Study on Mechanisms of Gene Silencing during Adult Stem Cell Differentiation

我们与加州大学洛杉矶分校牙科学院的西村一郎教授合作开展了这项研究项目。我们报道了小鼠骨髓基质干细胞（BMSC）在体外表达广泛的基因簇，这些基因簇是不同潜在终点的晚期表型的特征，并且引导分化通过沉默不一致的基因簇采用从广泛表型表达到特异性表型表达的独特转换（J Biol Chem 2005.）。此外，我们使用定制的细胞外基质相关基因聚焦微阵列来表征未诱导和成骨诱导的人BMSCs和脂肪组织来源的基质细胞（ASC）的基因表达特征。我们发现BMSCs和ASCs在成骨分化过程中都下调了离散的ECMG集，这表明成骨分化的BMSCs和ASCs从反映其多能性的多样化基因表达模式向特异性满足ECMG集的构型转变。 ...更多信息 目标谱系的要求。这种变化可能有助于使来自不同组织的干细胞混合群体中的基因表达正常化（Tissue Engineering，revision）。我们假设在成体干细胞分化过程中这种转录下调可能涉及表观遗传事件，如DNA甲基化。为了验证这一假设，我们进行了限制性标志基因组扫描（RLGS）方法来检测未诱导或成骨诱导的BMSCs基因组DNA上CpG岛内甲基化状态的变化。结果，大约1%的RLGS点改变了它们的存在，这意味着在成骨分化过程中一些CpG位点的甲基化状态发生了变化。进一步采用亚硫酸氢盐PCR方法分析未诱导和诱导成骨的BMSCs的骨钙素基因启动子区CpG甲基化状态。序列分析结果显示，在骨髓间充质干细胞向成骨细胞分化过程中，某些特定CpG位点的甲基化状态发生了改变，提示骨髓间充质干细胞向成骨细胞分化过程中下调了多组基因的表达，DNA甲基化和/或去甲基化参与了部分基因的沉默。少

We carried out this research project in collaboration with Professor Ichiro Nishimura in UCLA School of Dentistry. We reported that mouse bone marrow stromal stem cells (BMSCs) in vitro expresses a broad range of gene clusters characteristic of the advanced phenotypes of the diverse potential endpoints, and guided differentiation employ a unique conversion from broad to specific phenotype expression through silencing of discordant gene clusters (J Biol Chem 2005.).In addition, we used a custom-made extracellular matrix-related gene-focused microarray to characterize the gene expression signatures of uninduced and osteogenically-induced human BMSCs and adipose-tissue-derived stromal cells (ASCs). We found that BMSCs and ASCs both downregulate discrete ECMG sets during osteogenic differentiation, suggesting that osteogenically differentiating BMSCs and ASCs transition away from a diverse gene expression pattern reflecting their multipotency toward a configuration specifically meeting the … More requirements of the target lineage. This change may serve to normalize gene expression in mixed populations of stem cells derived from different tissues (Tissue Engineering, revision).We hypothesized that this transcriptional downregulation during adult stem cell differentiation may involve epigenetic events such as DNA methylation. To test this hypothesis, we carried out restriction landmark genomic scanning (RLGS) method to detect changes in methylation status within CpG islands on genomic DNA from uninduced or osteogenically-induced BMSCs. As a result, approximately 1% of RLGS spots changed their presence, implying methylation status of some CpG sites changed during osteogenic differentiation. We further analyzed CpG methylation status in osteocalcin gene promoter region of uninduced or osteogenically-induced BMSCs by bisulfite PCR method. The sequence data showed that methylation status of some specific CpG sites was altered during osteogenic differentiation.These results suggest that osteogenic differentiation of BMSCs downregulates sets of gene expressions, and DNA methylation and/or demethylation is involved in part in the gene silencing during the differentiation. Less