誘導性多潛能性幹細胞分化為感覺神經細胞的表型特性分析

Abstract

誘導性多潛能幹細胞 (Induced pleuripotent stem cell, IPSC)在近年來，逐漸成為一項相當蓬勃發展的研究工具，原因在於具有多重分化和自我更新的能力。在本篇研究中，我們將源自於三位健康個體的誘導性多潛能幹細胞分化為周邊神經。為了確認所分化出的神經細胞是屬於何種亞型的感覺神經細胞，我們使用免疫細胞化學染色和即時聚合酶連鎖反應 (Real-time Polymerase Chain Reaction, Real-time PCR)來檢測蛋白質標記的表現和基因的表現量。peripherin和neurofilament分別作為標定小直徑感覺神經元和大直徑感覺神經元的標記。基因，TrkA、TrkB、TrkC、PRPH和NF作為確認感覺神經分化是偏向何種譜系的指標。分化的感覺神經細胞以同時表現peripherin和neurofilament的細胞為主。在不同基因的表現量具有相當明顯的差異，像是TrkA和PRPH的基因表現量相較於TrkB、TrkC和NF要高出許多。我們調整分化用的培養基質條件，藉由減少CHIR99021，一種肝醣合成酶激酶-3的抑制劑的作用時間，並更進一步改變神經營養因子的添加種類。結果顯示利用不同分化條件所分化出來的細胞，會改變細胞增殖及neurofilament的表現量。本篇研究顯示誘導性多潛能幹細胞成功的分化出感覺神經細胞，並進一步探討小分子抑制劑和神經營養物質對於神經分化的影響。

Induced pluripotent stem cell (iPSC) has become a promising research tool in recent years due to the abilities of multi-differentiation and self-renewal. In this study, cell lineage of the iPSC from three healthy individuals were used to differentiate into peripheral sensory neurons. To confirm the phenotypes of differentiation, we applied immunocytochemistry and RT-PCR to examine protein and gene expressions. Peripherin and neurofilament were selected as markers for small diameter sensory neurons and large diameter sensory neurons respectively. TrkA, TrkB, TrkC, PRPH, and NF were chosen as indicators of differentiation lineage. The differentiated sensory neurons were mainly expressed peripherin(+)/neurofilament (+). There was a distinct pattern of gene expression i.e. much higher TrkA and PRPH expression than those of TrkB, TrkC, and NF. We modified the medium condition for differentiation by reducing the treatment duration of CHIR99021, a glycogen synthase kinase-3 inhibitor, and further modified the contents of trophic factors. The results indicated a change in cell proliferation and neurofilament expression according to different protocols. This study demonstrated successful differentiation of sensory neurons from iPSC and further investigation for the effect of small molecule inhibitor and neurotrophic factors in neuronal differentiation.