br Materials and methods br Acknowledgements

Materials and methods
Acknowledgements Mef2c-AHF-Cre mice were generously provided by Dr. Brian L. Black. We thank Mamen Martín and Sandra Rodríguez (Molecular Cytogenetics Unit, CNIO) for technical advice; and the Imaging Core Facility for the confocal analysis (CIMA, University of Navarra). We thank the “Pluripotency Group” at the Department of Cell Therapy (FIMA) for the daily input and support, especially to Xabier L. Aranguren, Juan R. Rodriguez, Adrian Ruiz-Villalba and Beatriz Pelacho. This work was supported by the “Ramón y Cajal” State Program, Ministry of Economy and Competitiveness (MINECO, RYC-2012-10981) to XCV; the “Retos de la Sociedad” State Program, MINECO (SAF2013-46142-R) to XCV; “Promoción del Talento y su Empleabilidad” State Program, MINECO (BES-2014-069226) to JLA; “Red de Terapia Celular”, Ministry of Health, Social Services and Equality-Institute of Health Carlos III (TERCEL ISCIII-RETIC RD12/0019/0031) to FP.
Resource table
Resource details A total of 9 embryos from in vitro congo red (IVF) and 97 embryos from preimplantation genetic diagnosis (PGD) were donated for research in accordance with the legal requirements of the country of origin by donors included in PGD program at Unidad de Gestión Clínica de Genética, Reproducción y Medicina Fetal (UGC) from University Hospital Virgen del Rocío. The donors gave written informed consent (Cortes JL et al., 2007 and Fernández et al., 2014). Human embryos were thawed using Thaw Kit 1TM de Vitrolife® and cultured using G2 medium (GIII series, Vitrolife®), inner cell mass (ICM) was mechanically isolated under stereomicroscope using 2 insulin syringe (25G) at “hatching blastocyst” stage (Ström et al., 2007) and plated onto mitomycin-C inactivated human newborn foreskin fibroblasts (hFFs), the resulting colonies displayed the typical morphology of hESCs (Fig. 1A) and are positive to alkaline phosphatase staining (Fig. 1B). The analysis of pluripotent markers was evaluated by RT–PCR, immunofluorescence and flow cytometry. Undifferentiated HVR1, HVR2 and HVR3 cells expressed Oct4, Sox2, Nanog and Telomerase (TERT) detected by RT–PCR (Fig. 1C), are positive to OCT4, SSEA4, TRA-1-60 and TRA-1-81 proteins assessed by immunostaining (Fig. 1D) and more than 90% of cells were positive to SEEA4 (Fig. 1E). The ability to differentiate into three germ layers was demonstrated by in vitro differentiation of embryoid bodies (EBs) and by in vivo teratoma formation after cells implantation into immune-deficient mice. Differentiated cells from HVR1, HVR2 and HVR3 expressed neuronal class III β-tubulin ectoderm marker (Tuj1), human cardiac troponin T mesoderm marker (cTnT) and human α-Fetoprotein endoderm marker (AFP) (Fig. 2A), while hematoxylin and eosin (H&E) and alcian blue contrastained sections from the teratomas indicated that the cell lines HVR1, HVR2 and HVR3 differentiated in vivo to all three germ layers (Fig. 2B). The karyotype analysis showed a stable karyotype 46,XY for HVR1 (Fig. 3A) and 46,XX for HVR3 (Fig. 3B), however HVR2 presented a karyotype with chromosome translocation 46,XY, t(9;15)(q34.3;q14) (Fig. 3C); this translocation has been also detected with FISH analysis (Fig. 3D).
Materials and methods
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Author disclosure statement
Acknowledgments This work was supported by a non-profit Foundation ‘Fundación Progreso y Salud’ of the Andalusian Regional Ministry of Health; Consejería de Economía y Conocimiento, Junta de Andalucía and Fondo Europeo de Desarrollo Regional (FEDER) (TCMR0021/06 and PI246-2008). Authors are supported by Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (FEDER) (RD12/0019/0028 and RD012/0036/0017; PI10/00964; PI11/02923 and PI14/01015); the Ministry of Health and Consumer Affairs (Advanced Therapies Program Grant TRA-120). Support from FSED and FAID allowed access to databanks. CIBERDEM and CIBERER are initiatives of the Instituto de Salud Carlos III.