摘要:單倍機能不全(Haplo-insufficient)之端粒酶基因包括TERT, TERC 及 DKC1,為主要造成人類先天性角化不全症與特發性肺纖維化等各種不同複雜疾病的致病原因,而因此類病人帶有一般性的端粒缺陷,故統稱為“端粒綜合症”。透過客製化全能性幹細胞的產製,則可提供治療此類疾病最獨特之病人特異性細胞來源。
由體細胞核移殖(somatic cell nuclear transfer, SCNT)所產製的客製化人類全能胚幹細胞(ntESCs)終於在2013年由 Mitalipov 所領導的研究團隊(Tachibana et al)成功地建立,此一企盼已久的結果可視為近年人類幹細胞研究及再生醫學領域最重要的突破性進展。本研究團隊續於2014年12月初,首先在Cell Reports 發表以端粒酶單倍機能不全的端粒綜合症小鼠模式,經過SCNT再程序化(reprogramming)後,發現端粒缺陷細胞不但可穩固地延長其端粒長度,並可建立高度全能性的客製化幹細胞,這是目前為止,第一且唯一篇文章報導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,此ㄧ重要進展目前仍未在最廣泛使用之“誘導性多功能幹細胞(induced pluripotent stem cell, iPSC)”的細胞再程序化方式於小鼠動物模式或病人細胞所報導。
本研究團隊擬延續此等重要研究項目,進一步探索有效修復端粒綜合症細胞的端粒再程序化關鍵因子,故本三年期計畫旨在:1) 建立端粒綜合症的 ntESCs 與 iPSCs 於長期培養狀態下的端粒再生動態變化過程,探討端粒綜合症全能幹細胞是否及其如何於長期培養狀態下維持全能性;2) 透過 RNA sequencing 分析比較端粒綜合症的 ntESCs 與 iPSCs 兩種細胞模式的差異,探討相關基因在端粒功能重編程過程中所扮演的角色;3) 建立端粒綜合症胚幹細胞Cas9 傳導的 Zscan4 基因致活系統,探討端粒長度如何透過非端粒酶機制維持其恆定性,此為基於我們近期在端粒綜合症胚幹細胞的研究顯示 Zscan4 於非端粒酶機制(telomerase-independent recombination-based)的端粒功能再生可能扮演關鍵性角色。
從幹細胞發展歷史的觀點上來說,由SCNT所衍生的理論知識,是為最初發展iPSC概念應用與技術成功的基礎,亦正如本計畫所提出由SCNT所致之端粒再程序化機制策略,洞悉優化病人特異性iPSC 端粒重編程的關鍵,尤其是對端粒綜合症病患更具意義。基於我們研究團隊及近來數篇知名國際期刊研究的報導,我們假設 ntESC 比起它的相對應細胞 — iPSC 具更佳的優勢,科學家應朝向提升 iPSC 的端粒長度至與 ntESC 相當的品質,持續注入更多相關的研究。本計畫研究團隊擁有於SCNT、核再程序化、全能幹細胞及端粒生物學相關之核心技術,透過本計畫之執行,除提供重要平台進行端粒生物學之基礎研究,尤其是在多種端粒綜合症幹細胞治療失敗與抗早衰的病理學機制的探索,於未來人類相關疾病治療與再生醫學發展應用上,冀此等重要訊息可提供優化 iPSC 或其他多能幹細胞的端粒再程序化所需之相關知識與技術,亦為轉譯醫學之發展邁入另一重要里程碑。
Abstract: Haplo-insufficiency of telomerase genes including TERT, TERC, and DKC1 in humans leads to a wide spectrum of diseases such as dyskeratosis congenital and idiopathic pulmonary fibrosis, collectively referred to as telomere syndromes because of the common telomere defects found in these patients’ cells. Generation of pluripotent stem cells from telomere syndrome patients’ own cells would provide unique opportunities towards the realization of patient-specific stem cell therapies in these patents.
In 2013, Tachibana et al reported the successful establishment of human embryonic stem cells (ntESCs) by somatic cell nuclear transfer (SCNT), a long-waited breakthrough in regenerative medicine. Recently, we reported in Cell Reports of the robust telomere elongation and high pluripotency in ntESCs of a mouse model of telomere syndromes. This is the first and the only report of functional repair of telomere defects in patient-specific stem cells using telomerase haplo-insufficient donor cells, a mission that has not been achieved by any other means including the widely used induced pluripotent stem cell (iPSC) method in mice and in humans.
In the present project, we propose to further advance this line of research with the ultimate goal to identify factors responsible for effective telomere reprogramming in Terc+/- ntESCs, and to develop protocols to improve telomere reprogramming in telomerase deficient iPSCs and other types of pluripotent stem cells (PSCs). In this three-year grant proposal, we aim to: (i) study whether and how telomerase deficient PSCs maintain their stemness and document the telomere regeneration dynamics in ntESCs and iPSCs of different Terc genotypes; (ii) directly compare ntESCs and iPSCs of different Terc genotypes by performing RNAseq to identify genes contributing to the successful telomere resetting process in Terc+/- ntESCs; and (iii) demonstrate the feasibility of modulating telomere homeostasis through telomerase independent mechanisms. Specifically, we propose to use the catalytically dead Cas9 (dCas9) system to modulate telomere homeostasis in Terc null stem cells by up-regulating Zscan4, a key player in telomerase-independent, recombination-based telomere elongation, which was differentially expressed in Terc deficient ntESCs.
Understanding SCNT-mediated telomere reprogramming, as proposed in the present project, would provide insights to how to optimally reset telomeres in patient-specific PSCs especially for telomere syndrome patients. Based on our findings and several recent major publications, we hypothesize that ntESCs have certain advantages over their iPSC counterparts, and advocate that at its minimum, researchers should aim to improve the iPSCs’ quality to the level of ntESCs. These ntESC lines are also valuable in vitro models to study telomere biology, especially considering the fact that stem cell failure is a common pathophysiological mechanism underling many of the telomere syndromes.