幹細胞分化成各種型態細胞的能力稱之為多能性，但終端分化的體細胞則失去了這些多能性。在近年的研究中發現，將體細胞大量表現特定的幹細胞轉錄因子 (Yamanaka 因子, YFs) ，可使細胞重新編寫(reprogram)成具有多能性的細胞，這些即所謂的誘導性幹細胞 (iPSC)。然而YFs通常是透過反轉錄病毒介導方式送入，細胞中大量表現，導致基因體的嵌入現象，引發插入性突變嚴重的關切。隨著iPSC研究的進步，漸漸發展出許多安全性誘導iPSC產生的reprogramming方式，其中像是(1) 利用具有可回復的短暫通透性細胞膜之細胞，再加入ES 細胞的萃取液培養，(2) 或是利用帶有細胞通透序列的YFs重組蛋白，來誘導iPSC的產生。雖然這兩種方式避免了基因體的嵌入性突變，但其產生iPSC的效率非常低。因此我們想要利用結合這兩種方式，將具有通透性的體細胞同時加入帶有細胞通透序列的YFs重組蛋白，來增加iPSC的產生效率。首先，建立攜帶由Oct4啟動子驅動的報導基因Oct4-puromycine 或 Oct4-luciferase-IRES-GFP的穩定纖維母細胞株，接著利用鏈球菌溶血素 (SLO) 將細胞膜產生短暫通透性，最後再加入YFs重組蛋白促使細胞reprogram並產生多能性，並且檢測puromycin或是GFP的表現篩檢reprogramming的能力。現階段利用細菌系統表現的GST-Oct4、GST-Nanog、Sox2 LpRH、Lin28 LpRH 和 c-Myc LpRH蛋白，同時加入小分子物質 (VPA、BIX-01294和 EVR) 來誘導通透性纖維母細胞的多能性，目前已進行數次reprogramming 的實驗，但成功的比率偏低。未來希望可利用昆蟲系統表現YFs重組蛋白，增加具有轉譯後修飾作用YFs。如果成功誘導iPSC產生後，可再進一步測定細胞的多能性，確認是否會產生類似ES cell的colony現象，以及判定內部的基因表現情形，或是胚胎體 (embroid body)形成的分化測試。Stem cells can differentiate into most cell types while differentiated somatic cells have lost this pluripotency. Recent studies have shown that somatic cells can be reprogrammed by over-expression of defined transcription factors (Yamanaka factors, YFs) to become pluripotent stem cell (called as induced pluripotent stem cells, iPSC). Over-expression of YFs is usually mediated by retrovirus and whose integration into genome raises serious concern about the insertional mutagenesis. To develop safer iPSC, one reprogramming method has incubated reversibly permeable target cells in ES cell extracts and another has used recombinant cell–penetrating YFs proteins to generated iPSC. Although these two methods avoid insertional mutagenesis, unfortunately, they generate iPSC with only low efficiency. Therefore, we like to combine these two approaches by treating somatic cells with permeable system and cell-penetrating YFs proteins simultaneously to increase the reprogramming efficiency. Firstly, stable clones of fibroblasts carrying Oct4 promoter driven puromycine or IRES-GFP reporter genes were established, then, they were treated with streptolysin-O (SLO) to make their plasma membrane transiently permeable. Finally, recombinant YFs proteins will be added to reprogram them to pluripotent state and the reprogramming efficiency will be examined by puromycin or GFP selection. Currently, the pluripotency of permeable fibroblasts were induced by bacterially expressed GST-Oct4、GST-Nanog、Sox2 LpRH、Lin28 LpRH and c-Myc LpRH proteins in the presence of small compounds (VPA、BIX-01294 and EVR). Unfortunately, the reprogramming efficiency is rather low. In the future, YFs proteins will be expressed by insect cells that allow post-translational modification of these factors. Once iPSC clones have been generated, their pluripotent state will be examined by ES-like colony formation, gene expression profiling assays, and embryoid body formation.
