2019-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/692574摘要：體細胞核移置（somatic cell nuclear transfer, SCNT）技術，亦稱為動物複製，可將體細胞轉錄體（transcriptome）於12小時內迅速進行再程序化(reprogramming)，為一最有效且突破性地證明已分化細胞可被重編程至全能性（totipotent）階段，此等概念後續亦引領人類與動物“誘導性多能幹細胞（induced pluripotent stem cell, iPSC）”的建立。因源自病人體細胞所產製的“複製-胚幹細胞(ntESCs)”或“誘導性多能幹細胞”可達客製化，即病人專一性，不但為未來再生醫學發展帶來無限希望，並為人類各種疾病的個人化醫療與精準醫療的應用提供最有價值的研究模式。 計畫主持人為世界上首次發表以終極分化的顆粒性白血球細胞產製複製小鼠，其後研究並證明可有效衍生ntESCs的產製。近期研究團隊亦在Cell Reports首次發表具功能性修復端粒缺陷的客製化端粒綜合症全能幹細胞模式，發現端粒缺陷細胞在經過SCNT再程序化後，不但可穩固地延長其端粒長度，並可建立高度全能性的ntESCs，此ㄧ重要進展目前仍是最廣泛使用相對應細胞-- iPSC再程序化方式所無法企及。故探索優化SCNT所衍生的學理機制，將可於未來促進各類多能幹細胞的蓬勃發展。 核再程序化研究領域中懸而未解之謎：為何成功的小鼠複製或ntESCs僅能從雜交品系小鼠的卵母細胞質作為受核細胞（recipient oocyte）所產製？自從1999年，Wakayama團隊首先以BDF1雜交品系小鼠的受核卵母細胞成功報導世界上第一隻複製小鼠，而目前為止，無任何團隊（包括本團隊）曾以近親品系小鼠的卵母細胞質成功進行SCNT的體細胞再程序化。因此我們假設在雜交品系小鼠的卵母細胞質中，應含有某種特殊的”再程序化促進因子”主導細胞進行重編程的能力。 故本三年期研究計畫旨在：(1)探究BDF1雜交品系小鼠的卵母細胞質中所含的”再程序化促進因子”；(2)以SCNT與iPSCs的體細胞重編程平台，於體外分析鑒定候選之”再程序化促進因子”功能特性；(3)由前述兩項研究所得之”再程序化促進因子”，將其導入無複製能力的C57BL/6 (B6)近親品系小鼠的受核卵母細胞質內，以SCNT平台驗證其是否可提升細胞重編程能力，藉以探索再程序化機制。 由本計畫所提之研究若成功被驗證，不僅將解悉近二十年來圍繞困惑科學界的謎題，更可提供特殊見解對於如何進一步提升SCNT細胞再程序化能力，尤其是在發展人類醫療性複製（therapeutic cloning）時所需的人類卵母細胞來源短缺問題，更重要的是冀此等關鍵訊息可提供優化 iPSCs 或其他多能幹細胞再程序化所需之相關知識與技術應用創新，亦將引領轉譯醫學、再生醫學與精準醫療研究之卓越發展。 <br> Abstract: Somatic cell nuclear transfer (SCNT), also known as “cloning”, rapidly reprograms somatic transcriptome within 12 hours; it is an efficient and the first working means to reprogram a differentiated cell to the totipotent state, a concept later led to the development of induced pluripotent stem cells (iPSCs) in animals and humans. Embryonic stem cells derived using cloned blastocyst (referred to as “ntESCs”) and iPSCs can be patient-specific, therefore represent a great promise in regenerative medicine and serve as valuable model systems to study many human diseases in a precision medicine approach. The PI of the present project is among the first to report cloned mice using terminally differentiated postmitotic granulocytes, followed by efficient derivation of mouse ntESCs. Most recently her team reported that the telomeres of haplo-insufficient cells are properly restored in derivative ntESCs but not in iPSCs, adding evidence to the notion that ntESCs have certain advantages over their iPSC counterparts, and that improving ntESC efficiency and quality will help improving those of iPSCs and other pluripotent stem cells (PCSs). One mystery remains to be solved is why only recipient eggs from hybrid strains can be used for successful SCNT and ntESC derivation in mouse. Since the first cloned mouse report in 1999 by Wakayama who used hybrid strain (B6D2F1: C57BL/6 x DBA/2, or call BDF1) mouse oocyte as the recipient cytoplasm, to our knowledge, no group including us has ever reported successful reprogramming of somatic cells using oocyte cytoplast from an inbred mouse strain. Behind this mystery, we hypothesize that novel reprogramming promoting factors (RPFs) exist in the cytoplasm of hybrid strain mouse oocytes that render them the superior reprogramming capacity. In this three-year grant proposal, our aims are: (1) Determine the RPFs in hybrid mouse strain that promote somatic cell reprogramming; (2) In vitro characterizations of candidate RPFs for somatic cell reprogramming by SCNT and iPSC approaches; and (3) Improve SCNT reprogramming using “non- clonable” inbred C57BL/6 (B6) mouse cytoplast by the RPFs identified in Aim 1 and 2. The proposed work, if successfully implemented, will have major impact in the cellular reprogramming research field by not only solving the mystery associated with SCNT for almost two decades, but also providing novel insights on how to further improve the SCNT reprogramming for therapeutic cloning. This is especially important considering the scarce of available human oocytes. Importantly, the work will shed light on how to improve iPSCs qualities, as well as other cellular reprogramming means.體細胞核移置再程序化卵母細胞質&#63613多能幹細胞小鼠somatic cell nuclear transferreprogrammingcytoplasmpluripotent stem cellsmouse