Oodi Arezoo, Noruzinia Mehrdad, Habibi Roudkenar Mehryar, Nikougoftar Mahin, Soltanpour Mohamad Soleiman, Khorshidfar Mona, Amirizadeh Naser
Blood Transfusion Research Center, High Institute for Education and Research in Transfusion Medicine, Tehran, Iran.
Hematology. 2012 Nov;17(6):334-40. doi: 10.1179/1607845412Y.0000000009. Epub 2012 Sep 12.
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.
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.
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.
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.
由于脐带血造血干细胞（CB - HSC）数量不足，这些细胞的扩增对于成人临床应用似乎很重要。细胞周期抑制剂是正常造血再生中的重要调节因子。在本研究中，使用细胞因子和与间充质干细胞（MSC）饲养层的共培养系统，在CB - HSC体外扩增过程中评估了p16的mRNA表达和启动子甲基化状态。
在三种培养条件下对CB - HSC进行14天的体外培养：含MSC饲养层的细胞因子培养、不含MSC饲养层的细胞因子培养以及不含细胞因子的与MSC共培养。扩增后，进行细胞总数、CD34 +细胞测量以及集落形成单位（CFU）测定。使用甲基化特异性聚合酶链反应（PCR）分析p16（INK4a）基因启动子的甲基化状态，并通过实时逆转录PCR评估p16 mRNA表达。
在扩增的第10天观察到最大的CB - HSC扩增。数据显示，10天后，在不含细胞因子的共培养系统中扩增细胞中的p16 mRNA表达高于新鲜CD34 +细胞（P < 0.01）；然而，在含和不含MSC饲养层的两种细胞因子培养中扩增细胞中的p16 mRNA表达均降低。在培养的第5天、第10天和第14天，在所有三种培养条件下，扩增的CD34 +细胞的p16基因启动子与新鲜CD34 +细胞一样保持未甲基化形式。
在与MSC饲养层共培养的10天扩增过程中，正常造血再生中重要的细胞周期调节因子p16（INK4a）在造血干细胞中的表达增加，其破坏与白血病细胞发育有关。此外，未观察到p16启动子的甲基化，p16启动子甲基化能够引发一些白血病细胞进展或造血再生破坏。
