Elucidating the Secretion Proteome of Human Embryonic Stem Cell-derived Mesenchymal Stem Cells

Transplantation of mesenchymal stem cells (MSCs) has been used to treat a wide range of diseases, and the mechanism of action is postulated to be mediated by either differentiation into functional reparative cells that replace injured tissues or secretion of paracrine factors that promote tissue repair. To complement earlier studies that identified some of the paracrine factors, we profiled the paracrine proteome to better assess the relevance of MSC paracrine factors to the wide spectrum of MSC-mediated therapeutic effects. To evaluate the therapeutic potential of the MSC paracrine proteome, a chemically defined serum-free culture medium was conditioned by MSCs derived from human embryonic stem cells using a clinically compliant protocol. The conditioned medium was analyzed by multidimensional protein identification technology and cytokine antibody array analysis and revealed the presence of 201 unique gene products. 86–88% of these gene products had detectable transcript levels by microarray or quantitative RT-PCR assays. Computational analysis predicted that these gene products will significantly drive three major groups of biological processes: metabolism, defense response, and tissue differentiation including vascularization, hematopoiesis, and skeletal development. It also predicted that the 201 gene products activate important signaling pathways in cardiovascular biology, bone development, and hematopoiesis such as Jak-STAT, MAPK, Toll-like receptor, transforming growth factor-β, and mTOR (mammalian target of rapamycin) signaling pathways. This study identified a large number of MSC secretory products that have the potential to act as paracrine modulators of tissue repair and replacement in diseases of the cardiovascular, hematopoietic, and skeletal tissues. Moreover our results suggest that human embryonic stem cell-derived MSC-conditioned medium has the potency to treat a variety of diseases in humans without cell transplantation. Transplantation of mesenchymal stem cells (MSCs) has been used to treat a wide range of diseases, and the mechanism of action is postulated to be mediated by either differentiation into functional reparative cells that replace injured tissues or secretion of paracrine factors that promote tissue repair. To complement earlier studies that identified some of the paracrine factors, we profiled the paracrine proteome to better assess the relevance of MSC paracrine factors to the wide spectrum of MSC-mediated therapeutic effects. To evaluate the therapeutic potential of the MSC paracrine proteome, a chemically defined serum-free culture medium was conditioned by MSCs derived from human embryonic stem cells using a clinically compliant protocol. The conditioned medium was analyzed by multidimensional protein identification technology and cytokine antibody array analysis and revealed the presence of 201 unique gene products. 86–88% of these gene products had detectable transcript levels by microarray or quantitative RT-PCR assays. Computational analysis predicted that these gene products will significantly drive three major groups of biological processes: metabolism, defense response, and tissue differentiation including vascularization, hematopoiesis, and skeletal development. It also predicted that the 201 gene products activate important signaling pathways in cardiovascular biology, bone development, and hematopoiesis such as Jak-STAT, MAPK, Toll-like receptor, transforming growth factor-β, and mTOR (mammalian target of rapamycin) signaling pathways. This study identified a large number of MSC secretory products that have the potential to act as paracrine modulators of tissue repair and replacement in diseases of the cardiovascular, hematopoietic, and skeletal tissues. Moreover our results suggest that human embryonic stem cell-derived MSC-conditioned medium has the potency to treat a variety of diseases in humans without cell transplantation. Mesenchymal stem cells (MSCs) 1The abbreviations used are: MSC, mesenchymal stem cell; hESC, human embryonic stem cell; hESC-MSC, hESC-derived MSC; ITS, insulin, transferrin, and selenoprotein; CM, conditioned medium; NCM, non-conditioned medium; SCX, strong cation exchange; qRT, quantitative RT; mTOR, mammalian target of rapamycin; MAPK, mitogen-activated protein kinase; STAT, signal transducers and activators of transcription; Jak, Janus kinase; TGF, transforming growth factor; DMEM, Dulbecco's modified Eagle's medium; PDGF, platelet-derived growth factor; FA, formic acid; GO, gene ontology; ECM, extracellular matrix; LOD, limit of detection. 