Proteomic and metabolomic analysis of H 2 O 2 -induced premature senescent human mesenchymal stem cells Ji-Soo Kim a , Eui-Jin Kim a , Hyun-Jung Kim a , Ji-Young Yang b , Geum-Sook Hwang b , Chan-Wha Kim a, ⁎ a School of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea b Korea Basic Science Institute, Seoul, Republic of Korea abstract article info Article history: Received 29 August 2010 Received in revised form 9 February 2011 Accepted 9 February 2011 Available online 4 March 2011 Section Editor: T.E. Johnson Keywords: Cellular senescence Mesenchymal stem cells Reactive oxygen species Proteomics Metabolomics Stress induced premature senescence (SIPS) occurs after exposure to many different sublethal stresses including H 2 O 2 , hyperoxia, or tert-butylhydroperoxide. Human mesenchymal stem cells (hMSCs) exhibit limited proliferative potential in vitro, the so-called Hayflick limit. According to the free-radical theory, reactive oxygen species (ROS) might be the candidates responsible for senescence and age-related diseases. H 2 O 2 may be responsible for the production of high levels of ROS, in which the redox balance is disturbed and the cells shift into a state of oxidative stress, which subsequently leads to premature senescence with shortening telomeres. H 2 O 2 has been the most commonly used inducer of SIPS, which shares features of replicative senescence (RS) including a similar morphology, senescence-associated β-galactosidase activity, cell cycle regulation, etc. Therefore, in this study, the senescence of hMSC during SIPS was confirmed using a range of different analytical methods. In addition, we determined five differentially expressed spots in the 2- DE map, which were identified as Annexin A2 (ANXA2), myosin light chain 2 (MLC2), peroxisomal enoyl-CoA hydratase 1 (ECH1), prosomal protein P30-33K (PSMA1) and mutant β-actin by ESI-Q-TOF MS/MS. Also, proton ( 1 H) nuclear magnetic resonance spectroscopy (NMR) was used to elucidate the difference between metabolites in the control and hMSCs treated with H 2 O 2 . Among these metabolites, choline and leucine were identified by 1 H-NMR as up-regulated metabolites and glycine and proline were identified as down-regulated metabolites. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Mesenchymal stem cells (hMSCs) are multipotent progenitors, which are able to self-renew and terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow (BM) stromal cells, fat, and other diverse connective tissues (Studeny et al., 2004). hMSCs also support the expansion of hematopoietic stem cells (HSCs) via cytokine expression and hematopoietic microenvironment recon- struction. Furthermore, they contribute to the homeostatic maintenance of many organs and tissues (Haynesworth et al., 1996; Majumdar et al., 1998; Sharpless and DePinho 2007). However, hMSCs exhibit a finite proliferative potential in vitro and undergo a limited number of po- pulation doublings (PD) before entering permanent growth arrest, which is referred to as “cellular senescence” (Campisi et al., 2001). Several studies have demonstrated that aging is, in part, associated with stem cell senescence that occurs as a result of extrinsic or intrinsic agents that cause DNA damage (Campisi and d'Adda di Fagagna 2007; Sharpless and DePinho 2007). Thus, it is important to investigate proteomic and metabolomic markers of cellular senescence in hMSCs for delaying senescence. Cellular senescence is an irreversible proliferation arrest that is triggered by activated oncogenes and, consequently, is an important tumor suppression process in vivo. Senescence is also caused by cellular stresses and inadequate growth condition (Ye et al., 2007). The rate of metabolism and cumulative oxidative damage to DNA and proteins, as well as genomic instability and mutations to mitochon- drial DNA, have all been implicated in determining the intrinsic rate of cell aging and ultimately, a species' life-span (Cevenini et al., 2008; Vijg and Campisi 2008). Similar to many proliferative cell types, including lung and skin human diploid fibroblasts (HDFs), human melanocytes, endothelial cells and human retinal pigment epithelial cells, hMSCs exposed to subcytotoxic stress (UV, tert-butylhydroperoxide (t-BHP), H 2 O 2 , ethanol, mitomycin C, hyperoxia, γ-radiations, homocysteine, hydroxyurea, etc.) undergo stress-induced premature senescence (SIPS) in vitro. H 2 O 2 has been the most commonly used inducer of SIPS, which shares several features of replicative senescence including a similar morphology, senescence-associated β-galactosidase activity, cell cycle arrest, etc. (Frippiat et al., 2001; Frippiat et al., 2002). Potential molecular markers for further investigation of senes- cence in hMSCs include p53, p16, pRB, Numb, NFk and ICAM-, all of which are known to be upregulated by senescence signals (Sethe Experimental Gerontology 46 (2011) 500–510 ⁎ Corresponding author at: School of Life Sciences and Biotechnology, Korea University, 1, 5-ka, Anam-dong, Sungbuk-ku, Seoul 136-701, Republic of Korea. Tel.: +82 2 3290 3439; fax: +82 2 3290 3957. E-mail address: cwkim@korea.ac.kr (C.-W. Kim). 0531-5565/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2011.02.012 Contents lists available at ScienceDirect Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero