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)，於未來提供人類各種疾病治療與再生醫學應用帶來無限希望。造血幹細胞(Hematopoietic stem cell﹐HSC)為目前研究最清楚之成體幹細胞(Adult Stem Cells﹐ASC)，並已成功應用於臨床上之治療。然而，細胞來源不足與移植後免疫排斥問題嚴重限制其臨床之廣泛發展。透過SCNT建立病人特異性胚幹細胞，於體外誘導再分化(Re-differentiation)成造血(幹)細胞，於臨床醫學應用上，將可提供充分之種子細胞來源，且免疫排斥問題亦迎刃而解。
吾等於近來研究發現，採用已分化之末端血球顆粒細胞(Granulocytes)，其複製成功效率遠高於未分化之HSC。然而，此等造血(幹)細胞之分化狀態是否可藉由後生遺傳修飾(Epigenetic modifications)機制進行修整，進而影響其後之複製胚生長潛能及ntESCs產製效率，仍待進一步釐清。其它研究證據顯示，ntESCs產製效率受其供核細胞之後生遺傳與分化狀態所左右。 Trichostatin A (TSA)，為一種組蛋白去乙醯轉移酶抑制劑，可增加組蛋白乙醯化及DNA去甲基化作用。因此，於SCNT試驗中，藉由此等後生遺傳試劑TSA之加入，期能改變細胞各分化階段特性，進而提高複製胚發育與其ntESCs產製效率。另一方面，此等源自造血細胞之ntESCs，經分化後，是否易被誘導而再分化成造血(幹)細胞？或以HSC為供核所產製的ntESCs，因HSC處於體細胞分化之源頭，於再程序化過程中，其“後生遺傳記憶” 是否可被完全磨滅？或其是否仍保有“歸航〞(Homing)之記憶而易被誘導分化成HSC？因此，本三年計劃擬由小鼠造血(幹)細胞模式–即長生命期造血幹細胞(long-term hematopoietic stem cell, LT-HSC)，短生命期造血幹細胞(short-term hematopoietic stem cell, ST-HSC)及分化末端之顆粒細胞探討：1. TSA是否可改變造血(幹)細胞特性，進而提升核移殖效率；2. 不同分化階段造血(幹)細胞對產製ntESCs效率之影響；3. 源自造血(幹)細胞之ntESCs，經分化後，被誘導再分化成造血(幹)細胞潛能。藉本計劃之執行，預期成果除包括提升整體ntESCs與其分化之造血(幹)細胞產製效率外，於基礎研究上，針對造血(幹)細胞核之再程序化(即去分化)及再分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: Therapeutic cloning or nuclear transfer stem cells, to produce patient-specific stem cells, hold great promise for the treatment of many human diseases. Previously, we surprisingly showed that differentiated cells in the same hematopoietic cell lineage are more efficient than adult stem cells as donors for somatic cell nuclear transfer (SCNT) based cloning. However, questions remained about whether epigenetic modification is responsible for differences in these cells’ different reprogramming capacity following SCNT for cloning development and derivation of ntESC lines. Trichostatin A (TSA), a histone deacetylase inhibitor (HADCi), increases histone acetylation and DNA demethylation. Thus, treating the haematopoietic cells with TSA at different differentiation stages may change the differences in their reprogramming capacity. Furthermore, if ntESC lines can be generated from different hematopoietic cell lineage such as long-term hematopoietic stem cells (LT-HSC), short-term HSC (ST-HSC), and terminally differentiated postmitotic granulocytes (Gr-1), it is still uncertain how different of ntESCs generation efficiency among these cell types, and whether HSC-derived ntESCs would amenable to differentiate into hematopoietic precursor cells following re-differentiation. The serial of experiments would need in order to understanding what kind of epigenetic modifications and mechanisms are involved in this epigenetic reprogramming and re-differentiation processes. Therefore, in this 3-year of research project is focus on using LT-HSC, ST-HSC, and Gr-1 cells derived from the same animal to investigate 1) whether TSA can change hematopoietic stem cells properties, with improved cloning efficiency; 2) the efficiency of ntESC derivation in different differentiation state of hematopoietic cell lineage; and 3) the potential of established ntESCs re-differentiated into transplantable HSC or more mature hematopoietic lineages following differentiation assays. The central hypotheses we want to test are that the TSA treatment can change the state of differentiation in hematopoietic cell lineage to improved cloning efficiency and created ntESCs eventually can be differentiate into transplantable HSC for treating blood disorders (i.e. bank for HSCs via cloning). Knowledge obtained from this system will not only answer the interesting question of whether “homing” capacity still remain in HSC-ntESCs that favor differentiation towards the hematopoietic precursor cells but also provide valuable knowledge to generate a limitless cell source that can be employed therapeutically.
hematopoietic stem cell