Extensive effort has been made to develop a three-dimensional (3D) system for the culture of pluripotent stem cells in human and model animals, which yields lots of benefits for monitoring cell-to-cell or cell-to-environment interaction and for suggesting alternative materials for clinical cases. Initial study using animal model moved toward supporting embryonic stem cells (ESCs) self-renewal in a synthetic scaffold conjugated with suitable peptide motifs. As results, the feeder-free, 3D acellular niche consisting of vinyl sulfone (VS)-functionalized polyethylene glycol (PEG)-based hydrogel binding with extracellular matrix analogs could support ESC self-renewal, but main stemness signals were switched in the 3D environment. We employed this PEG-based hydrogel for 3D culture of human ESCs and further adjustment of hydrogel constituent made it possible to support self-renewal of three ESC lines. In this study, we examined transcriptional and translational activity of integrin heterodimers for optimizing the 3D system by using peptide motifs and subsequently elucidated that transcription and translation of integrin α5β1, α6β1 and αVβ5 were stronger than other heterodimers in a referenced human ESC line.