Zhao Jinping, Tang Mingliang, Cao Jing, Ye Dan, Guo Xudong, Xi Jiajie, Zhou Yi, Xia Yuchen, Qiao Jing, Chai Renjie, Yang Xiaowei, Kang Jiuhong
Clinical and Translational Research Center of Shanghai First Maternity and Infant Health Hospital School of Life Science and Technology Tongji University Shanghai 200092 China.
Institute for Regenerative Medicine Shanghai East Hospital School of Materials Science and Engineering Tongji University Shanghai 200092 China.
Adv Sci (Weinh). 2019 Apr 17;6(12):1802136. doi: 10.1002/advs.201802136. eCollection 2019 Jun 19.
Culturing embryonic stem cells (ESCs) in vitro usually requires animal-derived trophoblast cells, which may cause pathogenic and immune reactions; moreover, the poor repeatability between batches hinders the clinical application of ESCs. Therefore, it is essential to synthesize a xenogeneic-free and chemically well-defined biomaterial substrate for maintaining ESC pluripotency. Herein, the effects of structurally tunable reduced graphene oxide (RGO) substrates with different physicochemical properties on ESC pluripotency are studied. Colony formation and CCK-8 assays show that the RGO substrate with an average 30 µm pore size promotes cell survival and proliferation. The unannealed RGO substrate promotes ESC proliferation significantly better than the annealed substrate due to the interfacial hydrophilic groups. The RGO substrate can also maintain ESC for a long time. Additionally, immunofluorescence staining shows that ESCs cultured on an RGO substrate highly express E-cadherin and β-catenin, whereas after being modified by Dickkopf-related protein 1, the RGO substrate is unable to sustain ESC pluripotency. Furthermore, the cell line that interferes with E-cadherin is also unable to maintain pluripotency. These results confirm that the RGO substrate maintains ESC pluripotency by promoting E-cadherin-mediated cell-cell interaction and Wnt signaling.
体外培养胚胎干细胞（ESC）通常需要动物来源的滋养层细胞，这可能会引发致病和免疫反应；此外，批次间较差的重复性阻碍了ESC的临床应用。因此，合成一种无外源物且化学性质明确的生物材料基质以维持ESC的多能性至关重要。在此，研究了具有不同物理化学性质的结构可调还原氧化石墨烯（RGO）基质对ESC多能性的影响。集落形成和CCK-8测定表明，平均孔径为30 µm的RGO基质促进细胞存活和增殖。由于界面亲水基团，未退火的RGO基质比退火基质更能显著促进ESC增殖。RGO基质还能长时间维持ESC。此外，免疫荧光染色显示，在RGO基质上培养的ESC高表达E-钙黏蛋白和β-连环蛋白，而用Dickkopf相关蛋白1修饰后，RGO基质无法维持ESC的多能性。此外，干扰E-钙黏蛋白的细胞系也无法维持多能性。这些结果证实，RGO基质通过促进E-钙黏蛋白介导的细胞间相互作用和Wnt信号传导来维持ESC的多能性。