1The abbreviations used are: MSC, mesenchymal stem cell; hESC, human embryonic stem cell; hESC-MSC, hESC-derived MSC; ITS, insulin, transferrin, and selenoprotein; CM, conditioned medium; NCM, non-conditioned medium; SCX, strong cation exchange; qRT, quantitative RT; mTOR, mammalian target of rapamycin; MAPK, mitogen-activated protein kinase; STAT, signal transducers and activators of transcription; Jak, Janus kinase; TGF, transforming growth factor; DMEM, Dulbecco's modified Eagle's medium; PDGF, platelet-derived growth factor; FA, formic acid; GO, gene ontology; ECM, extracellular matrix; LOD, limit of detection. are multipotent stem cells that have been used in clinical and preclinical applications to treat a wide range of diseases (1Le Blanc K. Pittenger M. Mesenchymal stem cells: progress toward promise.Cytotherapy. 2005; 7: 36-45Abstract Full Text Full Text PDF PubMed Google Scholar, 2Reiser J. Zhang X.Y. Hemenway C.S. Mondal D. Pradhan L. La Russa V.F. Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases.Expert Opin. Biol. Ther. 2005; 5: 1571-1584Crossref PubMed Scopus (164) Google Scholar) including musculoskeletal tissue bioengineering (3Hui J.H. Ouyang H.W. Hutmacher D.W. Goh J.C. Lee E.H. Mesenchymal stem cells in musculoskeletal tissue engineering: a review of recent advances in National University of Singapore.Ann. Acad. Med. Singapore. 2005; 34: 206-212PubMed Google Scholar, 4Caplan A.I. Review: mesenchymal stem cells: cell-based reconstructive therapy in orthopedics.Tissue Eng. 2005; 11: 1198-1211Crossref PubMed Scopus (659) Google Scholar) and heart disease (5Menasche P. The potential of embryonic stem cells to treat heart disease.Curr. Opin. Mol. Ther. 2005; 7: 293-299PubMed Google Scholar, 6Laflamme M.A. Murry C.E. Regenerating the heart.Nat. Biotechnol. 2005; 23: 845-856Crossref PubMed Scopus (801) Google Scholar). They are routinely isolated from adult tissues such as bone marrow and adipose tissues and expanded ex vivo. Ex vivo expanded MSCs have lineage-restricted differentiation potential and can be induced to differentiate into mesenchymal lineages such as osteoblasts, chondrocytes, adipocytes, myocytes, tendon-ligament fibroblasts, and cardiomyocytes. Transplantation of these MSCs has been shown to enhance repair of musculoskeletal injuries, reduce tissue damage and improve cardiac function in ischemic heart disease, and ameliorate severity of graft versus host disease (1Le Blanc K. Pittenger M. Mesenchymal stem cells: progress toward promise.Cytotherapy. 2005; 7: 36-45Abstract Full Text Full Text PDF PubMed Google Scholar). Unlike embryonic stem cells, these lineage-restricted stem cells have negligible risk of teratoma formation (7Kinnaird T. Stabile E. Burnett M.S. Epstein S.E. Bone-marrow-derived cells for enhancing collateral development: mechanisms, animal data, and initial clinical experiences.Circ. Res. 2004; 95: 354-363Crossref PubMed Scopus (241) Google Scholar). The therapeutic capacity of MSCs to treat a wide spectrum of diseases has been attributed to their potential to differentiate into many different reparative cell types. However, the efficiency of transplanted MSCs to differentiate into functional reparative cells in the injured tissues or organs and in therapeutically relevant numbers has never been adequately documented or demonstrated. Recent reports have suggested that some of these reparative effects are not mediated by differentiation of MSCs but rather by paracrine factors secreted by MSCs (8Caplan A.I. Dennis J.E. Mesenchymal stem cells as trophic mediators.J. Cell. Biochem. 