摘要：治療性複製(Therapeutic cloning)為應用體細胞核移殖(Somatic cell nuclear transfer，SCNT)技術將病人身上取出之已分化細胞再程序化(Reprogramming)至相當於受精胚階段具有全能性(Totipotency)之複製胚，由此衍生之囊胚進而發展成為病人特異性胚幹細胞(Patient-specific embryonic stem cells, ntESCs)，於未來人類各種疾病治療與再生醫學之應用具有無窮發展潛力。迄目前為止，已有12種哺乳動物可成功透過體細胞核移殖技術產生複製個體，然而，整體之低複製效率仍面臨極大的挑戰。一般體細胞核移殖效率取決於1. 核移植囊胚之發育率，2.將核移植胚胎進行胚移殖至子宮後之複製個體產出率，3. 自核移植囊胚分離培养胚胎幹细胞之效率。理論上，身體各部位之任一型態細胞皆可被應用於體細胞核移殖之供核細胞。據吾等之近期研究於小鼠造血幹細胞模式結果顯示，採用已分化之末端血球顆粒細胞(Granulocytes)為供核細胞，其成功產製核移植胚胎及複製小鼠效率遠高於其他未分化之造血幹細胞。血球顆粒細胞，為人體中分佈最廣之一種白血球細胞，一般於骨髓中可儲存數天，一旦釋放至周邊血液循環則僅能維持存活數小時。因此，延續先前試驗，當前首要之務，勢必釐清分化之末端血球顆粒細胞使否對產製胚幹细胞效率有何影響。更進一步，若能證明源自周邊血液循環之血球顆粒細胞與源自骨髓者無異，透過簡易之採血方式即可取得有效之供核細胞作為產製病人特異性胚幹細胞來源，再加上不需經過培養，無性別及年齡限制之優點，無異將顯著提升其臨床醫學之應用價值。因此，本計畫旨在研究：1. 應用SCNT比較源自骨髓與周邊血液循環而來之血球顆粒細胞，其體細胞核移植後之發育潛能，2. 探討源自周邊血液循環而來之血球顆粒細胞，於體細胞核移殖後之胚胎幹细胞分離培养效率。藉本計劃之執行，預期成果除包括提升整體複製產製效率外，於基礎研究上，針對造血細胞核之再程序化調控機制將有更深入詳實之了解；於臨床醫學應用上，將提供重要訊息發展充分具功能性之種子細胞來源，冀未來於人類疾病治療上，取得進一步突破性之發展。
Abstract: Nuclear reprogramming by somatic cell nuclear transfer (SCNT) to produce patient-specific embryonic stem (ES) cell, which is know as therapeutic cloning, hold great promise in the field of regenerative medicine. Successful reprogramming of differentiated cell by SCNT has been reported in a dozen of mammalian species that resulted in the live clones. However, cloning efficiency by SCNT still remains extremely low, it can be evaluated through three different levels, including 1) the rate of cloned blastocyst formation, 2) term development after cloned embryo transplanted into the uterus, and 3) the frequency of deriving ES cell lines after explanation of cloned embryo in culture. In theory, any type of cell in the body can be used as donor cell for SCNT. It has been reported that the differentiation state of the donor cells can strongly influence the cloning efficiency. As general hypothesis that tissue-specific stem cells might serve as efficient donors for nuclear transfer because of the undifferentiated state of their genome. However, the correlation between the state of differentiation and cloning efficiency still remains controversial. We found previously that highly differentiated granulocytes were more efficient than primarily isolated hematopoietic stem/ progenitor cells (HSC/HPC) in yield cloned blastocyst and pups. This was consistent with the report of Inoue et al (2006). Recent attempts by Blelloch et al (2007) shown contrastive tendency in using neural cell as donors for nuclear transplantation, compared to terminally differentiated neuronal cells, neural stem (NS) cell could direct higher efficiency of NTES cell derivation following NT. These data implicated that resulted cloned offspring and NTES cell lines might govern by different level of epigenetic modifications. According to our previously founding in mouse hematopoietic system demonstrated that bone marrow derived-granulocytes given highest rate in resulted cloned blastocyst, it is important to understanding the potential of NTES cell derivation by this cell types, as well as the epigenetic modification during this reprogramming process. Moreover, the granulocytes, especial the neutrophil granulocytes are the most abundant white blood cells in human. It will be critical to test whether blood derived-neutrophil granulocyte can lead similar cloning efficiency as it derived from bone marrow, the result will certainly provide an ideal donor cell type for SCNT because of easy to collect from blood. Therefore, the aim of this proposal is to investigate 1) the development potential of terminally differentiated neutrophil granulocytes derived from peripheral blood after SCNT, and 2) the efficiency of NTES cell derivation in both of bone marrow derived-granulocytes and blood derived- granulocytes. The results will provide an excellent practical application in the research of therapeutic cloning.
embryonic stem (ES) cell