Fig. 3. Maturation of definitive endoderm by the reduction of Nodal signaling. (A) Outline of the three-stage maturation protocol of definitive endoderm with the reduction of Nodal signaling and the formation of a 3D structure. (B) RT-PCR analysis of definitive endodermal gene expression by stages 2, 3a and 3b cells. (C) Immunofluorescence images of stage-3a cell aggregates were obtained by confocal microscopy and are representative of at least five samples for each probe (Foxa2, E-cadherin, panCK, TTF1, proSftpC, albumin and Pdx1). Original magnification was 400x; bars, 50 µm.