2006; 98: 1076-1084Crossref PubMed Scopus (2291) Google Scholar). These factors are postulated to promote arteriogenesis through paracrine mechanisms (7Kinnaird T. Stabile E. Burnett M.S. Epstein S.E. Bone-marrow-derived cells for enhancing collateral development: mechanisms, animal data, and initial clinical experiences.Circ. Res. 2004; 95: 354-363Crossref PubMed Scopus (241) Google Scholar); support the stem cell crypt in the intestine (9Leedham S.J. Brittan M. McDonald S.A. Wright N.A. Intestinal stem cells.J. Cell. Mol. Med. 2005; 9: 11-24Crossref PubMed Scopus (111) Google Scholar); protect against ischemic renal (10Togel F. Hu Z. Weiss K. Isaac J. Lange C. Westenfelder C. Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms.Am. J. Physiol. 2005; 289: F31-F42Crossref PubMed Scopus (1030) Google Scholar, 11Patschan D. Plotkin M. Goligorsky M.S. Therapeutic use of stem and endothelial progenitor cells in acute renal injury: ça ira.Curr. Opin. Pharmacol. 2006; 6: 176-183Crossref PubMed Scopus (51) Google Scholar), myocardial (12Miyahara Y. Nagaya N. Kataoka M. Yanagawa B. Tanaka K. Hao H. Ishino K. Ishida H. Shimizu T. Kangawa K. Sano S. Okano T. Kitamura S. Mori H. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction.Nat. Med. 2006; 12: 459-465Crossref PubMed Scopus (1022) Google Scholar, 13Gnecchi M. He H. Noiseux N. Liang O.D. Zhang L. Morello F. Mu H. Melo L.G. Pratt R.E. Ingwall J.S. Dzau V.J. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement.FASEB J. 2006; 20: 661-669Crossref PubMed Scopus (980) Google Scholar, 14Gnecchi M. He H. Liang O.D. Melo L.G. Morello F. Mu H. Noiseux N. Zhang L. Pratt R.E. Ingwall J.S. Dzau V.J. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells.Nat. Med. 2005; 11: 367-368Crossref PubMed Scopus (1354) Google Scholar, 15Mayer H. Bertram H. Lindenmaier W. Korff T. Weber H. Weich H. Vascular endothelial growth factor (VEGF-A) expression in human mesenchymal stem cells: autocrine and paracrine role on osteoblastic and endothelial differentiation.J. Cell. Biochem. 2005; 95: 827-839Crossref PubMed Scopus (266) Google Scholar), and limb tissue injury (16Nakagami H. Maeda K. Morishita R. Iguchi S. Nishikawa T. Takami Y. Kikuchi Y. Saito Y. Tamai K. Ogihara T. Kaneda Y. Novel autologous cell therapy in ischemic limb disease through growth factor secretion by cultured adipose tissue-derived stromal cells.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2542-2547Crossref PubMed Scopus (478) Google Scholar); support and maintain hematopoiesis (17Van Overstraeten-Schlogel N. Beguin Y. Gothot A. Role of stromal-derived factor-1 in the hematopoietic-supporting activity of human mesenchymal stem cells.Eur. J. Haematol. 2006; 76: 488-493Crossref PubMed Scopus (75) Google Scholar); and support the formation of megakaryocytes and proplatelets (18Cheng L. Qasba P. Vanguri P. Thiede M.A. Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34+ hematopoietic progenitor cells.J. Cell. Physiol. 2000; 184: 58-69Crossref PubMed Scopus (125) Google Scholar). This paracrine hypothesis will introduce a radically different dimension to the use of MSCs in regenerative medicine. Instead of using cells, repair of injured tissues will be mediated by enhancing endogenous tissue repair using biologics secreted by MSCs. This will bypass the present confounding issues associated with cell-based therapy, i.e. immune compatibility, tumorigenicity, xenozootic infections, costs, and waiting time for ex vivo expansion of autologous cell preparations. Such an approach will have a greater potential for the development of "off-the-shelf" MSC-based therapeutics at affordable costs and with better quality control and consistency. Numerous reports have invoked the secretion of paracrine factors as a mechanism for the reparative effects of MSCs on injured tissues (8Caplan A.I. Dennis J.E. Mesenchymal stem cells as trophic mediators.J. Cell. Biochem. 2006; 98: 1076-1084Crossref PubMed Scopus (2291) Google Scholar, 19Liu C.H. Hwang S.M. Cytokine interactions in mesenchymal stem cells from cord blood.Cytokine. 