As the number of human embryonic stem cell (hESC) lines increases, so does the need for systematic evaluation of each line's characteristics and potential. Comparisons between lines are complicated by variations in culture conditions, feeders, spontaneous differentiation, and the absence of standardized assays. These difficulties, combined with the inability of most labs to maintain more than a few lines simultaneously, compel the development of reference standards to which hESC lines can be compared. The use of a stable cell line as a reference standard offers many advantages. A line with a relatively unchanging hESC-like gene and protein expression pattern could be a positive control for developing assays. It can be used as a reference for genomics or proteomics studies, especially for normalizing results obtained in separate laboratories. Such a cell line should be widely available without intellectual property restraints, easily cultured without feeders, and resistant to spontaneous changes in phenotype. We propose that the embryonal carcinoma (EC) line 2102Ep meets these requirements. We compared the protein, gene, and microRNA expression of this cell line with those of hESC lines and alternative reference lines such as the EC line NTERA-2 and the karyotypically abnormal hESC line BG01V. The overall expression profiles of all these lines were similar, with exceptions reflecting the germ cell origins of EC. On the basis of global gene and microRNA expression, 2102Ep is somewhat less similar to hESC than the alternatives; however, 2102Ep expresses more hESC-associated microRNAs than NTERA-2 does, and fewer markers of differentiated fates. STEM CELLS 2007;25:437-446

The application of human embryonic stem (hES) cells in regenerative medicine will require rigorous quality control measures to ensure the safety of hES cell-derived grafts. During propagation in vitro, hES cells can acquire cytogenetic abnormalities as well as submicroscopic genetic lesions, such as small amplifications or deletions. Many of the genetic abnormalities that arise in hES cell cultures are also implicated in human cancer development. The causes of genetic instability of hES cells in culture are poorly understood, and commonly used cytogenetic methods for detection of abnormal cells are capable only of low-throughput analysis on small numbers of cells. The identification of biomarkers of genetic instability in hES cells would greatly facilitate the development of culture methods that preserve genomic integrity. Here we show that CD30, a member of the tumor necrosis factor receptor superfamily, is expressed on transformed but not normal hES cells, and that CD30 expression protects hES cells against apoptosis.

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. STEM CELLS 2006;24:531-546

BackgroundHuman embryonic stem cells (hESC) offer a renewable source of a wide range of cell types for use in research and cell-based therapies to treat disease. Inspection of protein markers provides important information about the current state of the cells and data for subsequent manipulations. However, hESC must be routinely analyzed at the genomic level to guard against deleterious changes during extensive propagation, expansion, and manipulation in vitro.ResultsWe found that short tandem repeat (STR) analysis, human leukocyte antigen (HLA) typing, single nucleotide polymorphism (SNP) genomic analysis, mitochondrial DNA sequencing, and gene expression analysis by microarray can be used to fully describe any hESC culture in terms of its identity, stability, and undifferentiated state.ConclusionHere we describe, using molecular biology alone, a comprehensive characterization of 17 different hESC lines. The use of amplified nucleic acids means that for the first time full characterization of hESC lines can be performed with little time investment and a minimum of material. The information thus gained will facilitate comparison of lines and replication of results between laboratories.