EpCAM 調控人類胚胎幹細胞之未分化表態之研究

Abstract

人類胚胎幹細胞 (簡稱為幹細胞) 為一群具多功能性的細胞，能自我更新並分化成三個胚層。到目前為止，仍須有更多明確的表面標記以用來鑑別幹細胞。上皮細胞黏著分子 (EpCAM) 為第一型穿膜醣蛋白，表達在一些前驅細胞或癌細胞，也曾被用來純化具癌症起始能力之癌細胞 (tumor-initiating cells)。本研究中，我們利用一株對抗EpCAM 單株抗體OC98-1，透過免疫螢光顯微、西方點漬法與流式細胞分析廣泛地研究EpCAM 的表現。我們發現EpCAM 高度專一地表達在未分化的幹細胞，而不表現在已分化的細胞中。其蛋白質與轉錄體 (transcript) 在幹細胞進行分化時即減少。此現象之背後調控機制與轉換組織蛋白 (histone) 的後修飾作用緊密相關。進一步地，我們證明兩個調控組織蛋白3離氨酸27 三甲基化 (lysine 27 trimethylation of histone 3 ; H3K27me3) 的分子，SUZ12 與JMJD3，可作用在EpCAM 起始子上。過去研究發現SUZ12 與JMJD3可維持幹細胞多功能性之恆定。此外，我們也利用核染質免疫沈澱分析 (chromatin immunoprecipitation; ChIP) 證明EpCAM 可直接調控數個改編基因 (reprogramming gene)，包括c-MYC、OCT-4、NANOG、SOX2 及KLF4，以利於維持幹細胞未分化的狀態。綜合以上結果，我們認為EpCAM 可作為幹細胞的表面標記，而EpCAM 在幹細胞內的表現，是透過表觀遺傳機制 (epigenetic mechanism) 所調控，並與維持幹細胞未分化特性緊密相關。

Human embryonic stem cells (hESCs) are pluripotent cells capable of selfrenewal and differentiation into all three germ layers. To date, more cell surface markers capable of reliably identifying hESCs are needed. The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein expressed in several progenitor cell populations and cancers. It has been used to enrich cells with tumorinitiating activity in xenograft transplantation studies. Here, we comprehensively profile the expression of EpCAM by immunofluorescence microscopy, Western blotting, and flow cytometry using an anti-EpCAM monoclonal antibody (MAb) OC98-1. We found EpCAM to be highly and selectively expressed by undifferentiated rather than differentiated hESCs. The protein and transcript level of EpCAM rapidly diminished as soon as hESC had differentiated. This silencing was closely and exclusively associated with the radical transformation of histone modification at the EpCAM promoter. Moreover, we demonstrated that the dynamic pattern of lysine 27 trimethylation of histone 3 (H3K27me3) was conferred by the interplay of SUZ12 and JMJD3, both of which were involved in maintaining hESC pluripotency. In addition, we used chromatin immunoprecipitation (ChIP) analysis to elucidate EpCAM’s direct regulation of several reprogramming genes, including c-MYC, OCT-4, NANOG, SOX2and KLF4, to help maintain the undifferentiation of hESCs. Collectively, our results suggest that EpCAM might be used as a surface marker for hESC. The expression of EpCAM may be regulated by epigenetic mechanisms, and it is strongly associated with the maintenance of the undifferentiated state of hESCs.