2005; 32: 270-279Crossref PubMed Scopus (191) Google Scholar). However, there has been no systematic or comprehensive profiling of the paracrine proteome that will enable an adequate assessment of the general validity of the paracrine hypothesis and the relevance of this paracrine hypothesis to the wide spectrum of MSC-mediated therapeutic effects. In the present study we assessed the secretion proteome of MSCs by performing multidimensional protein identification technology (20Washburn M.P. Wolters D. Yates III, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4062) Google Scholar) and cytokine antibody array analysis on a chemically defined culture medium conditioned by the HuES9.E1 MSC line, one of three lines that we have previously derived from a human embryonic stem cell (hESC) line using a clinically compliant protocol (21Lian Q. Lye E. Suan Yeo K. Khia Way Tan E. Salto-Tellez M. Liu T.M. Palanisamy N. El Oakley R.M. Lee E.H. Lim B. Lim S.K. Derivation of clinically compliant MSCs from CD105+, CD24− differentiated human ESCs.Stem Cells. 2007; 25: 425-436Crossref PubMed Scopus (248) Google Scholar). These hESC-derived MSC (hESC-MSC) lines fulfilled the minimal criteria of a multipotent MSC and share the basic distinctive characteristics of adult tissue-derived MSCs, i.e. these cell lines are plastic-adherent when maintained in standard culture conditions; express CD105, CD73, and CD90 and lack expression of CD45 and CD34; and can differentiate into osteoblasts, adipocytes, and chondroblasts (21Lian Q. Lye E. Suan Yeo K. Khia Way Tan E. Salto-Tellez M. Liu T.M. Palanisamy N. El Oakley R.M. Lee E.H. Lim B. Lim S.K. Derivation of clinically compliant MSCs from CD105+, CD24− differentiated human ESCs.Stem Cells. 2007; 25: 425-436Crossref PubMed Scopus (248) Google Scholar, 22Dominici M. Le Blanc K. Mueller I. Slaper-Cortenbach I. Marini F. Krause D. Deans R. Keating A. Prockop D. Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.Cytotherapy. 2006; 8: 315-317Abstract Full Text Full Text PDF PubMed Scopus (12259) Google Scholar). Although hESC-derived MSCs resemble adult bone marrow-derived MSCs, there are differences. For example, genes that were preferentially expressed in the HuES9.E1 MSC line are associated with embryonic processes such as proliferation and early developmental processes of embryogenesis and segmentation, whereas those in bone marrow-derived MSCs were over-represented in biological processes associated with more mature cell types such as metabolic processes, cell structure, and late developmental processes of skeletal development and muscle development (21Lian Q. Lye E. Suan Yeo K. Khia Way Tan E. Salto-Tellez M. Liu T.M. Palanisamy N. El Oakley R.M. Lee E.H. Lim B. Lim S.K. Derivation of clinically compliant MSCs from CD105+, CD24− differentiated human ESCs.Stem Cells. 2007; 25: 425-436Crossref PubMed Scopus (248) Google Scholar). This phenomenon is consistent with the observation that the biology of MSCs isolated from fetal and adult tissues is different and is characteristic of the developmental stage of their tissue of origin (24Gotherstrom C. West A. Liden J. Uzunel M. Lahesmaa R. Le Blanc K. Difference in gene expression between human fetal liver and adult bone marrow mesenchymal stem cells.Haematologica. 2005; 90: 1017-1026PubMed Google Scholar, 25Le Blanc K. Immunomodulatory effects of fetal and adult mesenchymal stem cells.Cytotherapy. 2003; 5: 485-489Abstract Full Text PDF PubMed Scopus (454) Google Scholar, 26Zhang H. Fazel S. Tian H. Mickle D.A. Weisel R.D. Fujii T. Li R.K. Increasing donor age adversely impacts beneficial effects of bone marrow but not smooth muscle myocardial cell therapy.Am. J. Physiol. 2005; 289: H2089-H2096Crossref PubMed Scopus (116) Google Scholar). In general, MSCs from younger donors or developmentally less mature tissues are more proliferative and have a more robust differentiation potential and greater therapeutic efficacy (24Gotherstrom C. West A. Liden J. Uzunel M. Lahesmaa R. Le Blanc K. Difference in gene expression between human fetal liver and adult bone marrow mesenchymal stem cells.Haematologica. 2005; 90: 1017-1026PubMed Google Scholar, 25Le Blanc K. Immunomodulatory effects of fetal and adult mesenchymal stem cells.Cytotherapy. 