Figure 5.

Reprogrammed Cells Exhibit Stem Cells Features

(A–C) Long-term established reprogrammed clones present a more compact morphology as shown by lower SSC and FSC density plot for 1D and 3E clones compared with CEF by flow cytometry analysis. The reprogrammed clones (1A, 1D, 3E, and 3F), the GFP-labeled homologs (1D, GFP; 3E, GFP), and the established cESCs as controls for avian stem cells were positive for SSEA-1 and EMA-1, as detected by both fluorescence-activated cell sorting analysis (B) and confocal microscopy after immunofluorescence (C). Scale bars, 5 μm.

(D) Endogenous telomerase activity was measured in the same cells and in CEFs as negative control cells used as the substrates for reprogramming. CTR+ is the positive control provided by the manufacturer.

(E) Analysis of cell-cycle phases reveals a stem cell profile with a short G2/M phase and a long S phase for the chicken reprogrammed clones (1A, 1D, 3E, and 3F), the GFP-labeled homologs (1D, GFP; 3E, GFP), and for the established cESCs and mESCs as controls for avian and murine ESCs, and in CEFs and DEFs as somatic negative control cells.

(F) Reprogrammed cells (1D and 3E) were probed for the presence of large nuclear foci of trimethylated histone H3 on lysine 27, which is typical for cESCs and not observed in CEFs. These foci which colocalize with heterochromatin foci containing trimethylated histone H3 on lysine 9 were present in 66%–98% of the nuclei, depending on the cell clone. Scale bars, 5 μm.

(G) The karyotype analysis of the reprogrammed clones (1D and 3E) reveals a normal chicken karyotype with macrochromosomes and minichromosomes.