Expression and promoter methylation changes of the P15INK4b during ex vivo cord blood CD34+ cell expansion following co-culture with mesenchymal stromal cells.

BACKGROUND

Because of the 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 p15(INK4b) were evaluated during CB-HSC ex vivo expansion using cytokines and in co-culture system with a mesenchymal stem cells (MSCs) feeder layer.

METHODS

ex vivo cultures of CB-HSCs were performed in three culture conditions for 14 days: cytokines with an MSCs feeder layer, cytokines without a MSCs feeder layer, and co-culture with MSCs without cytokines. After expansion, measuring the total number of cells, CD34(+) cells, and CFU assay was performed. Methylation status of the p15(INK4b) gene promoter was analyzed using methylation-specific polymerase chain reaction and p15 mRNA expression was evaluated by real-time reverse transcriptase polymerase chain reaction.

RESULTS

Maximum CB-HSC expansion was observed on day 10 of expansion. The data showed that after 10 days, p15 mRNA expression in the expanded cells in all the three culture conditions was higher than in CD34(+) fresh cells (P < 0.01). p15 gene promoter of expanded CD34(+) cells remained in an unmethylated form just like fresh CD34(+) cells in all the three culture conditions at days 5, 10, and 14 of culture.

CONCLUSIONS

Expression of p15(INK4b) in HSCs was not decreased during ex vivo expansion. Also, no methylation of p15 promoter was observed, otherwise it would be capable of initiating some leukemic cell progression or disruption in hematopoietic regeneration.