Characterization of functional differentiation of organoids.

<p>Immunostaining analysis by confocal microscopy of 28–30 day organoids for cytodifferentiation and functional differentiation markers. (<b>A and D</b>) Luminal cell cytodifferentiation markers AR (green) and (<b>B and E</b>) CK8/18 (red) and (<b>C and F</b>) merged images with DAPI (blue) reveals most luminal epithelial cells contain nuclear AR. (<b>G</b>) Merged NKX3.1 (green), a prostate specific epithelial cell marker, with CK8/18 (red), a luminal epithelial cell marker and DAPI (blue) shows nuclear NKX3.1 in all epithelial cells suggesting prostatic nature. Functional differentiation markers PSA (<b>H and I</b>) and androgen regulated gene TMPRSS2 (<b>J</b>) indicate the ability of cytodifferentiated luminal cells to produce secretory proteins specific to the prostate. Laminin (<b>K</b>), a basement membrane marker, delineates the normal acinar organization of the organoids. Staining for Vimentin (<b>L</b>) confirms the extra-acinar cells are derived from mesenchymally differentiated hESC cells, forming a stromal compartment. Normal IgG as negative controls for each probe is shown in insets. Lumens are indicated by white dotted lines (H-L) and epithelial ducts are outlined by green dotted lines (D-F). Scale bars represent 20 μm.