• Cell strainer with 70 mm mesh size (Cat. No. 353350, BD Biosciences)

• Fluorescence-activated cell sorter

1 Harvest hES cell colonies using dispase as described above (Protocol 3). Wash colonies with PBS ", and dissociate using trypsin/EDTA solution into a single cell suspension.

2 Stop trypsin action with 1 ml SSM, and harvest cells by microcentrifugation at 500 g for 2 min. Resuspend cells in 50 ml SSM.

3 Fix cells by transferring them into 5 ml ice-cold, 100% methanol, and incubate for 30 min on ice.a

4 Centrifuge cells for 2 min at 500 g, and wash with 500 ml of SSM.

5 Resuspend cells in 300 ml mixture of mouse monoclonal antibody supernatants: GCTM-2 IgM (1:2 dilution), mouse TG30 IgG2a (1:2 dilution), and anti-Oct-4 IgG2b (1:50 dilution), or a mixture of class-matched negative-control antibodies, for 30 min on ice.

6 Centrifuge cells for 2 min at 500 g, and wash with 1 ml SSM.

7 Pellet the cells again by centrifugation at 500g for 2 min, and remove supernatant medium. Resuspend the cells in 300 ml biotinylated rabbit anti-mouse IgM (1:125 dilution), and incubate for 30 min on ice.

8 Centrifuge cells for 2 min at 500 g, and wash in 500 ml SSM before resuspending in a mixture of goat anti-mouse IgG2a-AF488 (1:1000 dilution), goat anti-mouse IgG2b AF647 (1:1000 dilution), and streptavidin-PE (1:1000 dilution) for 30 min on ice.

9 Wash the cells again in 1 ml SSM prior to resuspending in 500 ml of the same. Filter cells through a cell strainer to remove clumps.

10 Analyse cells on a flow cytometer with initial gating using forward and right-angle light scatter; and collect AF488, AF647, and PE fluorescence signals. Human ES cells analysed by this method should be compared to single colour controls for TG30, GCTM-2, and Oct-4.

a Live, non-fixed hES cells also may be analysed for the presence of the cell surface markers, KSPG and CD9, recognized by antibodies GCTM-2 and TG30.

In Protocol 7 we describe a rapid and convenient technique for estimating the proportion of undifferentiated hES cells in a culture, by double labelling for cell-surface epitopes KSPG and CD9, combined with fluorescence-activated cell sorting (FACS®) analysis.

5.2 FACS® analysis of hES-cell antigen expression

Flow cytometry provides quantitative information on the proportion of hES cells in a culture expressing particular surface markers. Because the technique requires dissociation of hES-cell colonies into single cell suspensions, which is only poorly tolerated at best, it is important to monitor cell viability and to bear in mind the possibility that some subpopulations of cell types within a hES cell culture may be more susceptible to dying following dissociation than others.

Protocol 7 describes the method of simultaneous immunostaining cells in single-cell suspension for the stem-cell markers KSPG, CD9, and Oct-4, and subsequent quantitative analysis by flow cytometry.

5.3 Immunomagnetic isolation of viable hES cells

Flow cytometry requires dissociation of hES colonies into single cells, a procedure that results in loss of viability and differentiation. Therefore, isolation of viable stem cells by fluorescence-activated cell sorting (FACS®) is problematic. An alternative approach is to immunoisolate small clusters of cells using magnetic beads. In the case of the surface marker defined by antibody GCTM-2, positive cells and negative cells generally appear to segregate spatially within a colony and, as a consequence, the clusters of cells isolated using this method are usually homogeneous with respect to antigen expression and show much better viability than single cells.

5.4 Gene expression in hES cells

Examination of transcription in mouse and human ES cells using conventional methods and microarray profiling have supported the concept of a network of genes characteristic of pluripotent stem cells in both species (3, 13). Molecular embryology in the mouse has also defined many genes that are characteristically expressed in cells that have undergone commitment to specific differentiation lineages, and of course there are vast numbers of genes expressed in particular

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