title: Optimized protocol for derivation of human embryonic stem cell lines.
abstract: For the past 12 years, the biology and applications of human embryonic stem cells (hESCs) have received great attention from the scientific community. Derivatives of the first hESC line obtained by J. Thomson's group (Science 282(5391):1145-1147, 1998) have been used in clinical trials in patients with spinal cord injury, and other hESC lines have now been used to generate cells for use in treating blindness (Lancet 379(9817):713-720, 2012). In addition to the classical protocol based on mouse or human feeder layers using open culture methods (In Vitro Cellular & Developmental Biology - Animal 46(3-4):386-394, 2010; Stem Cells 23(9):1221-1227, 2005; Nature Biotechnology 24(2):185-187, 2006; Human Reproduction 21(2):503-511, 2006; Human Reproduction 20(8):2201-2206, 2005; Fertility and Sterility 83(5):1517-1529, 2005), novel hESC lines have been derived xeno-free (without using animal derived reagents) (PLoS One 5 (4):1024-1026, 2010), feeder-free (without supporting cell monolayers) (Lancet 365(9471):1601-1603, 2005), in microdrops under oil (In Vitro Cellular & Developmental Biology - Animal 46(3-4):236-41, 2010) and in suspension with ROCK inhibitor (Nature Biotechnology 28(4):361-4, 2010). Regardless of the culture system, successful hESC derivation usually requires optimization of embryo culture, the careful and timely isolation of its inner cell mass (ICM), and precise culture conditions up to the establishment of pluripotent cell growth during hESC line derivation. Herein we address the crucial steps of the hESC line derivation protocol, and provide tips to apply quality control to each step of the procedure.
authors.affiliation: Caubet-Cimera Fundation, Centre for Advanced Respiratory Medicine, Recinte Hospital Joan March, Ctra Sóller km 12, 07110 Bunyola, Illes Balears, Mallorca, Spain. telomerasi@yahoo.es
authors.forename: María Vicenta
authors.initials: MV
authors.lastname: Camarasa
authors.forename: Víctor Miguel
authors.initials: VM
authors.lastname: Galvez
authors.forename: Daniel Roy
authors.initials: DR
authors.lastname: Brison
authors.forename: Daniel
authors.initials: D
authors.lastname: Bachiller
mesh_terms: D000818:Animals; D053624:Blastocyst Inner Cell Mass / Q000166:cytology; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D018920:Coculture Techniques; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology*; D061252:Feeder Cells; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology; D006801:Humans; D051379:Mice; D016685:Mitomycin / Q000494:pharmacology; D011247:Pregnancy
chemical_list: D016685:Mitomycin