Method

1 To precoat the culture dishes with gelatin, add sufficient gelatin solution to cover their bases, incubate for 1 h to overnight at 4 °C, and aspirate the residual solution.b

2 Into each gelatin-coated dish dispense an appropriate volume of Differentiation Medium I (i.e. 5 ml per 60 mm dish, 2 ml per 35 mm dish, and 1 ml per well of a 24-well plate), followed by EBs at the following densities: plate 20-30 EBs per 60 mm dish; 5-10 EBs per 35 mm dish; and one EB per well of a 24-well plate.

3 Incubate EB cultures, changing medium every second or third day, until 9 d after EB plating (i.e. 5 + 9 d in total from the start of aggregation); and proceed to lineage-specific differentiation (Protocols 4 and 5).

a Multi-well plates are convenient to use for the morphological analysis of EB outgrowths.

b For immunofluorescence analysis, spread sterile, glass coverslips over the bases of the dishes before gelatinization.

For EB formation, mES cells may be induced to aggregate either by the 'hanging-drop' method (18) or by 'mass culture' in bacteriological-grade dishes ((19); and see Chapter 6, Protocol 3). The advantages of the 'hanging-drop' method, employed here, include the low variation in size of EBs produced due to a defined number of mES cells in the starting aggregates, as well as greater reproducibility of differentiation. Incubating EBs for 5 d prior to plating ensures the development of progenitor cells deriving from all three primary germ layers.

3.2 Induction of lineage-specific differentiation

Although EB-mediated differentiation of mES cells generates cellular derivatives of all three primary germ layers, the proliferation and differentiation of heterogeneous lineage-specific progenitor cells thus generated are considerably enhanced by their culture in the presence of defined growth- and differentiation-inducing factors. In addition, the following parameters are known to affect the efficiency of differentiation of mES cell-derived progenitors into pancreatic and hepatic cells in such systems, and should therefore be taken into consideration when devising and optimizing in vitro differentiation experiments:

(a) Dissociation of the compact structure of EB outgrowths. Cell-to-cell interactions within the complex and heterogeneous structure of EB outgrowths may influence the fate of progenitor cells.

(b) A suitable, adhesive substratum. ECM factors determine adhesion, proliferation, and migration of specific progenitor cells after replating of dissociated EBs.

(c) Cell density after replating. To support the proliferation and migration of a specific progenitor population, the concentration of cells should be optimal to prevent overgrowth resulting in metabolic starvation, necrosis, and cell death. However, if the initial density of cells is too low, the level of autocrine factors may not reach a required, threshold level; and consequently the low growth-factor activity and reduced cell-to-cell contacts may result in poor efficiency of differentiation of the desired cell types.

3.2.1 Induction of pancreatic differentiation

The medium used for the differentiation of pancreatic cells from EB-derived progenitor cells contains factors required for pancreatic-cell survival, such as progesterone, putrescine, insulin, sodium selenite, and transferrin, in addition to factors promoting pancreatic differentiation, such as nicotinamide (20) and lami-nin (21). This pancreatic differentiation medium (PDM) is serum-free. The addition to PDM of other substances promoting pancreatic differentiation, such as glucagon-like peptide-1 (GLP-1, (22)) and growth inhibitors (23), also may prove effective. The precoating of tissue-culture dishes with poly-L-ornithine and lami-nin is necessary for pancreatic differentiation to occur. After replating and culture of EBs under these conditions for 7 d, pancreatic progenitor cells with a characteristic morphology (Figure 1A) become apparent. Continued incubation and concomitant differentiation results in the formation of typical islet-like cell clusters (Figure 1B; and see (24)).

3.2.2 Induction of hepatic differentiation

Mouse ES cells are induced to differentiate into hepatic cells when incubated in Hepatocyte Culture Medium (HCM). HCM contains factors required for hepatic-cell survival, such as insulin and transferrin, as well as hepatic differentiation factors, including hydrocortisone and epidermal growth factor (EGF). Supplementation of HCM with FCS also is required for cell survival; and the culture dishes are precoated with collagen Type I (or collagen I), which further promotes

Figure 1 Morphology of pancreatic (A-C) and hepatic (D-F) cells differentiating from mES cells in vitro, and viewed by light (A, B, D, E) and confocal (C, F) microscopy. (A) Pancreatic progenitor cells 7 d after induction of pancreatic differentiation in plated EBs, that is at stage 5 +16 d (according to Protocol 4). (B) Morphology of an islet-like cluster, at stage 5 + 28d; and (C) confocal image of immunolabelled, C-peptide-positive cells constituting an islet-like cluster. (D) Hepatic progenitor cells 10d after induction of hepatic differentiation in plated EBs, that is at stage 5 + 19d (according to Protocol 5). Continued culture in HCM leads to the generation of further differentiated and specialized cell types: (E) Morphology of cuboidal, hepatocyte-like (and frequently binucleated) cells, at stage 5 + 39d; and (F) confocal image of immunolabelled, a1-antitrypsin-positive cells. Scale bars = 20mm.

Figure 1 Morphology of pancreatic (A-C) and hepatic (D-F) cells differentiating from mES cells in vitro, and viewed by light (A, B, D, E) and confocal (C, F) microscopy. (A) Pancreatic progenitor cells 7 d after induction of pancreatic differentiation in plated EBs, that is at stage 5 +16 d (according to Protocol 4). (B) Morphology of an islet-like cluster, at stage 5 + 28d; and (C) confocal image of immunolabelled, C-peptide-positive cells constituting an islet-like cluster. (D) Hepatic progenitor cells 10d after induction of hepatic differentiation in plated EBs, that is at stage 5 + 19d (according to Protocol 5). Continued culture in HCM leads to the generation of further differentiated and specialized cell types: (E) Morphology of cuboidal, hepatocyte-like (and frequently binucleated) cells, at stage 5 + 39d; and (F) confocal image of immunolabelled, a1-antitrypsin-positive cells. Scale bars = 20mm.

hepatic differentiation (9). After replating and culture of EBs under these conditions, hepatic progenitor cells develop with characteristic morphology (Figure 1D). With continued incubation and further differentiation, large cuboidal and epithelioid, hepatocyte-like cells become apparent, a proportion of which are binucleated (Figure 1E; and see (25)).

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