Uncovering the secretes of mesenchymal stem cells

Biochimie xxx (2013) 1e10

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Mini-review

Uncovering the secretes of mesenchymal stem cells Jessie R. Lavoie, Michael Rosu-Myles* Biologics and Genetic Therapies Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 May 2013 Accepted 18 June 2013 Available online xxx

Mesenchymal stem cells (MSC) show great promise in a wide array of therapeutic applications due mainly to their capacity to suppress immune and inflammatory reactions and instigate normal tissue repair processes. The secretion of bioactive factors is thought to play a predominant role in the mechanisms of action for these clinically relevant functions. As such, a large body of MSC research has focussed on characterization of the MSC secretome; including both soluble factors and factors released in extracellular vesicles (e.g., exosomes and microvesicles). This review provides an overview of our current knowledge of the MSC secretome in the context of determining the clinical relevance of these cells. In addition, the review summarizes various approaches that have been utilized to identify proteins secreted by MSC and discusses the advantages and limitations of different proteomic methods. Finally, we discuss issues that must be addressed before the clinical relevance of research into the MSC secretome can be realized. Crown Copyright Ó 2013 Published by Elsevier Masson SAS. All rights reserved.

Keywords: Mesenchymal stem cells Secretome Soluble factors Proteomics Exosomes Microvesicles

1. Introduction Mesenchymal stem cells (MSC) (also referred to as mesenchymal or multipotent stromal cells) were first discovered in the bone marrow in the early 1970s [1]. After more than three decades of research and an ever increasing number of published reports, MSC have been subsequently found in many tissues (umbilical cord blood, adipose, brain, liver, lungs [2e6]) and are seen as promising cells for the treatment of a wide variety of disorders, including cardiovascular, neurodegenerative and autoimmune diseases [7,8]. The therapeutic potential of MSC is well described through several pre-clinical studies using various animal models of disease, with recent investigations being more intensely focused on mechanisms of effect. Three main functions of MSC have been associated with their therapeutic effects; tissue replacement via multipotent differentiation [9,10], immunomodulatory and anti-inflammatory effects [11,12] and the secretion of molecules that instigate or assist in tissue repair (i.e., paracrine activity) [13,14]. Early studies of MSC focused primarily on their potential to differentiate into multiple tissue types. To date, there is a large body of evidence suggesting that MSC may have the capacity to form tissues from each of the three germ layers including adipocytes, osteocytes, myocytes (mesoderm), hepatocytes (endoderm) [3] and * Corresponding author. 251 Sir Frederick Banting Driveway, Tunney’s Pasture, Locator 2201E, Ottawa, Ontario, K1A 0K9, Canada. Tel.: þ1 613 952 8034; fax: þ1 613 941 8933. E-mail address: [email protected] (M. Rosu-Myles).

neurons (ectoderm) [15,16] (Fig. 1). However, with the possible exception of bone and cartilage formation, the differentiation of MSC into multiple cell types appears to be a rare event in vivo that is only modestly enhanced in some disease models. As well, biodistribution and engraftment studies in animal models of disease show that, regardless of the mode of administration or disease phenotype, MSC are relatively short-lived after injection, being detectable in various organs and tissues for periods ranging from 48 h to as long as 3 months [17,19]. Taken together, the current body of research seems to argue that multipotent differentiation contributes minimally to the beneficial effects attributed to MSC while immune/inflammatory suppression and paracrine activity play a more predominant role. The secretion of bioactive factors is thought to play a critical role in MSC-mediated immune/inflammatory suppression and paracrine activity. To gain a deeper understanding of the MSC secretome, and its potential relevance in a clinical setting, several comprehensive proteomic studies have recently been conducted. The majority of these studies have utilized either shotgun broad range scanning or targeted proteomic approaches (i.e., candidatebased) to uncover soluble or extracellular vesicle (EV) bound factors present in MSC conditioned media (CM). A subset of investigations have conducted cell-free-based animal studies with MSC-CM or CM-derived EVs (e.g., exosomes and microvesicles) to further demonstrate the therapeutic relevance of their findings. In this review, we summarize the current body of research regarding the MSC secretome and its potential relevance in a clinical setting, with an emphasis on bone marrow-derived MSC

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Please cite this article in press as: J.R. Lavoie, M. Rosu-Myles, Uncovering the secretes of mesenchymal stem cells, Biochimie (2013), http:// dx.doi.org/10.1016/j.biochi.2013.06.017

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Adipogenesis Adipocyte (Adipose tissue) Hepatogenesis Hepatocyte (Liver)

MSC

Epidermogenesis Keratinocyte (Skin)

Self-renewal

Central nervous system (Neurons, glial cells)

Chondrogenesis Chondrocyte (Cartilage)

Myogenesis Myotube (Muscle)

Ectoderm

Mesoderm

Osteogenesis Osteocyte (Bone)

Marrow stroma Stromal cell (Marrow) Tendogenesis/ligamentogenesis Fibroblast (Tendon/ligament)

Fig. 1. A schematic model of the differentiation potential of mesenchymal stem cells.

