In vivo and in vitro differentiation of hESCs maintained in DC-HAIF. (A) Analysis of teratomas from BG02 DC-HAIF p25 cells demonstrated pluripotent differentiation potential to ectoderm, mesoderm, and endoderm. NSE indicates neuron-specific enolase; SMA, smooth-muscle actin; and pCK, pan-cytokeratin. Scale bars equal 20 μm (NSE, p63, 100× oil objective), 50 μm (SMA, Ducts, pCK, 20× objective), and 200 μm (Bone, 10× objective). (B) Differentiation of BG02 DC-HAIF p5 cells to ectoderm (βIII tubulin⁺), mesoderm (SMA⁺), and endoderm (alphafetoprotein⁺ [αFP]) lineages in embryoid bodies (20× objective, same scale as panel D). (C,D) Directed differentiation of BG02 DC-HAIF p48 cells (HAIF) to definitive endoderm (d3) and foregut endoderm (d6) using defined medium differentiation conditions (Document S1). (C) qPCR analyses showed down-regulation of OCT4 expression and minimal PAX6 or CDX2 expression in differentiated samples, suggesting differentiation of pluripotent cells and lack of substantial neuroepithelial or trophectodermal differentiation. The generation of definitive endoderm at day 3 was confirmed by up-regulation of SOX17, CXCR4, and CER expression, and foregut endoderm at day 6 by up-regulation of FOXA2, HNF1β, and HNF4α. Similar results were observed with CyT49 cells. Error bars are plus or minus SD. (D) Immunostaining analyses confirmed homogenous expression of OCT4 and lack of SOX17 expression in undifferentiated cultures (HAIF). After 3 days, most cells express SOX17 with only pockets of OCT4⁺ cells remaining (d3). After 6 days, the differentiation to foregut endoderm was confirmed by expression of HNF1β and HNF4α (d6). Only rare OCT4⁺ cells were present at d6 (not shown). Scale bar equals 50 μm (20× objective).