Characterization of cES cell lines

3.1 Expression of cES-cell markers and karyotype analysis

Those ES cell lines established to date from diverse primates have been characterized with respect to cell-surface markers by immunocytochemistry, and alkaline phosphatase activity by chromogenic enzyme detection assay. In common with hES cells, monkey (specifically marmoset, rhesus, and cynomolgus) ES cells express SSEA-4, TRA1-60, and TRA1-81 cell-surface antigens, but not SSEA-1; and whereas human, marmoset, and rhesus monkey ES cells have been reported to express also SSEA-3 at variable levels, we have found that cES cells are negative by immunostaining, which may reflect a species-specific difference. (In contrast mES cells express SSEA-1, but neither SSEA-3 nor SSEA-4.) The expression of genes characteristic of pluripotent cells, such as Oct-3/4, Rex-1,

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antiserum complement

Figure 2 Morphology of cES cells during sequential stages of routine culture on feeder layers, viewed by light microscopy. (A) Sub-confluent culture of cES cells on a feeder layer, with several cES-cell colonies of various sizes. (B) Morphology of cES-cell colonies during treatment with dissociation solution: feeder cells have become rounded and are detaching from the substratum whilst the two cES-cell colonies remain intact, but are beginning to detach. (C) Clusters of cES cells obtained after dissociation of colonies by pipetting, each containing an optimal 50-100 cells. (D) Appearance of cES-cell colonies 1day after subculturing on a fresh feeder layer. (E) A typical spontanteously differentiated colony of cES cells, showing epithelial morphology and surrounded by feeder cells. (F) A simple embryoid body generated by cES cells in suspension. Scale bars: 750mm for (A); 300 mm for (B), (C), (D) and (E); and 150 mm for (F).

Figure 2 Morphology of cES cells during sequential stages of routine culture on feeder layers, viewed by light microscopy. (A) Sub-confluent culture of cES cells on a feeder layer, with several cES-cell colonies of various sizes. (B) Morphology of cES-cell colonies during treatment with dissociation solution: feeder cells have become rounded and are detaching from the substratum whilst the two cES-cell colonies remain intact, but are beginning to detach. (C) Clusters of cES cells obtained after dissociation of colonies by pipetting, each containing an optimal 50-100 cells. (D) Appearance of cES-cell colonies 1day after subculturing on a fresh feeder layer. (E) A typical spontanteously differentiated colony of cES cells, showing epithelial morphology and surrounded by feeder cells. (F) A simple embryoid body generated by cES cells in suspension. Scale bars: 750mm for (A); 300 mm for (B), (C), (D) and (E); and 150 mm for (F).

and Nanog, are detectable in undifferentiated cES cells by RT-PCR (unpublished). These ES cell-specific markers are summarized in Table 1.

Male and female cES cell lines can be isolated at equal frequencies. Usually, at least 70% of chromosome spreads demonstrate the euploid chromosome

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