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In vitro transformation of mesenchymal stem cell : the exploration of molecular mechanism and the development of monitoring system

间充质干细胞体外转化：分子机制探索及监测系统开发

基本信息

批准号：
18390414
负责人：
TOGUCHIDA Junya
金额：
$ 10.47万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18390414/
关键词：
Mesenchymal stem cell Transformation Mutation Methylation p16 発現誘導システム

项目摘要

Clinical applications of mesenchymal stem cells (MSC), one of tissue stem cells with multidirectional differentiation potential, have been conducted in a various fields of regeneration medicine. Transformation of transplanted MSC-derived cells is the most serious complication, for which no definite guideline has been established to monitor the process. In this research project, we aimed to establish the proper monitoring system to detect cells with genomic or epigenomic alterations at the earliest stage. At first, we have analyzed the growth profile of 29 cases of bone-marrow derived human MSCs. Median value of the culture period and the final population doubling (PD) was 151 days and 27 PD, respectively. The average length of telomere at the initial passage and the age of donor correlated with the number of final PD. Among cell cycle regulators, the expression level of p16 was closely associated with the number of PD and the morphological sign of cellular senescence. The inhibition of p16 expression successfully enabled cells to regain the growth potential and escape from senescence. Among 29 cases, the silencing of p16 expression took place during in vitro expansion in four cases, which was caused by the methylation of the core promoter region. One of these four cases, which continued to proliferate over 300 days, contained a chromosomal aberration. These results indicated the methylation of the p16 gene should be monitored as an early epigenetic alteration in ex vivo culture of MSC. Then we have established the rapid and sensitive method to detect the methylation of p16 gene by the combination of methylation specific PCR and Real time PCR detector. The entire process will be finished within 12hours and the sensitivity was 0.01%, meaning one cell with methylated p16 gene could be detected among 10,000 cells with unmethylated p16 gene.

间充质干细胞（MSC）是一种具有多向分化潜能的组织干细胞，其临床应用已在再生医学的各个领域进行。移植的MSC衍生细胞的转化是最严重的并发症，对此没有明确的指导方针来监测这一过程。在本研究项目中，我们旨在建立适当的监测系统，以在最早阶段检测具有基因组或表观基因组改变的细胞。首先，我们分析了29例骨髓来源的人MSCs的生长情况。培养期和最终群体倍增（PD）的中位值分别为151天和27 PD。首次传代时的平均端粒长度和供体年龄与最终PD数相关。在细胞周期调控因子中，p16的表达水平与PD的数量和细胞衰老的形态标志密切相关。抑制p16的表达成功地使细胞恢复生长潜能并逃避衰老。在29例中，4例p16表达沉默发生在体外扩增过程中，这是由核心启动子区甲基化引起的。这四个病例中有一个持续增殖超过300天，含有染色体畸变。这些结果表明，p16基因的甲基化应监测作为一个早期的表观遗传学改变在体外培养的MSC。建立了甲基化特异性PCR与真实的实时PCR检测仪相结合的p16基因甲基化检测方法。整个过程在12小时内完成，灵敏度为0.01%，即在10，000个p16基因未甲基化的细胞中可以检测到1个p16基因甲基化的细胞。