Proteomic identification of overexpressed adenomatous polyposis coli and cyclin B3 during endoderm differentiation from human embryonic stem cells.

OBJECTIVES

This study was aimed to investigate important proteins associated with endoderm differentiation by pancreatic derivation protocol from human embryonic stem cells (hESCs).

METHODS

Comparative proteomic analysis of endoderm cells differentiated from hESCs by activin A and low serum was performed. Proteins with altered expression levels during endoderm differentiation were investigated by 2-dimensional gel electrophoresis (2-DE) with mass spectrometric analysis.

RESULTS

Thirty-four protein spots with significantly changed intensities were identified. These were functionally annotated based on gene ontology. The messenger RNA expression levels of 5 genes, APC, CCNB3, HSPA9, CCT2, and YWHAE, were correlated with 2-DE analysis. We further validated the protein expression levels of adenomatous polyposis coli (APC) and cyclin B3 (CCNB3) by using Western blot analysis and immunocytochemistry. They are involved in the regulation of cell cycle, thus, cyclins and cyclin-dependent kinases, which regulate the cell cycle, were examined. Cyclin A1, cyclin D2, and cyclin E2 were upregulated, and other cyclins and cyclin-dependent kinases were downregulated in endoderm cells.

CONCLUSIONS

The increase in expression of APC and CCNB3 suggests that these proteins will be important markers for identifying endoderm cells differentiated from hESCs, and they can play important roles in the differentiation of endoderm cells from hESCs or in human endoderm development for pancreas.