ES Cell Culture
The ES cells maintained on feeder layers of MEF formed colonies with individual cells containing large nuclei, prominent nucleoli, and distinctive cell boundaries (Fig. 2A). The cell line R366.4 has a 42,XY karyotype and was at passage numbers 31-36 when placed in culture, where it has been maintained for more than 8 mo in vitro. Maintenance of ES cells in an undifferentiated state was monitored by visual observations of their morphology and the expression of primate ES cell-specific antigens TRA-1-60, TRA-1-81, and SSEA-4 (Fig. 2, B-D). In contrast to mouse ES cells, monkey ES cells did not express SSEA-1 (Fig. 2E). Monkey ES cells also expressed alkaline phosphatase (Fig. 2F) and the POU domain gene product, Oct-4 (Fig. 2G). buy nexium online
The initial step in spontaneous ES cell differentiation was colony removal from the feeder layer with transference of isolated ES cell clumps to hanging drops of ES medium containing serum. Efforts to completely disassociate ES cells into single cells (n = 50, two replicates) before culture in hanging drops were unsuccessful, because the dissociated cells failed to aggregate and degenerated. After 2-3 days of culture in hanging drops, 76% ± 6% (271/357, three replicates) of the ES cell clumps had aggregated into early or simple EBs (Fig. 3A) as defined by their uniform appearance and lack of a central cavity.
FIG. 1. Schematic diagram illustrating the sequential steps in the neural differentiation of monkey ES cells.
FIG. 2. Characterization of undifferentiated rhesus monkey embryonic stem (ES) cells. Phase-contrast micrographs depicting undifferentiated ES cells with large nuclei, distinctive nucleoli, and cell boundaries (A); expression of primate-specific ES cell surface markers TRA-1-60 (B), TRA-1-81 (C), and SSEA-4 (D), but not the mouse-specific marker SSEA-1 (E). Also depicted is alkaline phosphatase positivity (F) and staining for the POU domain gene product, Oct 4 (G). Original magnification X400 (A) and X200 (B-G).