Human mesenchymal stem or stromal cell (hMSC) therapies have promise across a wide range of diseases. However, inefficient cell delivery and low cell survival at injury sites reduce efficacy and are the major barriers in hMSC-based therapy. Formation of three-dimensional (3D) hMSC aggregates has been found to activate hMSC functions from enhancing secretion of therapeutic factors for improving cell migration and survival. As the stromal cells in bone marrow, hMSCs are significant sources of extracellular matrix (ECM) proteins and growth factors, which form an interactive microenvironment to influence hMSC fate via paracrine and autocrine actions. To date, however, the impact of the extracellular microenvironment on hMSC properties in the aggregates remains unknown. In the present study, we investigated the role of endogenous ECM matrices on hMSC aggregate formation and survival under ischemic stress. The results demonstrated that the preservation of endogenous ECM in the aggregates formed by thermal lifting (termed TLAs) as opposed to the aggregates formed by enzymatically detached hMSCs (termed EDAs) enhanced cell proliferation, multilineage potential, and survival under ischemic stress. The improved cell proliferation and viability in the TLAs is attributed to the incorporation of endogenous ECM proteins in the TLAs and their promitotic and antioxidant properties. The results demonstrate a novel method for the formation of hMSC aggregates via thermal responsive surface and highlight the significant contribution of the ECM in preserving hMSC properties in the 3D aggregates. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 441451, 2013