2003; 5: 485-489Abstract Full Text PDF PubMed Scopus (454) Google Scholar). Therefore by extrapolation, MSCs derived from embryonic hESC lines will be more proliferative and have a more robust differentiation potential and greater therapeutic efficacy than those derived from adult tissues. The use of these hESC-MSCs to characterize the secretory proteome of MSCs offers several advantages over the use of MSCs derived from adult tissues. 1) hESC-MSCs can be stably propagated in culture for at least 80 population doubling time as monitored by genome-wide gene expression profile and karyotyping using spectral karyotyping analysis (21Lian Q. Lye E. Suan Yeo K. Khia Way Tan E. Salto-Tellez M. Liu T.M. Palanisamy N. El Oakley R.M. Lee E.H. Lim B. Lim S.K. Derivation of clinically compliant MSCs from CD105+, CD24− differentiated human ESCs.Stem Cells. 2007; 25: 425-436Crossref PubMed Scopus (248) Google Scholar), thereby ensuring a stable and renewable source of cells for repeated verifications during proteomics profiling. This advantage is further bolstered by the reproducible generation of highly similar MSC cultures from either the same hESC line or different hESC lines (21Lian Q. Lye E. Suan Yeo K. Khia Way Tan E. Salto-Tellez M. Liu T.M. Palanisamy N. El Oakley R.M. Lee E.H. Lim B. Lim S.K. Derivation of clinically compliant MSCs from CD105+, CD24− differentiated human ESCs.Stem Cells. 2007; 25: 425-436Crossref PubMed Scopus (248) Google Scholar). 2) The use of hESCs in contrast to the use of adult tissues, e.g. bone marrow as the tissue source of MSCs, virtually ensures an infinitely renewable and consistent tissue source and enhances the reproducible and consistent batch to batch preparation of MSCs and therefore secretory products. 3) We have also demonstrated that the hESC-MSC lines derived using our protocol are highly similar to single cell-derived hESC-MSC cultures and are therefore homogenous (21Lian Q. Lye E. Suan Yeo K. Khia Way Tan E. Salto-Tellez M. Liu T.M. Palanisamy N. El Oakley R.M. Lee E.H. Lim B. Lim S.K. Derivation of clinically compliant MSCs from CD105+, CD24− differentiated human ESCs.Stem Cells. 2007; 25: 425-436Crossref PubMed Scopus (248) Google Scholar). Analysis of the secretory proteome of hESC-MSCs revealed a total of 201 unique gene products. 29 of these gene products have been previously reported to be secreted by adult tissue-derived MSCs. Four other proteins that were reported to be secreted by adult tissue-derived MSCs, namely IGFBP3, MIP3α, Oncostatin M, and TGFβ3, were not present in our list of 201 gene products. The spectrum of secreted gene products is consistent with the reported paracrine effects of MSCs on different diverse cellular systems and diseases (9Leedham S.J. Brittan M. McDonald S.A. Wright N.A. Intestinal stem cells.J. Cell. Mol. Med. 2005; 9: 11-24Crossref PubMed Scopus (111) Google Scholar, 10Togel F. Hu Z. Weiss K. Isaac J. Lange C. Westenfelder C. Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms.Am. J. Physiol. 2005; 289: F31-F42Crossref PubMed Scopus (1030) Google Scholar, 11Patschan D. Plotkin M. Goligorsky M.S. Therapeutic use of stem and endothelial progenitor cells in acute renal injury: ça ira.Curr. Opin. Pharmacol. 2006; 6: 176-183Crossref PubMed Scopus (51) Google Scholar, 12Miyahara Y. Nagaya N. Kataoka M. Yanagawa B. Tanaka K. Hao H. Ishino K. Ishida H. Shimizu T. Kangawa K. Sano S. Okano T. Kitamura S. Mori H. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction.Nat. Med. 2006; 12: 459-465Crossref PubMed Scopus (1022) Google Scholar, 13Gnecchi M. He H. Noiseux N. Liang O.D. Zhang L. Morello F. Mu H. Melo L.G. Pratt R.E. Ingwall J.S. Dzau V.J. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement.FASEB J. 2006; 20: 661-669Crossref PubMed Scopus (980) Google Scholar, 14Gnecchi M. He H. Liang O.D. Melo L.G. Morello F. Mu H. Noiseux N. Zhang L. Pratt R.E. Ingwall J.S. Dzau V.J. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells.Nat. Med. 2005; 11: 367-368Crossref PubMed Scopus (1354) Google Scholar, 15Mayer H. Bertram H. Lindenmaier W. Korff T. Weber H. Weich H. Vascular endothelial growth factor (VEGF-A) expression in human mesenchymal stem cells: autocrine and paracrine role on osteoblastic and endothelial differentiation.J. Cell. Biochem. 