上皮黏附因子與Oct4或Klf4可以透過活化轉錄激活蛋白3與低氧誘導蛋白2a生成誘導性多能幹細胞

EpCAM/EpEX and Oct4 or Klf4 alone are sufficient to generate induced pluripotent stem cells through STAT3 and HIF2a

管奕奕(I-I Kuan)

指導教授 ： 吳漢忠

國立臺灣大學/生命科學院/生命科學系/博士(2017年)

https://doi.org/10.6342/NTU201700408

摘要

人類胚胎幹細胞和誘導型多能幹細胞（induced pluripotent stem cells, iPSC）是能夠自我更新和分化成三種胚層的多能幹細胞。iPSCs在再生醫學上可能是一個功能強大的工具。傳統的誘導型多能幹細胞是由四個轉錄因子：Oct4 (O), Sox2 (S), Klf4 (K), c-Myc (M) 誘導細胞而成。O，S，K，M現在被稱為山中伸彌因子，誘導機制仍不清楚。越來越多研究致力於尋求更容易，更好的重編程策略。但是，鮮少有人研究是否有其他分泌蛋白可以取代四個山中伸彌因子。我們研究發現，上皮細胞粘附分子（EpCAM）高度表現在未分化的胚胎幹細胞，而不會表現在已經分化的細胞。此外，EpCAM能直接調控多能性基因，包括c-Myc、Oct4、Nanog、Sox2、Klf4，有助於維持胚胎幹細胞的多能性。在本研究我們發現EpCAM能促進小鼠纖維母細胞重編程。EpCAM和其胞外結構 (EpEX) 有助於提升iPSCs集落形成和提高重編程的效率。我們進一步發現EpCAM結合單一山中伸彌因子Oct4，或Klf4，並與EpEX共同刺激，可以成功地重編程小鼠纖維母細胞成為iPSC細胞。我們進一步鑑定誘導型多能性幹細胞的多能性，OE + EpEX和KE + EpEX-iPSC基因表現與小鼠幹細胞相同。此外，在嵌合小鼠中證實OE + EpEX和KE + EpEX-iPSC可以分化成多種組織器官。EpCAM和EpEX可以透過STAT3-HIF2α的訊息傳遞促使HIF2α結合到山中伸彌因子的啟動子並增加小鼠纖維母細胞的重編程，研究成果有助於開發更高效率的重編程策略和維持幹細胞的多能性。這些新的發現可能在未來幹細胞研究，組織工程與臨床應用之發展有極大助益。

關鍵字

上皮細胞粘附分子 ； 誘導型多能性幹細胞 ； 山中伸彌因子 ； 重編程

並列摘要

Induced pluripotent stem cells (iPSCs) are generated from somatic cells, and have pluripotency and self-renewal ability. iPSCs holds great promising for its multiple application including drug screening, disease remolding, and even the possibility of autologous transplantation. However, until now, most of the iPSCs are generated by the transduction of four Yamanaka factors. The epithelial cell adhesion molecule (EpCAM), a type I transmembrane glycoprotein, is a potent stem cell surface marker. The intracellular domain of EpCAM (EpICD) upregulates reprogramming genes, including Oct4 (O), Sox2 (S), Klf4 (K), c-Myc (M), and Nanog (N), through association with their promoters. Therefore, we are prompted to investigate the functional roles of EpCAM in reprogramming of fibroblasts into iPSCs and the underlying molecular mechanisms. Here, we found that OSKM with EpCAM/EpEX increased the iPSC formation up to 2.4-fold compared to OSKM iPSCs. Interestingly, single Yamanaka factor, Oct4 or Klf4, but not Sox2, can successfully form iPSCs with the presence of EpCAM and EpEX. The characteristics of iPSCs generated by one Yamanaka factor with EpCAM (E) + EpEX were further confirmed by in vitro and in vivo assays. Moreover, these iPSCs could differentiate into three germ layers in teratoma and contribute to germline transmission in chimera mice. Furthermore, we demonstrated that EpEX and EpCAM can initiate reprogramming via activation of STAT3 signaling and nuclear-translocation of HIF2a, thereby providing a positive feedback loops for pluripotent circuit. Through our study, we present a new way of triggering reprogramming by membrane protein and soluble factor with single Yamanaka factor, and highlight the novel finding of EpCAM/EpEX-STAT3-HIF2a signals. Understanding these signals is of great interests for enhancing our fundamental knowledge of EpCAM in reprogramming process and establishing a new reprogramming strategy by delivery of membrane protein and soluble factors.

並列關鍵字

EpEX ； EpCAM ； induced pluripotent stem cells ； reprogramming

參考文獻

Suhr, S.T., Chang, E.A., Tjong, J., Alcasid, N., Perkins, G.A., Goissis, M.D., Ellisman, M.H., Perez, G.I., and Cibelli, J.B. (2010). Mitochondrial rejuvenation after induced pluripotency. PloS one 5, e14095.

Aasen, T., Raya, A., Barrero, M.J., Garreta, E., Consiglio, A., Gonzalez, F., Vassena, R., Bilic, J., Pekarik, V., Tiscornia, G., et al. (2008). Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nature biotechnology 26, 1276-1284.

Abramovich, A., Muradian, K.K., and Fraifeld, V.E. (2008). Have we reached the point for in vivo rejuvenation? Rejuvenation research 11, 489-492.

Agger, K., Cloos, P.A., Christensen, J., Pasini, D., Rose, S., Rappsilber, J., Issaeva, I., Canaani, E., Salcini, A.E., and Helin, K. (2007). UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature 449, 731-734.

Amoury, M., Kolberg, K., Pham, A.T., Hristodorov, D., Mladenov, R., Di Fiore, S., Helfrich, W., Kiessling, F., Fischer, R., Pardo, A., et al. (2016). Granzyme B-based cytolytic fusion protein targeting EpCAM specifically kills triple negative breast cancer cells in vitro and inhibits tumor growth in a subcutaneous mouse tumor model. Cancer letters 372, 201-209.