Structurally Tunable Reduced Graphene Oxide Substrate Maintains Mouse Embryonic Stem Cell Pluripotency

Abstract

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.