By qPCR we confirmed that

By qPCR we confirmed that DLL4 mRNA levels increase dramatically as the control shSCR cells undergo differentiation, but in shDLL4 cells the DLL4 mRNA is efficiently suppressed (Fig. 2E, left panel). To confirm that DLL4 mRNA suppression had a functional effect we analyzed the mRNA levels of two well known targets of DLL4-mediated Notch activation, HEY1 and HEY2 (Benedito et al., 2008; Gale et al., 2004). We saw that in control shSCR cells both HEY1 and HEY2 mRNAs are induced during differentiation following a similar pattern as DLL4 mRNA. However, in shDLL4 cells both genes are induced at a lower level, especially at day 8 of differentiation, when DLL4 suppression is more acute (Fig. 2E, center and right panels).
Materials and methods
Resource table. Resource details Human ESC culture and processing were performed in a grade A tissue culture cabinet in a grade B clean room environment monitored for particulate and microbiological contamination during cell processing in accordance with Rules and Guidance for Pharmaceutical Manufacturers and Distributors — The Orange Guide, compiled by the UK Medicines Healthcare Products Regulatory Authority (go to: https://www.gov.uk/guidance/good-manufacturing-practice-and-good-distribution-practice). Accordingly, the facility was operating under a mature Quality Management System, compliant with ISO9001:2008 standards. HESC derivation was performed under licensure from the UK HFEA (R0136 to centre 0202) and HTA (Licensing Number 22631). By flow cytometry, RCe019-A (RC-15) expressed the pluripotency makers Oct-4, Tra-1-60 and SSEA-4 (87.4%, 80.7% and 94.6%, respectively), whereas low expression of the differentiation marker SSEA-1 (2.4%) was observed (Fig. 1, Fig. 2). Differentiation to the three germ layers, endoderm, ectoderm and mesoderm, was demonstrated using embryoid body formation in vitro, and expression of the germ layer markers α-fetoprotein, β-tubulin and muscle pyruvate dehydrogenase kinase inhibitor was observed (Fig. 3). A microsatellite PCR profile has been obtained for the cell line, and HLA Class I and II typing is available (Table 1). Blood group genotyping gave the blood group O1O1 (Table 1).
Materials and methods
Acknowledgements Research culminating in the derivation of this line was funded by a grant (PM07321) from the Scottish Enterprise Economic Development Agency to PDS, MB, and AC.
Resource table
Resource details Human ESC culture and processing were performed in a grade A tissue culture cabinet in a grade B clean room environment monitored for particulate and microbiological contamination during cell processing in accordance with Rules and Guidance for Pharmaceutical Manufacturers and Distributors — The Orange Guide, compiled by the UK Medicines Healthcare Products Regulatory Authority (go to: https://www.gov.uk/guidance/good-manufacturing-practice-and-good-distribution-practice). Accordingly, the facility was operating under a mature Quality Management System, compliant with ISO9001:2008 standards. hESC derivation was performed under licensure from the UK HFEA (R0136 to centre 0202) and HTA (Licensing Number 22631). By flow cytometry, RCe017-A (RC-13) expressed the pluripotency makers Oct-4, Tra-1-60 and SSEA-4 (80.8%, 88.3% and 79.6%, respectively), whereas low expression of the differentiation marker SSEA-1 (0.7%) was observed (Figs. 1 and 2). Differentiation to the three germ layers, endoderm, ectoderm and mesoderm, was demonstrated using embryoid body formation in vitro, and expression of the germ layer markers α-fetoprotein, β-tubulin and muscle actin was observed (Fig. 3). A microsatellite PCR profile has been obtained for the cell line, and HLA Class I and II typing is available (Table 1). Blood group genotyping gave the blood group AA (Table 1).
Verification and authentication The cell line was analysed for genome stability by G-banding and showed a mixed male genotype. Trisomy 12 (47XY, +12) was present in 26 of 30 cells analysed, but a subpopulation (4 of 30 cells) has an additional copy of chromosome 1 (48XY, +1, +12) (Fig. 4). The cell line is free from mycoplasma contamination as determined by RT-qPCR.