- 學位論文
探討小鼠胚胎幹細胞與誘導式多能幹細胞之分化差異
Study of Differences in Differentiation between Mouse Embryonic Stem Cells and Induced Pluripotent Stem Cells
摘要
胚胎幹細胞是一種從囊胚時期內細胞團所分離出來的多能性幹細胞，擁有自我複製及分化成三胚層所有細胞種類的能力。而誘導式多能幹細胞是利用四個轉錄因子Oct4、Sox2、Klf4、c-Myc轉染到體細胞中，利用後續的再程序化作用使體細胞回到原先類似胚胎幹細胞的狀態。由於誘導式多能幹細胞是從體細胞轉變而來的，取得比較便利同時也免除了倫理道德上的爭議，因此在發育學研究、藥物研發與篩選以及運用在自體細胞移植上，要比胚胎幹細胞更具有優勢。 在本研究中，我們探討小鼠胚胎幹細胞與誘導式多能幹細胞在神經分化上的差異性。在體外分化試驗中，我們利用了神經分化的誘導物“維他命A酸”來誘導兩株幹細胞分化成神經細胞。在經過維他命A酸處理過後，我們發現小鼠胚胎幹細胞比誘導式多能幹細胞分化出更多具有類似神經突出的細胞。為了釐清這種現象，我們利用即時定量聚合酶連鎖反應以及免疫細胞化學染色法分析兩種幹細胞所分化出來的細胞。分析結果顯示在維他命A酸處理過後，誘導式多能幹細胞比胚胎幹細胞分化出更多的中胚層的細胞。初步顯示誘導式多能幹細胞比胚胎幹細胞更容易往中胚層分化。另一方面我們也探討了維他命A酸受體在兩株幹細胞間的表現量，透過西方墨點法的分析，我們發現維他命A酸α型受體以及維他命A酸β型受體在兩株幹細胞間的表現量有很大的差異。在利用維他命A酸誘導分化之前與之後，維他命A酸α型受體在胚胎幹細胞的表現量皆高於誘導式多能幹細胞。而維他命A酸β型受體的表現量則是誘導式多能幹細胞高於胚胎幹細胞，然而在維他命A酸處理過後，維他命A酸β型受體在誘導式多能幹細胞中的表現量明顯地下降許多，並在誘導分化後第四天跟胚胎幹細胞相比達到顯著差異。為了更深入探討維他命A酸受體在神經分化上的作用，我們利用維他命A酸α型受體的抑制劑來抑制維他命A酸α型受體的活性。經過維他命A酸α型受體抑制劑的處理之後，我們發現在兩株幹細胞中維他命A酸α型受體及神經細胞特有的骨架蛋白 “β-微管蛋白III” 的基因表現量皆有下降的趨勢。並且在誘導分化後第四天和單獨處理維他命A酸的實驗組別相比，達到統計上的顯著差異。 總結研究結果，我們認為誘導式多能幹細胞具有較易往中胚層分化的特性，而維他命A酸α及β型受體在兩株幹細胞內表現量的差異，直接或是間接地影響這兩株幹細胞的神經分化。
並列摘要
Pluripotent stem cells possess the powerful ability to replicate indefinitely and can differentiate into various cell types derived from three germ layers. It has been reported that induced pluripotent stem cells (iPSCs) reprogrammed from somatic fibroblasts have been generated by transfecting four transcription factors including Oct4, Sox2, Klf4 and c-Myc. After the reprogram, iPSCs has provided great advantages in many applications, such as developmental studies, drug screening, and autologous cell transplantation. In this study, we compared the neural differentiation ability between two pluripotent stem cells, mouse embryonic stem cells (mESCs) and mouse induced pluripotent stem cells (miPSCs) with the neural inducer “retinoic acid (RA)” treatment. After RA-induction, both mESC- and miPSC-derived cells exhibited neuron-like processes. The potency of neurodifferentiation was different between the mESCs and the miPSCs after RA-induction. In order to clarify this phenomenon, we further characterized the mESC- and miPSC-derived cells by reverse transcription polymerase chain reaction (RT-PCR)/quantitative polymerase chain reaction (qPCR) and immunocytochemical approaches. More mesodermal lineage cells could be found from the miPSCs after RA-induction. Protein levels of retinoic acid receptors (RARs) involved in RA signaling pathway were also examined in both stem cells. The protein level of RARα in the mESCs was higher than that in the miPSCs. Using the RARα antagonist to attenuate RARα activity resulted in down-regulation of RARα and β-tubulin III in both stem cells. On the other hand, the level of RARβ was higher in the miPSCs before RA treatment, but was dramatically down-regulated after RA-induction in comparison with the mESCs. Our data indicated that the propensity of neuroectodermal differentiation could be correlated with the different distributions of RARα and RARβ induced by RA treatment between the mESCs and the miPSCs we used. We suggested that the cell memory of the miPSCs could be one of the key factors triggered the mesodermal differentiation. The neuroectodermal differentiation could be easily induced by RA treatment via RAR signal pathway in the pluripotent mESCs.