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The article examines the role of cell surface glycosphingolipids stage-specific embryonic antigen 3 (SSEA-3) and SSEA-4 in human embryonic stem cell (hESC) pluripotency. The results demonstrated that glycosphingolipids do not play a critical functional role in maintaining the pluripotency of hESC but suggested roles for the molecules during cellular differentiation.

Isolation of human embryonic stem cells (hESC) from the inner cell mass of surplus in vitro fertilization embryos has allowed them to be characterized and manipulated in vitro. While such manipulations allow for the development of cell lines that are easier to culture, and have therapeutic potential, methods are needed to determine their effects on pleuripotency. A method for histological and immunohistochemical characterization of formalin fixed teratomas induced in SCID mice by inoculation with cells from a stable karyotypic variant hESC line (BG01V) was developed. The teratomas exhibited primitive tissue differentiation including cartilage, bone, villi, glands, skin and various types of epithelia. Nuclear staining with antibodies to OCT-¾ was used to identify undifferentiated pleuripotent cells. Epithelium, of either endodermal or ectodermal origin, was pancytokeratin positive, while ectoderm, but not endoderm, had nuclear staining for p63. Mesodermal smooth muscle was detected with antibodies against smooth muscle actin. Neuroectoderm was identified using antibodies against neuron specific enolase and S100. This method of characterization can be used to confirm and compare the in vivo pleuripotency of stem cell lines that have been manipulated in vitro. [ABSTRACT FROM AUTHOR]

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

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.