间充质干细胞遗传稳定性研究

Despite mesenchymal stem cells can be easily isolated from many tissues，have multiple differentiation potential and extensive clinical value, the safety of its clinical applications have been questioned. Recent studies have found that long-term culture of human mesenchymal stem cells showing significant genomic variation [Cell Stem Cell 9:97], malignant transformation, and increased proliferative capacity, reduced differentiation potential [Cancer Res 69: 5331; Stem Cells 24:1095].In clinical practices, people usually use early passaged cells for treatment. A key question is: are short-term cultured mesenchymal stem cells safe? Whether stemness is still maintained? What tissue-derived mesenchymal stem cells safer? All these are important means to systematic evaluate mesenchymal stem cell genetic stability. In previous work, we have established 10 different mesenchymal stem cell lines derived from human bone marrow, umbilical cord, amniotic fluid respectively， prepared genomic DNA and RNA from generation cultured cells and did exome sequencing and RNA-seq sequencing. These experiments show that human mesenchymal stem cells from the early passaged cells do acquire dynamic genomic changes throughout their expansion in culture . Based on our previous preliminary work，in this application we will systematic study the genetic stability of short-term cultured (less than 15 generations) MSCs from different tissue sources. We plan to examine genomic mutation and transcriptome variation of MSCs using high throughput exome sequencing and RNA-seq sequencing，analyze the somatic mutation pattern, modification and reconstruction of key network, further predict the influences of these changes on MSCs tumorigenicity and stemness, and finally conduct experiments to examine the tumorigenicity of MSCs. The aim of this study is to provide guidance for MSCs clinical applications.

间充质干细胞来源广泛、具多向分化潜能和广泛临床应用价值, 但人们对其临床应用的安全性一直存在质疑。最近研究发现，长期培养人间充质干细胞系发生了明显的基因组变异[Cell Stem Cell 9:97], 间充质干细胞中会出现恶性转化细胞，其增殖能力变强，分化潜能降低[Cancer Res 69: 5331; Stem Cells 24:1095]。在临床试验中，常使用传代早期细胞进行治疗。然而关键问题是: 短期培养的间充质干细胞是否安全？干细胞干性是否维持？什么组织来源的间充质干细胞更安全? 我们已合作建立了骨髓、脐带、羊水等不同组织来源的间充质干细胞株，预试验发现其遗传稳定性在体外培养过程中呈动态变化。本申请拟在前期工作基础上，系统研究短期（15代内）培养的不同组织来源的间充质干细胞遗传稳定性以及遗传稳定性对间充质干细胞细胞命运和功能的影响，为间充质干细胞临床应用安全性评估提供理论基础。

间充质干细胞 (Mesenchymal stem cells, MSCs) 是一种多能成体干细胞，其来源广泛，免疫原性低，易于体外培养，具有多向分化潜能、免疫调节功能和营养因子分泌作用，在组织损伤、免疫疾病、退行性疾病等多种疾病的治疗方面有着有极高的临床应用潜力和价值，而对于间充质干细胞的临床应用，其安全性研究亟待解决。本研究项目利用了全外显子组和转录组测序技术，对4株脐带来源和3株羊水来源的间充质干细胞系在P0、P2、P5、P10、P15这五个培养代数采集样本并进行高通量测序，获得了大量间充质干细胞基因组和转录组测序数据。对不同时间点、不同来源样本的数据比较分析表明：1）间充质干细胞在中短期体外培养的过程中，基因组基本保持稳定；2）间充质干细胞具有异质性，细胞亚群在传代过程中发生动态变化；3）间充质干细胞高表达外分泌相关通路基因，基因表达模式在不同来源细胞间存在差异；4）不同来源的间充质干细胞具有特异性的细胞表面标志物，研究发现了一些可以作为鉴定间充质干细胞来源的候选标志物。此外，评估培养代数有利于早期传代，因为一些重要基因如HOX和gene 26在该过程中减少并且可能影响细胞分化和粘附功能。本研究首次将基因组与转录组相结合，在宏观角度对体外培养的不同来源间充质干细胞进行了遗传稳定性评价，对未来间充质干细胞临床应用的安全指导具有重要意义。