Figure 1 : A. iPSCs express DE markers SOX17 and CXCR4 to a similar extent when
differentiated using
Protocol I (Activin A) or Protocol II (GDF8+GSK3i+B27), using F1089A
cell line as an example.

B. Hindgut CDX2 positive immunostaining is expressed in developing cellular clumps (spheroids) after
protocol II incubation (KGF and RA) where nuclear (DAPI blue) and CDX2 (red) -immunoreactivity overlap
(example shown by arrow) C. Representative images of morphology at different stages of A1145A. Protocol
I (top) showed large elongated and dense structures at Stage 2, excised with some budding hollow
structures that were (e) that were excised. Protocol II (bottom) showed spheroids at Stage 2 (e), In both
protocols, HIO developed with a large number of budding hollow spheroids (e.g. arrow) during
differentiation in 3D matrigel culture. 4X magnification. D. Representative images of morphology at different
stages of F1089A. Protocol I at Stage 2 resulted in large compact spheroids that could be excised (e) and
developed into dense HIO with ill-defined outgrowths during Stage 3 differentiation in 3D matrigel. Protocol
II (bottom) showed spheroids at Stage 2, which developed into HIO with a large number of budding hollow
structures that were excisable (e) as well as and floating (f) spheroid structures. 4X magnification.

Hornby et al.Stem Cell Biology and Research  2015 2:1DOI : 10.7243/2054-717X-2-1