Background: Because of insufficient number of cord blood hematopoietic stem cells (CB-HSC), expansion of these cells seems to be important for clinical application in adults. Cell cycle inhibitors are important regulators in normal hematopoietic regeneration. In this study, mRNA expression and promoter methylation status of p16 were evaluated during CB-HSC ex vivo expansion using cytokines and a co-culture system with mesenchymal stem cells (MSCs) feeder layer.
Methods: ex vivo cultures of CB-HSCs were performed in three culture conditions for 14 days: cytokines with MSCs feeder layer, cytokines without MSCs feeder layer, and co-culture with MSCs without cytokine. After expansion, measuring total number of cells, CD34+ cells and colony-forming unit (CFU) assay was performed. Methylation status of the p16(INK4a) gene promoter was analyzed using methylation-specific polymerase chain reaction (PCR), and p16 mRNA expression was evaluated by real-time reverse transcriptase-PCR.
Results: Maximum CB-HSC expansion was observed in day 10 of expansion. The data showed that after 10 days, p16 mRNA expression in the expanded cells at the co-culture system without cytokine was higher than in CD34+ fresh cells (P < 0.01); however, p16 mRNA expression in the expanded cells at both cytokine cultures with and without MSCs feeder layer was decreased. p16 gene promoter of expanded CD34+ cells remained in unmethylated form just like fresh CD34+ cells in all the three culture conditions at days 5, 10, and 14 of culture.
Conclusion: Expression in HSCs of p16(INK4a), an important cell cycle regulator in normal hematopoietic regeneration disruption of which is involved in leukemic cell development, was increased during 10 days of expansion in co-culture with MSCs feeder layers. Also, no methylation of p16 promoter was observed, which is capable of initiating some leukemic cell progression or disruption in hematopoietic regeneration.