2005; 95: 827-839Crossref PubMed Scopus (266) Google Scholar, 16Nakagami H. Maeda K. Morishita R. Iguchi S. Nishikawa T. Takami Y. Kikuchi Y. Saito Y. Tamai K. Ogihara T. Kaneda Y. Novel autologous cell therapy in ischemic limb disease through growth factor secretion by cultured adipose tissue-derived stromal cells.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2542-2547Crossref PubMed Scopus (478) Google Scholar, 17Van Overstraeten-Schlogel N. Beguin Y. Gothot A. Role of stromal-derived factor-1 in the hematopoietic-supporting activity of human mesenchymal stem cells.Eur. J. Haematol. 2006; 76: 488-493Crossref PubMed Scopus (75) Google Scholar, 18Cheng L. Qasba P. Vanguri P. Thiede M.A. Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34+ hematopoietic progenitor cells.J. Cell. Physiol. 2000; 184: 58-69Crossref PubMed Scopus (125) Google Scholar, 27Kinnaird T. Stabile E. Burnett M.S. Shou M. Lee C.W. Barr S. Fuchs S. Epstein S.E. Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms.Circulation. 2004; 109: 1543-1549Crossref PubMed Scopus (1114) Google Scholar) and provides a molecular basis for the use of hESC-MSC-conditioned medium in local or systemic treatment of diseases including repair of the heart after myocardial infarction. HuES9.E1 cells were cultured as described previously (21Lian Q. Lye E. Suan Yeo K. Khia Way Tan E. Salto-Tellez M. Liu T.M. Palanisamy N. El Oakley R.M. Lee E.H. Lim B. Lim S.K. Derivation of clinically compliant MSCs from CD105+, CD24− differentiated human ESCs.Stem Cells. 2007; 25: 425-436Crossref PubMed Scopus (248) Google Scholar). 80% confluent HuES9.E1 cell cultures were washed three times with PBS and cultured overnight in a chemically defined medium consisting of DMEM without phenol red (catalog number 31053, Invitrogen) and supplemented with insulin, transferrin, and selenoprotein (ITS) (Invitrogen), 5 ng/ml FGF2 (Invitrogen), 5 ng/ml PDGF AB (Peprotech, Rocky Hill, NJ), glutamine-penicillin-streptomycin, and β-mercaptoethanol. The cultures were then rinsed three times with PBS, and then fresh defined medium was added. After 3 days, the medium was collected and centrifuged at 500 × g, and the supernatant was filtered using a 0.2-μm filter. For LC-MS/MS analysis, the conditioned medium was placed in dialysis cassettes with molecular weight cutoff of 3500 (Pierce), dialyzed against three changes of 10 volumes of 0.9% NaCl, then concentrated 20 times using Slide-A-Lyzer concentrating solution, and then dialyzed against 10 changes of 100 volumes of 0.9% NaCl before filtering with a 0.2-μm filter. The same volume of non-conditioned medium was dialyzed and concentrated in parallel with the conditioned medium. 1 ml of conditioned or non-conditioned medium was assayed for the presence of cytokines and other proteins using RayBio® Cytokine Antibody Arrays according to manufacturer's instructions (RayBio, Norcross, GA). Proteins in 2 ml of dialyzed conditioned medium (CM) or non-conditioned medium (NCM) were reduced, alkylated, and digested with trypsin as described previously (20Washburn M.P. Wolters D. Yates III, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4062) Google Scholar). The samples were then desalted by passing the digested mixture through a conditioned Sep-Pak C18 solid phase extraction cartridge (Waters, Milford, MA), washed twice with a 3% ACN (J. T. Baker Inc.) and 0.1% formic acid (FA) buffer, and eluted with a 70% ACN and 0.1% FA buffer. The eluted samples were then dried to about 10% of their initial volumes by removing organic solvent in a SpeedVac. The samples were kept at 4 °C prior to LC-MS/MS analysis. The desalted peptide mixture was analyzed by multidimensional protein identification technology with an LC-MS/MS system (LTQ, ThermoFinnigan, San Jose, CA). The sample was loaded into a strong cation exchange (SCX) column (Biobasic SCX, 5 μm, Thermo Electron, San Jose, CA) and fractionated by six salt steps with 50 μl of buffers (0, 2, 5, 10, 100, and 1000 mm ammonium chloride in 5% ACN and 0.1% FA) in