探討免疫分化基因之表現：比較人類體細胞、多能性幹細胞與分化後細胞之差異性

Exploration of global immune-related genes of human somatic cells, pluripotent stem cells and their differentiated derivatives

吳佳恩(Chia-Eng Wu)

指導教授 ： 何弘能

國立臺灣大學/醫學院/免疫學研究所/博士(2017年)

https://doi.org/10.6342/NTU201701802

摘要

人類多能性幹細胞包括胚胎幹細胞(human embryonic stem cell; hESC)與誘導型多能幹細胞(induced pluripotent stem cell; iPSC)，過去被視為是臨床應用上分化成各種細胞的主要來源。人類胚胎幹細胞經分離囊胚細胞當中的內細胞群得以建立，而誘導型多能幹細胞為利用送入特定因子：Oct4, Sox2, Klf4, 與 c-Myc於體細胞中，使該細胞變成多能性幹細胞。之前研究顯示，人類多能性幹細胞可能透過減少主要組織相容性複合蛋白達成逃避免疫及降低免疫反應。然而研究多能性幹細胞是否引起免疫反應的結果具有爭議性。部分研究指出，多能性幹細胞確實在小鼠模式中引起免疫反應。為了提升多能性幹細胞的臨床應用，更重要的課題是探討已分化幹細胞的免疫性。本研究比較人類胚胎幹細胞、誘導型多能幹細胞與體細胞的免疫相關基因表現，並從中發現可能影響引起免疫反應的候選基因。藉由比較已分化幹細胞、未分化幹細胞以及包括真皮乳頭細胞、卵巢顆粒細胞與包皮纖維細胞等三種人類體細胞的免疫相關基因表現。結果顯示以下基因：CD24, GATA3, PROM1, THBS2, LY96, IFIT3, CXCR4, IL1R1, FGFR3, IDO1和KDR，其表現量之多寡與我們所選定的免疫相關基因相符。其中CD24, PROM1, FGFR3, IDO1和KDR基因於未分化幹細胞表現量較體細胞高，LY96, IFIT3和IL1R1基因於未分化幹細胞表現量較體細胞低。GATA3, CXCR4, PROM1, FGFR3和KDR基因於分化幹細胞表現量較體細胞高，THBS2基因於分化幹細胞表現量較體細胞低。雷帕黴素複合物的哺乳動物靶(mammalian target of rapamycin; mTOR)扮演調控人類免疫重要角色，雷帕黴素(rapamycin)更是器官移植時重要的免疫抑制劑。我們進一步利用雷帕黴素(rapamycin)探討雷帕黴素複合物的哺乳動物靶在已分化幹細胞中的角色。結果顯示抑制雷帕黴素複合物的哺乳動物靶訊息傳遞會導致對已分化幹細胞之免疫反應顯著降低。雷帕黴素處理之已分化幹細胞使CD24表現量降低，此結果顯示抑制雷帕黴素複合物的哺乳動物靶訊息傳遞調控CD24基因表現。進一步應用慢病毒轉導shRNA抑制CD24與GATA3表現導致已分化幹細胞中不同程度的調控免疫相關基因表現。此外，我們以混合淋巴細胞反應法探討對已分化幹細胞引起之免疫反應，結果顯示包含神經細胞等缺乏CD24與GATA3之已分化幹細胞，活化人類淋巴球能力較差。藉由分析免疫相關基因轉錄組，我們發現人類多能性幹細胞有分化為具有免疫表現型細胞的趨勢。因此，本研究發現的免疫相關候選基因，例如本研究提出的CD24與GATA3，可作為日後臨床應用之重要標的。

關鍵字

人類多能性幹細胞 ； 免疫性 ； 雷帕黴素複合物的哺乳動物 ； 移植

並列摘要

Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSCs), represent a potentially unlimited cell sources for clinical applications. hESCs are derived from the inner cell mass (ICM) of the early stage human blastocyst. The iPSCs are reprogrammed from somatic cells to pluripotent cells by transduction of the factors Oct4, Sox2, Klf4, and c-Myc. Previous studies have suggested that hPSCs may benefit from immune privilege and limited immunogenicity reflected by the reduced expression of major histocompatibility complex class (MHC)-related molecules. However, the immunogenicity of PSCs is still controversial. Some evidence shows that PSCs do activate immune response in immunocompetent mice. Moreover, before the application of hPSCs for clinical application, the most important issue is the immunogenicity of cells derived from PSCs. Here, we investigated the global immune-related gene expression profiles of hESCs, hiPSCs and somatic cells, and identified candidate immune-related genes that may alter their immunogenicity. The expression levels of global immune-related genes were determined by comparing undifferentiated and differentiated stem cells and in three types of human somatic cells: dermal papilla cells, ovarian granulosa cells, and foreskin fibroblast cells. We identified the differentially-expressed genes CD24, GATA3, PROM1, THBS2, LY96, IFIT3, CXCR4, IL1R1, FGFR3, IDO1, and KDR which overlapped with selected immune-related gene lists. Among these genes, CD24, PROM1, FGFR3, IDO1 and KDR were consistently significant upregulated, and LY96, IFIT3 and IL1R1 were consistently significant downregulated in undifferentiated stem cells compared with somatic cells. The genes GATA3, CXCR4, PROM1, FGFR3 and KDR were consistently upregulated, and only THBS2 was uniformly downregulated in 15-day differentiated stem cells compared with somatic cells. The mammalian target of rapamycin complex (mTOR) signaling pathway has been identified as a key mediator of human immunity and has been leveraged as a therapeutic strategy in tissue transplantation by using rapamycin. In further analyses, mTOR signaling was investigated in the differentiated stem cells following treatment with rapamycin. We found that the inhibition of mTOR signal pathways significantly downregulated immunogenicity of differentiated stem cells. CD24 expression was significantly reduced by rapamycin treatment in the differentiated stem cells. The data indicated mTOR pathway regulated CD24 in stem cell derivatives. Furthermore, reduced expression of CD24 and GATA3 by lentiviral transduction with specific shRNAs in stem cell derivatives leaded to differential regulation of multiple immune-related genes. We also tested the immune responses induced by differentiated stem cells by mixed lymphocyte reactions. We found that CD24- and GATA3-deficient differentiated stem cells including neural lineage cells had limited abilities to activate human lymphocytes. By analyzing the transcriptome signature of immune-related genes, we showed a tendency of the hPSCs to differentiate toward an immune cell phenotype. Taken together these data indicated candidate immune-related genes, in our case, CD24 and GATA3 that might constitute a valuable target for clinical applications.

並列關鍵字

Human pluripotent stem cells ； immunogenicity ； mTOR ； transplantation

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