發展人類胚胎幹細胞株無餵養細胞培養系統 及比較其基因表現之研究

Development of feeder-free culture system for human embryonic stem cell lines and comparation of their expression profilings of mRNAs and microRNAs

蔡宗芸(Zong-Yun Tsai)

指導教授 ： 李水龍

高雄醫學大學/生命科學院/醫藥暨應用化學研究所/碩士(2008年)

https://doi.org/10.6832/KMU.2008.00086

摘要

人類胚胎幹細胞(hES cells)，為一種具備在體內體外自我更新及分化成三胚層能力的細胞，在組織修復移植、藥物篩檢研發、毒性測試、功能基因研究方面皆有應用潛力。根據之前的研究指出，人類胚胎幹細胞維持需依賴小鼠胚胎母細胞(MEF)作為餵養細胞，以維持細胞於未分化狀態。然而這樣的培養方式卻是隱藏許多危機，如異種動物帶來的病源體感染及用於移植時免疫排斥等問題需克服，此外調控人類胚胎幹細胞維持多功能分化能力的分子機制也尚未釐清，因此需要一個無餵養細胞的培養系統，進行相關的研究。此次研究首先是發展無餵養細胞系統，以人類胚胎幹細胞分化的類纖維母細胞作為餵養細胞及培養在細胞外基質上觀察人類胚胎幹細胞的生長並比較人類胚胎幹細胞(包括T3MF，正常核型的人類胚胎幹細胞培養在MEF上；T3CM，正常核型的人類胚胎幹細胞以MEF-conditioned medium培養在Matrigel上；T3BA，正常核型的人類胚胎幹細胞以添加4 ng/ml bFGF與5 ng/ml Activin A的培養基培養在Matrigel上等)在不同培養條件下基因(mRNAs 與microRNAs)的表現量。我們先以RT-PCR，real time qPCR與免疫螢光染色法比較了不一樣的培養條件，發現皆有OCT4與NANOG的表現，再將其中三種(T3MF,T3CM,T3BA)送至台大進行微矩陣基因表現分析，發現培養在無餵養細胞系統中以conditioned medium 培養的細胞(T3CM)表現相似於培養在MEF上的細胞(T3MF)。更重要的是在同時添加Activin A與bFGF的細胞(T3BA)中發現幾個up-regulated microRNAs，包括四個人類胚胎幹細胞特異的microRNAs：miR-372、302d,、367與200c，及三個非人類胚胎幹細胞特異的microRNAs：miR-199a、19a與217；而因Activin A表現量降低的down-regulated miRNAs有：miR-19b、221、 222,、let-7b與 let-7c，並找到幾個屬於miR-372, 302, 367, 217, 與19a的標的基因：NR4A2、ERBB4、 CXCR4、PCDH9、TMEFF2、 COX6A1 與 CD24。經由表現量較高的mRNA分析之間的關係，可以看出Activin A在細胞增殖與分化中的調控，這發現有助於釐清人類胚胎幹細胞維持型態與功能的途徑。

關鍵字

人類胚胎幹細胞 ； 無餵養細胞培養系統 ； 基因表現

並列摘要

Human embryonic stem (hES) cells have the capacities to be propagated for extended periods and to differentiate into cell types from all three germ layers both in vitro and in vivo. These characteristics of self-renewal and pluripotency enable hES cells having the potential to provide an unlimited supply of different cell types for tissue replacement, drug screening, and functional genomics studies. Previous reports have shown that maintenance of undifferentiated hES cells requires culturing on mouse embryonic fibroblast (MEF) feeders. However, this culture system contains animal-derived components that bear a risk of transmitting animal pathogens and incorporation of non-human immunogenic molecules to hES cells. Besides, the molecular mechanisms underlying pluripotency are not yet fully understood. Here a feeder-free culture system was developed and the expression profilings of mRNAs and mircoRNAs of hES cells cultured in different feeder-free conditions were compared with those cultured on MEF feeder. The hES-T3 cells with normal female karyotype cultured on MEF feeder (T3MF) and on Matrigel in MEF-conditioned medium (T3CM) were found to express very similar profiles of mRNAs and microRNAs, indicating that the unlimited self-renewal and pluripotency of hES cells can be maintained by continuing culture on either MEF in hES medium (containing 4 ng/ml bFGF) or feeder-free Matrigel in MEF-conditioned medium (supplemented with additional 4 ng/ml bFGF). However, the expression profiles, especially microRNAs, of the hES-T3 cells cultured on Matrigel in defined medium supplemented with 4 ng/ml bFGF and 5 ng/ml Activin A (T3BA) was found to be different from those of T3MF and T3CM cells. In T3BA cells, seven microRNAs (four hES cell-specific microRNAs miR-372, 302d, 367, 200c and three other microRNAs miR-199a, 19a and 217) were found to be up-regulated, whereas five miRNAs (miR-19b, 221, 222, let-7b and let-7c) were down-regulated by Activin A. Furthermore, seven abundantly (more than 3-fold of overall mean) differentially (more than 3-folds of changes) expressed mRNAs of NR4A2, ERBB4, CXCR4, PCDH9, TMEFF2, COX6A1 and CD24 genes targeted by miR-372, 302d, 367, 200c, 199a, 19a and/or 217 were identified by inverse expression levels of these seven microRNAs to their target mRNAs in T3MF, T3CM and T3BA cells. The abundantly expressed mRNAs in T3MF, T3CM and T3BA cells were also analyzed for the network and signaling pathways, and roles of Activin A in cell proliferation and differentiation were found. These findings will help elucidate the complex signaling network which maintains the hES cell phenotype and function.

並列關鍵字

human embryonic stem cells ； feeder-free ； microRNA

參考文獻

Alison, M.R., Poulsom, R., Forbes, S., and Wright, N.A. (2002). An introduction to stem cells. The Journal of pathology 197, 419-423.

Ambros, V. (2004). The functions of animal microRNAs. Nature 431, 350-355.

Amit, M., and Itskovitz-Eldor, J. (2006). Feeder-free culture of human embryonic stem cells. Methods in enzymology 420, 37-49.

Barnes, D.W., and Loutit, J.F. (1967). Haemopoietic stem cells in the peripheral blood. Lancet 2, 1138-1141.

Beattie, G.M., Lopez, A.D., Bucay, N., Hinton, A., Firpo, M.T., King, C.C., and Hayek, A. (2005). Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem cells (Dayton, Ohio) 23, 489-495.