the first dimension. The peptides eluted from the SCX column were concentrated and desalted in a Zorbax peptide trap (Agilent Technologies, Palo Alto, CA). The second dimensional chromatographic separation was carried out with a home-packed nanobored C18 column (75-μm inner diameter x 10 cm, 5-μm particles) directly into a PicoFrit nanospray tip (New Objective, Woburn, MA) operating at a flow rate of 200 nl/min with a 120-min gradient. The LTQ was operated in a data-dependent mode by performing MS/MS scans for the three most intense peaks from each MS scan. The mass spectrometer was operated at a nanospray voltage of 1.8 kV, without sheath gas and auxiliary gas flow, at an ion transfer tube temperature of 180 °C, at a collision gas pressure of 0.85 millitorr, and with normalized collision energy at 35% for MS2. The ion selection threshold was set to 500 counts for initiating MS2; activation quadrupole electric field was set to 0.25, and activation time was set to 30 ms. The MS scan range was 400–1800 m/z. Dynamic exclusion was activated with an exclusion duration of 1 min. For each experiment, MS/MS (dta) spectra of the six salt steps were extracted from the raw data files by the extract_msn program in Bioworks 3.2 (ThermoFinnigan). The extracted dta files were combined into a single file with Mascot generic file format by a home-written program. Except the conversion of precursor mass from MH+ in dta to m/z in the Mascot generic file, the fragment ion m/z and intensity values were used as is without any manipulation. Protein identification was achieved by searching the combined data against the Internation Protein Index (IPI) human protein database (version 3.15, 58,009 entries) via an in-house Mascot server (version 2.01). The search parameters were as follows: a maximum of three missed cleavages using trypsin; fixed modification, carbamidomethylation; and variable modification, oxidation of methionine. The mass tolerances were set to 2.0 and 0.8 Da for peptide precursor and fragment ions, respectively. The averaged Mascot identity score with significance threshold p < 0.05 is 41. A protein was accepted as a true positive if two or more different peptides from the same protein were found with ion scores greater than their Mascot identity score. The validated proteins were collated by removing the background proteins identified in the non-conditioned medium. The IPI identifier of each protein was then converted to the gene symbol by using the protein cross-reference table. Gene products were classified into the different biological processes or pathways of the gene ontology (GO) classification system using GeneSpring GX7.3 Expression Analysis software (Agilent Technologies), the frequency of genes in each process or pathway was compared with that in the GenBank™ human genome database, and those processes of pathways with significantly higher gene frequency (p < 0.05) were assumed to be significantly modulated by the secretion of MSC. Total RNA was extracted from HuES9.E1 cells with TRIzol reagent (Invitrogen) and purified over a spin column (Nucleospin RNA II System, Macherey-Nagel GmbH and Co., Düren, Germany) according to the manufacturer's protocol. 1 μg of total RNA was converted to cDNA with random primers in a 50-μl reaction volume using a High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). The cDNA was diluted with distilled water to a volume of 100 μl. 1 μl was used for each primer set in pathway-specific RT2 Profiler PCR Arrays (SuperArray, Frederick, MD) according to the manufacturer's protocol. The plates used for the analysis were Chemokines and Receptors PCR Array (catalog number APH-022), NFκB Signaling Pathway PCR Array (catalog number APH-025), Inflammatory Cytokines and Receptors PCR Array (catalog number APH-011), Common Cytokine PCR Array (catalog number APH-021), and JAK/STAT Signaling Pathway PCR Array (catalog number APH-039). To ensure that there was minimal contamination of conditioned medium by medium supplements such as serum replacement medium, HuES9.E1 MSCs were grown to about 80% confluency, washed three times with PBS, and incubated overnight in a chemic