studies along with studies from other types of MSC. The review highlights proteins and other molecules identified to have a potential role in mediating the beneficial therapeutic effects demonstrated for MSC in pre-clinical studies. Further, we discuss current proteomics based methods for characterizing secreted proteins and outline some of the limitations that must be addressed before the therapeutic value of the MSC secretome can be realized. 2. Definition and biological characteristics of MSC MSC form a heterogeneous population of non-hematopoietic cells capable of self-renewal (i.e., multipotent) and differentiation into distinct mesodermal, endodermal and ectodermal cell lineages under appropriate culture conditions [9,20e22]. They reside in many adult tissues, such as bone marrow, adipose tissue, umbilical cord blood, liver, thymus, spleen and placentas [4,16,23e27]. MSC can be harvested from their resident tissues and expanded in culture over several passages, a property that contributes greatly to their clinical potential [3,23,28e30]. To foster a more uniform characterization of MSC across different tissue types and various culture conditions and to facilitate the comparison of data between research groups, the International Society for Cellular Therapy (ISCT) has proposed a minimal set of criteria to define MSC. These criteria include: (1) Plasticadherence when maintained in standard culture conditions; (2) The expression of CD105, CD73 and CD90 (95% positive) and lack of expression of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR surface markers (2% positive), as measured by flow cytometry; (3) In vitro trilineage differentiation to adipogenic, chondrogenic and osteogenic cells [31,32]. In addition, a recent publication by the ISCT and the International Federation of Adipose Therapeutics provided initial guidance for the scientific community regarding the minimal properties expected for adipose-derived cells (i.e., both stromal cells from the stromal vascular fraction (SVF) of the adipose tissue and the adipose tissue-derived stromal cells (ASC)) [33]. For instance, in the SVF, cells are identified phenotypically by a set of markers (CD45, CD235a, CD31, CD34þ) and functionally by the fibroblastoid colony-forming unit assay. For identification of the ASC, the authors recommend this set of markers in common with other MSC (CD90þ, CD73þ, CD105þ,

CD44þ, CD45, CD31) as well as two markers to distinguish them from BM-MSC (CD36þ, CD106). As for the other MSC, the trilineage differentiation assays (i.e., adipocyte, chondroblast and osteoblast) is recommended for cell identification and potency assessment in conjunction with a quantitative evaluation of the differentiation either biochemically or by reverse transcription polymerase chain reaction [33]. The implementation of these criteria internationally has greatly furthered the field of MSC research; however they do not reflect the true level of heterogeneity that exists in stromal cell cultures that contain MSC [24]. Thus, the ISCT criteria have become challenged of late by current broad based proteomic and transcriptomic approaches that may require more specific means of standardization to allow data interpretation and comparison among groups. MSC are ideal candidates for regenerative medicine, since they can be easily obtained from tissues currently used in clinical settings, such as bone marrow, adipose and umbilical cord blood. They can also be expanded in culture to provide sufficient numbers for clinical use. In addition, they possess low immunogenicity, likely due to a lack of HLA-DR and diminished HLA-I expression, thus negating the need for HLA matching and expanding their potential utility [34]. Hence, clinical interest in MSC has risen considerably over the last two decades, as shown by the increasing number of clinical trials registered in the public clinical trials database at ClinicalTrials.gov (http://clinicaltrials.gov). At the time of writing, the registry contained over 300 trials aimed at testing the safety and efficacy of MSC in treating a myriad of diseases, and one MSCbased health product (Prochymal [remestemcel-L]) has been given conditional market approval in Canada for treating pediatric acute graft versus host disease. An overview of the recent clinical findings related to MSC therapeutic effects have been thoroughly reviewed previously and the reader is referred to these comprehensive reports for further details [35e37]. 3. Functional roles of the MSC secretome 3.1. Immune and inflammatory modulation The ability of MSC to modulate the immune system seems to play a role in almost all treatment effects attributed to these cells.

Please cite this article in press as: J.R. Lavoie, M. Rosu-Myles, Uncovering the secretes of mesenchymal stem cells, Biochimie (2013), http:// dx.doi.org/10.1016/j.biochi.2013.06.017

