We further compared the significance of differentially expressed proteins in our early and later passage hESCs in an optimized culture condition since we wished to exclude the influence of other changes associated with the in vitro culture adaptation. Our data showed relatively stable expression of HDAC2 in long-term in vitro cultures of normal hESCs, but displayed increasing levels during tumorigenesis followed with increased histone deacetylation. These observations supported the idea that enhanced HDAC2 expression may be associated with malignant transformation by regulating the architecture of chromatin. Thus HDAC2 could serve as a potential marker for abnormal hESCs with a tendency of initiating progression to the malignant state. Also, our data showed increased levels of proteins associated with DNA methylation, including DNMT3A, DNMT3B, AZD6244 DNMT1 and KPNB1. These proteins might contribute to the increased methylation of CpG islands and silencing of affected target genes, which are frequently found in human cancer. Though DNMT3B increases in karyotypically abnormal hESCs, in much the way as HDAC2, expression of DNMT3B was also enhanced in long-term in vitro and optimal culture condition, which was consistent with the gene mRNA expression levels. Recent studies have also shown that DNA methyltransferases, and DNMT3B, were correlated with HDAC1 and HDAC2 and were involved in the epigenetic regulation by silencing transcription and promoting cell proliferation and tumorigenesis. DNMT3B can also interact with HDACs 1/2 and other components of the epigenetic machinery to establish the chromatin environment. Peter Andrews reported that hESCs might undergo culture adaptation in long-term in vitro culture and that variations in gene expression might reflect the aberrant karyotype of the cells or might result from karyotypically silent epigenetic changes, implying that adaptation reflects an alteration in the balance between self-renewal and differentiation. Longterm in vitro cultured hESCs show a high risk of genomic instability due to culture conditions. Thus, we optimized our culture system including the preparation of feeder cells by irradiation, control of the density of feeder cells and passage by manual cutting. In optimized conditions,BAY-60-7550 hESCs show a stable normal karyotype, even after more than two years of in vitro culture. The increased expression of DNMT3B in culture suggested that it might be relevant to culture adaptation and reflect the progressive adaptation of self-renewing cells to their culture conditions. However, the possibility of tumorigenesis can not be excluded, and further research is required in future work to empirically demonstrate or disprove this. Moreover, aberrant expression of CTNNB1, which is the epigenetically modified protein, induces malignant pathways in normal cells and abnormal activity of CTNNB1 also exists in malignant progression. In stem cells, expression of CTNNB1 might serve as a multifunctional protein with a central role in stem cell renewal and differentiation. Recent studies have shown that CTNNB1 could regulate Tert expression through the interaction with Klf4, and thereby telomere length, which could be critical in human cancer. Here, enhanced expression of CTNNB1 and increased expression target genes accompanied with more serious transformation in hESCs implied its role in stem cell-derived tumor initiation and progression.