• Differentiation Medium I (Protocol 1)

• Suspension-culture dishes (Protocol 2)

• Gelatin-coated, 60 mm tissue-culture dishes (Protocol 2)

1 Add 5 x 105 mES cells to a 10 cm suspension-culture dish containing 10 ml Differentiation Medium I, and incubate at 37 °C.b

2 After 2 d, tilt the plate to one side and allow cell aggregates to settle. Carefully remove medium and replace with 10 ml Culture Medium.

3 Change medium every second day, supplementing the cell suspension with 5 ml Culture Medium on intervening days.

4 On day 7, plate EBs onto gelatin-coated tissue-culture dishes (at densities given in Protocol 2) in Culture Medium, and incubate. Spontaneously beating cardiomyocytes should become visible 1-2 d after plating. Because of the large number of EBs that can be plated in this way it is essential to change medium regularly, otherwise changes in pH will adversely affect the longevity of the mES cell-derived cardiomyocytes.

a Generally, 5 x 105 to 2 x 106 mES cells (depending on the cell line) are seeded into suspension-culture dishes in the appropriate medium.

b Spinner flasks also can be employed for the generation of very large numbers of differentiating cells (14; and see Chapter 11 for human ES cells).

Figure 3 Cardiomyocyte differentiation in genetically modified mES cells. (A) to (C) Stages in the modification and differentiation of a mES cell line transformed with a puromycin-selection cassette controlled by the rat MLC2v promoter (MLC2v-pacr/PGK-neor). After introduction of the construct into mES cells by electroporation, cells were treated with G418 to select for transformants. Following selection and expansion of undifferentiated, G418-resistant colonies (A), cells from a clonal line were induced to differentiate via the hanging-drop method into cardiomyocytes. Day 7 EBs in suspension culture are shown in (B). After EB plating at day 7 and following 3d of culture, puromycin was added to the medium for a period of 4d, and cells were stained by the periodic acid-Schiff (PAS) reaction to visualize areas of cardiogenic induction (C) (31). (D) to (H) Cardiomyocyte differentiation in clonal mES cell lines transformed with a neomycin-resistance cassette (NCX1-neor; D, E) or a puromycin-IRES-EGFP cassette (NCX1-pacr-IRES-EGFP; F- H) underthe control of the rat NCX1 promoter (35). (D), (E) A cluster of differentiated NCX1-neor mES cells selected in G418 for 8d to eliminate non-cardiomyocytes; (D) phase-contrast image, and (E) fluorescent-antibody staining with the anti-MHC antibody, MF20 (adapted from (24)). (F) Area of beating cardiomyocytes expressing EGFP within a mixed population of differentiated NCX1-pacr-IRES-EGFP mES cells. After dissection (Protocol 6), individual cardiomyocytes continue to express EGFP (inset). (G) Phase-contrast image of an NCX1-pacr-IRES-EGFP mES cell line following differentiation into cardiomyocytes and selection with puromycin: these cells express EGFP (H). Scale bars = 20 mm.

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