Human iPSC-derived mesenchymal stem cells (iMSCs) are an alternative to primary mesenchymal stem cells (MSCs), which have been a limited supply, and have attracted a great deal of interest as a promising cell source in cell-based therapy. However, despite their enormous therapeutic potential, it has been difficult to translate this potential into clinical applications due to the short viability duration of transplanted iMSCs. Therefore, to maximize the therapeutic effects of iMSCs, it is extremely important to extend their retention rate during and even after the transplantation. In this study, we developed a new extracellular matrix (ECM)-coating method involving the mild reduction of the cell surface. The reduction of disulfide bonds around the cell membrane enhanced the coating efficiency without a decrease in the viability and differentiation potential of iMSCs. We then induced ECM-coated single iMSCs to form three-dimensional spheroids via self-assembly of the aggregates within a physically confined microenvironment. The spheroids exhibited longer maintenance of the survival rate. Nanometric ECM coating of the cell membrane is a new approach as a key for resolving the conventional challenges of cell-based therapy.