EGFP-positive cells do not always directly differentiate from DsRed-positive cells.

<p>(A) DsRed, EGFP, phase contrast, and merged images of transgenic HepaRG cells at the growing period (G). Undifferentiated transgenic HepaRG cells are blastic and DsRed-positive. Arrow indicates DsRed-negative cells. The size of the scale bar, 100 µm. (B) DsRed, EGFP, phase contrast, and merged images of transgenic HepaRG cells at D5d. EGFP-positive cells appear after most DsRed-positive cells have disappeared. Double-positive cells are rarely seen at D5d. Arrow indicates an exceptional double-positive cell. The size of the scale bar, 100 µm. Asterisk indicates a degenerated intracellular structure. (C) Sequential dual-color images from movies of HepaRG C3 cell differentiation between D6d and D14d. (a) A DsRed-positive cell (R+) becomes non-fluorescent (R-) and then EGFP-positive (G+); (d) a non-fluorescent cell divides into two cells, both of which are EGFP-positive. (D) A model for the course of hepatic cell differentiation from CYP3A7-expressing hepatoblastic precursors. Following differentiation of transgenic HepaRG cells, there is a colorimetric shift from red to green, which is separated by a period in which cells are not fluorescent without (a) or with (c and d) cell division. (E) Frequencies of EGFP-positive cells that differentiated through process a, b, c, or d between D6d and D8d or between D11d and D14d.