The analysis of BORIS expression demonstrated that we successfully enriched the cell population that expressed BORIS mRNA. In our experiments we used NCCIT cell line, classified as germ-cell tumor or GSK2118436 embryonic cancer cells. Due to its higher BORIS expression compared to the other tumor cell lines, the NCCIT cell line provides a good model for our studies, especially for the feasibility of isolation of BORIS positive cells by MB technology. In normal human cells, telomerase is generally absent in somatic cells but remains active in germ cells, progenitor cells and some adult stem cells. It has been shown that telomerase is reactivated in a majority of tumors and in clinical studies its reactivation is associated with poor outcomes of different tumors. In addition, current literature supports the evidence that CSCs express telomerase and its inhibition suppresses the self-renewal of CSCs. All these discoveries, together with our observations, indicate that BORIS could play an important and direct role in tumor malignancies by up-regulation of the hTERT telomerase gene. Consistent with this, we notably observed that the BORIS-positive/hTERT-high isolated cells expressed also high levels of the most important stem cell markers. The embryonic carcinoma cells that we investigated, indeed, provide a good model system to study the stem cell concept of cancer. They are stem cells derived from a teratocarcinoma and are also the malignant transformed embryonic stem cells. They show gene expression profiles close to those of human embryonic stem cells. Therefore, we analyzed the association of BORIS-positive/hTERT-high cells with the expression of the key-regulator genes of embryonic cells and with some of the most known specific markers of CSCs. CSCs are cancer cells characterized by stem cell properties and represent a small population of tumor cells that drives tumor development, progression, metastasis and drug resistance. Interestingly, a correlation of the isolated BORISpositive/hTERT-high cells with high expression of stem cell markers was observed. These findings were confirmed by BORIS silencing studies. Stable BORIS knockdown NCCIT-derived cells were generated by an efficient system of inducible-shRNA lentivirus. After BORIS silencing, a significant decrease of hTERT expression was observed, as well as a down-regulation of telomerase activity, which is strictly regulated by hTERT gene transcription. CTCF expression was also decreased. This is consistent with our previous results and confirms a role of BORIS in the transcriptional regulation of CTCF. This correlation between CTCF and BORIS was not observed in the expression analysis of the isolated BORIS-positive cells. This discrepancy could be due to the different experimental conditions. Indeed, in the expression analysis of the isolated BORIS-positive cells, CTCF was analyzed at steady state, while in BORIS silencing studies what we observed is the result of cellular and genetic modifications. Importantly, after BORIS knockdown a decrease of expression of stem cell and CSC marker genes was also observed. These results highlight the importance of BORIS in malignant disease and its possible critical role on cancer growth and progression.