Clinical and industrial applications require large quantities of human induced pluripotent stem cells (hiPSCs); however, little is known regarding the mechanisms governing aggregate formation and stability in suspension culture. To address this, we determined differences in growth processes among hiPSC lines in suspension culture. Using an hiPSC aggregate suspension culture system, hiPSCs from different lines formed multicellular aggregates classified as large compact or small loose based on their size and morphology. Time-lapse observation of the growth processes of two different hiPSC lines revealed that the balance between cell division and the extent of subsequent cell death determined the final size and morphology of aggregates. Comparison of the cell survival and death of two hiPSC lines showed that the formation of small, loose aggregates was due to continued cell death during the exponential phase of growth, with apoptotic cells extruded from growing hiPSC aggregates by the concerted contraction of their neighbors. Western blot and immunofluorescent staining revealed that aggregate morphology and proliferative ability relied to a considerable extent upon secretion of the extracellular matrix (ECM). hiPSCs forming large compact and stable aggregates showed enhanced production of collagen type I in suspension culture at 120 h. Furthermore, these aggregates exhibited higher expression of E-cadherin and proliferation marker Ki-67 as compared with levels observed in small and loose aggregates at 120 h. These findings indicated that differences in both aggregate formation and stability in suspension culture among hiPSC lines were caused by differences in ECM secretion capacity. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.