Pluripotent human embryonic stem cells (hES) can proliferate indefinitely in vitro and differentiate in all three germ layers and represent therefore a valuable tool for drug discovery, cell replacement therapy and regenerative medicine. Therefore, it is fundamental to understand the genetic network that regulates' pluripotency. A microarray experiment carried out in our laboratory compared the expression profile of undifferentiated versus differentiated hES cells. The results highlighted the down regulation upon differentiation of both already known master genes of pluripotency, like OCT3/4 and NANOG, and others, some of which were so far not associated with the undifferentiated state. We focused investigations on DPPA4, a SAP domain protein that has been associated with pluripotency, but remains poorly understood. SAP domain proteins are mainly implicated in chromosomal organisation, DNA repair and RNA metabolism. The aim of this study was to determine whether DPPA4 maintains pluripotency. Using RNAi we demonstrated that DPPA4 is not regulating the pluripotent state. Morphological differentiation and an accompanied reduced growth curve were induced, however, key markers of pluripotency, eg the transcription factors NANOG, OCT3/4 and SOX2 were unaffected. Unexpectedly, CDX2 was upregulated, but not hCG, both are markers of the trophoectodermal lineage. The cell surface antigens SSEA3 and TRA1-60, which are expressed in the undifferentiated state and downregulated upon differentiation were unaffected upon DPPA4 knock down. However, SSEA1 another cell. surface antigen, that has a - reciprocal expression pattern to SSEA3, was upregulated. Expression analysis in human cell lines, including hES and hEC cells, using a generated polyclonal peptide anti-DPPA4 antibody showed that all pluirpotent stem cells expressed DPPA4. However, it was also expressed in nullipotent hEC cells and on lower levels in choriocarcinoma cells, indicating that DPPA4 does not induce pluripotency. In localization studies using a DPPA4-EGFP fusion protein and immunohistochemistry experiments we found that DPPA4 is associated with chromatin, thus narrowing the potential role of this SAP domain protein. Taken together, we hypothesize that DPPA4 is playing a role in chromatin remodelling of pluripotent cells and some germ cell tumours.