mRNA expression and promoter DNA methylation status of CDKi p21 and p57 genes in ex vivo expanded CD34+ cells following co-culture with mesenchymal stromal cells and growth factors

Background: Several studies indicate that ex vivo cytokine-based expansion of cord blood (CB) CD34(+) cells can induce cell cycle abnormality in expanded cells. Cycline-dependent kinase inhibitors, p21 and p57, are critical regulators of cell cycle. We investigated mRNA expression and promoter DNA methylation status of p21 and p57 genes in ex vivo expanded CD34(+) cells in different culture conditions. Materials and methods: Human CB CD34(+) cells were cultured either in cytokine-supplemented liquid culture or in co-culture with mesenchymal stromal cells (co-culture system) in presence and absence of cytokine. After 14 days, expansion fold in total nucleated cells (TNCs), CD34(+) cells, CD34(+)/CD38(-) cells and colony-forming units in culture (CFU-C) count was determined. Moreover, mRNA expression level and promoter DNA methylation status of p21 and p57 genes in expanded CD34(+) cells were analyzed by realtime reverse transcriptase-polymerase chain reaction (PCR) and methylation-specific PCR technique, respectively. Results: Maximum expansion of TNC, CD34(+) cells, CD34(+)/CD38(-) cells and CFU-C was seen at day 10 in all three culture conditions. p21 and p57 mRNA expression level decreased in both cytokine liquid culture and co-culture system with cytokine, while in the co-culture system without cytokine, the mRNA expression of p21 and p57 genes relatively increased. In addition, promoter DNA methylation status of p21 and p57 genes did not change during expansion in all three culture conditions. Conclusions: Co-culture of CB CD34(+) cells with mesenchymal stromal cells, especially in the absence of cytokines, maintained mRNA expression of p21 and p57 genes in ex vivo expanded CD34(+) cells. Moreover, transcriptional repression of p21 and p57 genes in ex vivo expanded CD34(+) cells was not mediated by promoter DNA methylation.