臺灣博碩士論文加值系統

建立一有效率的分化平台是利用萬能性幹細胞(pluripotent stem cells)進行細胞移植的必要先決條件。不論是發育中的胚胎進行神經轉換或以人類胚幹細胞(human embryonic stem cells, hESCs)進行神經誘導(neural induction, NI)其過程已知需活化纖維母細胞生長因子(fibroblast growth factor, FGF)訊號和抑制activin/nodal路徑。Wnt 訊號和Oct4的表現對於人類胚幹細胞維持萬能性(pluripotency)的狀態扮演著決定性的角色，然而此兩者在細胞分化上的機制卻仍有許多不明瞭之處。在我們的研究中證實了利用可以活化纖維母細胞生長因子訊號的人類鹼性纖維母細胞生長因子 (rh-basic FGF, FGF2)和抑制activin/nodal 路徑的activin抑制劑(inhibitor) SB431542一併投與具有Wnt致效劑(agonist)的小分子化合物BIO可將所有進行實驗測試的人類胚幹細胞有效率且快速的引發神經誘導。而某些人類誘導式萬能幹細胞(induced pluripotent stem cells, iPSCs)無法藉由活化鹼性纖維母細胞生長因子(FGF2)進行有效神經轉化的細胞也能在BIO/SB431542/ FGF2作用後高效率的分化出表現神經細胞早期標記Sox1的細胞。此外，在BIO與FGF2和SB431542的作用中BIO於神經誘導的初期階段具有促進分化中的細胞提升細胞存活率及短暫持續Oct4表現的作用。有趣的是在經由BIO/SB431542/ FGF2作用後的人類胚幹細胞於神經誘導後期階段其Oct4 表現的水平會迅速下降而引發具有前腦神經元(forebrain-neuron)特性的神經玫瑰環(neural rosettes)細胞形成。此研究亦證實Wn的活化在神經誘導時期於不同階段會有不同作用產生，亦即在神經誘導初期階段活化Wnt可維持人類胚幹細胞和誘導式萬能幹細胞於萬能性狀態，而這些細胞於神經誘導後期階段可能參與神經細胞譜系的發生。

Establishing an efficient differentiation procedure is prerequisite for the cell transplantation of pluripotent stem cells. Activating fibroblast growth factor (FGF) signals and inhibiting activin/nodal pathway are both conserved principles to direct the neural induction (NI) of developing embryos and human embryonic stem cells (hESCs). Wnt signal and Oct4 expression are critical for the hESC pluripotency, however, their roles in cell differentiation are largely unclear. We demonstrate that in the presence of FGF2 and activin inhibitor SB431542, applying a small-molecule Wnt-agonist, BIO, efficiently and rapidly steers the NI of all our tested hESCs. A human induced pluripotent stem cell (iPSC), which is refractory for efficient neural conversion by FGF2, effectively differentiated to Sox1+ cells after the BIO/SB431542/FGF2 treatment. In addition, BIO promoted cell survival and transiently sustained Oct4 expression at the early NI stage with FGF2 and SB431542. Interestingly, at the late NI stage, the Oct4 level rapidly declined in the treated hESCs and consequently initiated the formation of neural rosettes with forebrain-neuron characteristics. This study demonstrates the distinct effects of Wnt activation on maintaining pluripotency and committing neural lineages at the early and late NI stages of hESCs and iPSCs, respectively.

中文摘要………………………………………………………………………………I
英文摘要……………………………………………………………………………...II
目錄…………………………………………………………………………….……III
圖表目錄………………………………………………………………………….....V
第一章 前言與文獻探討 1
第一節 前言 1
第二節 文獻探討 2
(一) 人類胚幹細胞 2
(二) 誘導式萬能幹細胞 3
(三) 誘導式萬能幹細胞的運用 4
(四) 特定神經元的誘導 6
(五) 古典Wnt訊號路徑 11
(六) Activin/nodal inhibitor SB431542 12
第二章 實驗材料與方法 13
第一節 人類胚幹細胞和誘導式萬能幹細胞的培養 13
第二節 神經誘導(Neural induction, NI) 15
第三節 神經誘導效率分析…………………………………………………17
第四節 特定神經細胞的誘導 18
第五節 高效率神經誘導機制的探討 21
第六節 Trisomy21 AF iPS的神經誘導 22
第七節 實驗技術 24
第三章 結果 29
第一節 具有萬能性的人類胚幹細胞和誘導式萬能幹細胞 29
第二節 BiSF神經誘導獲得高度表現神經早期標記的神經前驅細胞 29
第三節 BiSF協同作用下快速又有效率的神經誘………………………30
第四節 BiSF適用於萬能幹細胞進行有效率的神經誘導 32
第五節 BiSF誘導的神經前驅細胞具有勝任繼續分化的能力 33
第六節 BIO促進Oct4表現促進了人類胚幹細胞的神經誘導 36
第七節 Trisomy21 AF iPS的神經誘導 38
第四章 討論 39
第五章 結論與未來計畫 43
第七章 參考文獻 44
第八章 圖表 55
附錄…………………………………………………………………………………..72

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