Stem cells have tremendous promise to treat intractable diseases. Notably, adipose-derived stem cells (ADSCs) are actively being investigated because of ease of sampling and high repopulation capacity in vitro. ADSCs can exert a therapeutic effect through differentiation and paracrine potential, and these actions have been proven in many diseases, including cutaneous and inflammatory diseases. Transplantation of ADSCs necessitates therapeutic quantities and thus, long term ex vivo culture of ADSCs. However, this procedure can impair the activity of ADSCs and provoke cellular senescence, leading to low efficacy in vivo. Accordingly, strategies to restore cellular activity and inhibit senescence of stem cells during ex vivo culture are needed for stem cell-based therapies. This study evaluated a potential supplementary role of Substance P (SP) in ADSC ex vivo culture. After confirming that the ADSC cell cycle was damaged by passage 6 (p6), ADSCs at p6 were cultured with SP, and their proliferation rates, cumulative cell numbers, cytokine profiles, and impact on T/endothelial cells were assessed. Long-term culture weakened proliferation ability and secretion of the cytokines, transforming growth factor-beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), and stromal cell derived factor-1 alpha (SDF-1alpha) in ADSCs. However, SP treatment reduced the population doubling time (PDT), enabling gain of a sufficient number of ADSCs at early passages. In addition, SP restored cytokine secretion, enhancing the ADSC-mediated paracrine effect on T cell and human umbilical vein endothelial cells (HUVECs). Taken together, these results suggest that SP can retain the therapeutic effect of ADSCs by elevating their proliferative and paracrine potential in ex vivo culture. (C) 2017 Elsevier Inc. All rights reserved.