Using homologous recombination in human ESC, we inserted an enhanced green fluorescent protein (eGFP) transgene into a locus encoding a postulated marker of human endoderm, SOX17 in H9 human embryonic stem cells. This allowed purification of SOX17+ hESC endodermal progeny by fluorescence activated cell sorting (FACS) to generate microarray gene expression profile. Using Wnt3 and Activin to differentiate hSOX17-2 to stage 1 cells, and subsequently FGF10 and cyclopamine to stage 2 cells, we isolated eGFP+ cells by FACS at each stage, performed microarray analysis.

Identify a cell surface marker signature for human endodermal cells

Gene expression patterns of undifferentiated hS17 ESCs and their FACS-sorted eGFP+ progeny at stage 1 and stage 2 were compared by microarray analysis. Three replicates for each sample were analyzed using the Affymetrix Human Genome U133 Plus 2.0 arrays. To identify new cell surface markers for endoderm, transmembrane proteins (GO:000588 'integral to plasma membrane') were selected whose expression changed in SOX17-eGFP+ cells at stage 1 and 2. Findings were validated using commercially available antibodies for cluster of differentiation (CD) antigens.

A total of 13,209 genes showed statistically significant differential expression among the three samples. These included 608 genes encoding adhesion molecules, receptors, transporters, and ion channels, which were differentially expressed between undifferentiated hESCs and SOX17-eGFP+ endodermal cells. Antibodies to three molecules induced in stage 2 SOX17-eGFP+ cells were identified: CD49e (biolegend: 328005), CD141 (BD: 559780 and 559781), and CD238 (AbDSerotec:MCA1987T).

Microarray-based analysis of SOX17-eGFP+ cells identified cell surface markers for isolating endodermal progeny from hESC cultures.

• ES cell differentiation: 2800, 3365, 3425, 3668, 4216, 4314, 4574,