4, Nanog Tra-1-60 and Tra-1-81, but not the differentiation marker SSEA-1 using immunocytochemistry (Table 1, Fig. 1). By flow cytometric analysis, expression of the pluripotency makers SSEA-4, Tra-1-60 and Tra-1-81 was 81.8%, 55.0% and 43.6%, respectively, whereas low expression of the differentiation marker SSEA-1 (2.3%), was observed at passage 6 (Fig. 2). Differentiation to the three germ layers, endoderm, ectoderm and mesoderm, was demonstrated using embryoid body formation with all three germ layers present as shown by expression of α-fetoprotein, β-tubulin and muscle actin (Fig. 3). A microsatellite PCR profile has been obtained for the cell line, and HLA Class I and II typing is available (Table 2). Blood group genotyping gave the blood group BO1 (Table 2).
Verification and authentication The cell line was analysed for genome stability by G-banding (Fig. 4) and showed an abnormal 47XY, +12 male genotype in all 20 cells analysed. The cell line is free from mycoplasma contamination as determined by RC-qPCR. Microsatellite PCR DNA profiling for cell identity is available.
Materials and methods
Acknowledgements Research culminating in the derivation of this line was funded by a grant from Scottish Enterprise Economic Development Agency (PM07321) to PDS, MB, and AC.