Characterization of hES cells

The current definition of the hES-cell phenotype relies on immunocytochemical characterization combined with analysis of gene expression, and biological assays to demonstrate pluripotentiality. In addition to these criteria, regular

Figure 2 Subculture and growth of hES-cell colonies. (A-H) Colonies grown In serum-supplemented medium with MEF feeder-layer support, at sequential stages following transfer by mechanical dissection. (A) Colony at day 7 of culture, ready for subculture. (B) Colony in (A) scored mechanically for transfer, avoiding the central, differentiated area (denoted by flattened morphology). (C) Colony at day 1 of culture following transfer. (D) Colony at day 3. (E) Colony at day 3 with differentiated area at the top left. (F) Colony at day 4. (G, H) Colony at day 5 with typical smoothened morphology in centre and jagged, defined cell borders at the periphery. (I-L) colonies grown in medium supplemented with proprietary serum replacement and FGF2, with MEF feeder-layer support, and following transfer by enzymatic dissociation. (Feeders are at one-third the density used in serum-based cultures, and the hES colonies are less compact.) (I, J) Colonies at day 1 of culture following transfer, the colony in centre of (J) is differentiated (arrow). (K) Colony at day 3. (L) Colony at day 5. Magnifications: (C, E, F, G, I, and J) as shown in (G); (A, B, H, K, and L) as shown in (H).

Figure 2 Subculture and growth of hES-cell colonies. (A-H) Colonies grown In serum-supplemented medium with MEF feeder-layer support, at sequential stages following transfer by mechanical dissection. (A) Colony at day 7 of culture, ready for subculture. (B) Colony in (A) scored mechanically for transfer, avoiding the central, differentiated area (denoted by flattened morphology). (C) Colony at day 1 of culture following transfer. (D) Colony at day 3. (E) Colony at day 3 with differentiated area at the top left. (F) Colony at day 4. (G, H) Colony at day 5 with typical smoothened morphology in centre and jagged, defined cell borders at the periphery. (I-L) colonies grown in medium supplemented with proprietary serum replacement and FGF2, with MEF feeder-layer support, and following transfer by enzymatic dissociation. (Feeders are at one-third the density used in serum-based cultures, and the hES colonies are less compact.) (I, J) Colonies at day 1 of culture following transfer, the colony in centre of (J) is differentiated (arrow). (K) Colony at day 3. (L) Colony at day 5. Magnifications: (C, E, F, G, I, and J) as shown in (G); (A, B, H, K, and L) as shown in (H).

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