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a Stem Cell Center, American Type Culture Collection, Manassas, Virginia, USA;
b Laboratory of Neuroscience, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA;
c Cellular Neurobiology Branch, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland, USA;
d BresaGen, Inc., Athens, Georgia, USA;
e Department of Veterinary Pathology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia, USA;
f Department of Pathology, Oncology, and Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
Key Words. Embryonic stem cell • hESC karyotype • Characterization • BG01V • NTERA-2
Correspondence: Jonathan Auerbach, Ph.D., Stem Cell Center, American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, USA. Telephone: 703-365-2809; Fax: 703-365-2790; e-mail: jauerbach{at}atcc.org
Received July 12, 2005;
accepted for publication November 11, 2005.
Human embryonic stem cells (hESCs) offer a renewable source of a wide range of cell types for use in research and cell-based therapies. Characterizing these cells provides important information about their current state and affords relevant details for subsequent manipulations. For example, identifying genes expressed during culture, as well as their temporal expression order after passaging and conditions influencing the formation of all three germ layers may be helpful for the production of functional beta islet cells used in treating type I diabetes. Although several hESC lines have demonstrated karyotypic instability during extended time in culture, select variant lines exhibit characteristics similar to their normal parental lines. Such variant lines may be excellent tools and abundant sources of cells for pilot studies and in vitro differentiation research in which chromosome number is not a concern, similar to the role currently played by embryonal carcinoma cell lines. It is crucial that the cells be surveyed at a genetic and proteomic level during extensive propagation, expansion, and manipulation in vitro. Here we describe a comprehensive characterization of the variant hESC line BG01V, which was derived from the karyotypically normal, parental hESC line BG01. Our characterization process employs cytogenetic analysis, short tandem repeat and HLA typing, mitochondrial DNA sequencing, gene expression analysis using quantitative reverse transcription-polymerase chain reaction and microarray, assessment of telomerase activity, methylation analysis, and immunophenotyping and teratoma formation, in addition to screening for bacterial, fungal, mycoplasma, and human pathogen contamination.
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