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Suppression of cell-mediated immune reactions was first reported by Di Nicola et al. by co-culturing irradiated bone marrow derived MSC with mixed lymphocyte reaction (MLR) or dendritic cell stimulated T-cells [38]. MSC were found to inhibit the proliferation of both CD4þ and CD8þ T-cells and were able to do so in the absence of direct cell contact. This study has been subsequently repeated by others who have further demonstrated that the function of both Th1 and Th2 CD4þ T-cell subsets as well as dendritic cells and natural killer cells could be suppressed by MSC [11]. Overall, three major aspects of the immunomodulatory action mediated by MSC have been reported: cell-to-cell contact, production of inhibitory molecules and induction of regulatory T-cells. The release of soluble immunosuppressive factors has been shown in a few studies to play a major role and has led investigators to assess the therapeutic potential of MSC-derived molecules in immunological conditions. For instance, using computed tomography of adoptively transferred leukocytes, Parekkadan et al. have shown in a rat model of fulminant hepatic failure, that conditioned media from cultured MSC can functionally divert immune cells from the injured organ [39]. This data indicates that factors secreted by MSC can alter immune reactions in the diseased liver by reducing leukocyte migration to the tissue. In several human trials, MSC-induced suppression of T-cellmediated immunity has been demonstrated to have clinical significance for treatment of graft versus host disease (GvHD) [40e 42]. Unfortunately, positive clinical outcomes remain inconsistent and the complete mechanism of how MSC are able to affect cellmediated immunity is still not fully understood [43e45]. Further mechanistic studies and proteomic characterization of MSC may be useful for improving the efficacy of MSC-based products for treating GvHD. Innate/inflammatory immune responses are the body’s first line of defence against infection or tissue damage. Excessive or inappropriate inflammation, such as that which occurs during sepsis or severe allergic reactions, can also have detrimental effects on various organs and tissues. In 2009, the Mezey group at the US National Institutes of Health performed an elegant set of experiments to show that bone marrow-derived MSC were capable of suppressing sepsis-induced severe inflammatory responses in mice. In these experiments, MSC were found to reduce sepsis-related mortality by acting directly on macrophages through IL-10, Toll-like receptor 4 and prostaglandin E2-dependent mechanisms [46]. These studies have since been repeated by other groups [47,48] and have lead to the initiation of clinical trials to determine the suitability of using MSC for treating sepsis [49]. MSC have been shown to suppress inflammatory reactions in a variety of different disease states or damaged tissues [18,50e53]. Interestingly, the mechanisms by which MSC suppress inflammation seem to be specific to the cause of inflammation. In the case of ragweed-induced asthma, for example, MSC suppressed Th2mediated inflammation in a manner that involved TGF-beta secretion and the activation of IL-4- and IL-13-induced STAT6 pathway [52]. In the environment set by interstitial lung disease, inflammation was suppressed by MSC through mechanisms involving TNF-alpha and IL1R [51] while in a mouse model of acute myocardial infarction (MI), cardiac inflammation was suppressed through MSC production of TSG-6 [18]. Even during tissue repair, MSC-mediated immune modulation participates in the overall effect by reducing tissue damage and scarring. A large number of studies have provided strong evidence that MSC contribute to the regeneration of damaged organs in vivo, in part due to modulation of the host immune response. This process has been reviewed extensively by others [54,55].

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3.2. Paracrine effects on tissue repair In animal models, MSC have been clearly shown to promote regeneration of damaged organs and tissues without a high incidence or duration of engraftment [56,57]. This body of work supports the paracrine hypothesis which is the idea that MSC secrete bioactive molecules that promote a regenerative microenvironment. Direct evidence that paracrine activity plays a role in mediating tissue repair by MSC has been generated from studies on MSC-CM [13,14,53,58]. To date, the therapeutic response of CM has been tested in disease models for lung injury [13], chronic kidney disease [53] and liver injury [14,58], among others, and demonstrate that MSC-CM alone is sufficient to mediate lasting therapeutic effects. For instance, Van Poll et al. provided clear evidence that MSC-CM therapy in a D-galactosamine-induced rat model of acute liver injury could provide a significant survival benefit, a 90% reduction of apoptotic hepatocellular death and an increase in the number of proliferating hepatocytes, which altogether create a new avenue for the treatment of fulminant hepatic failure [58]. In a mouse model of excisional wound healing, Chen et al. demonstrated that CM from murine BM derived MSC accelerated wound healing though promoting endothelial progenitor cell growth and macrophage recruitment [59]. Recent reports have also demonstrated that EVs produced by MSC, also participate in the tissue repair. Specifically, using a rat-based middle cerebral occlusion model of stroke Xin et al. demonstrated that MSC secrete exosomes containing miR-133b RNA that significantly enhance brain remodelling and recovery by regulating gene expression in astrocytes and neurons [60]. In all, there is a plethora of information demonstrating that soluble factors and EVs within the secretome provide a major contribution to the paracrine activity attributed to MSC. It is evident from the current body of research that immune/ inflammatory modulation and paracrine factors contained within the MSC secretome function in a co-operative manner to generate a tissue microenvironment that is more permissive of repair/regeneration (Fig. 2). In the last decade, investigators have made progress towards characterizing CM and EVs derived from MSC. These studies have identified numerous candidate modulators of paracrine effects and cell-mediated/inflammatory suppression and are described in detail in the sections below and in Table 1. 4. Proteomic studies on MSC-CM To better understand the content of the secretome contributing to therapeutic effects, investigators have begun to characterize CM from MSC using various proteomic approaches. In the following sections, we highlight some of the published literature regarding this work. For ease, we have categorized these studies into two groups: those that have used a targeted proteomic approach and those that have used a shotgun-based proteomic approach (i.e., proteomic screen) (Fig. 3). Secretion of bioactive factors EV

Immune/inflammatory modulation

Soluble factors

Co-operative effects

Paracrine activity

Tissue repair/regeneration Fig. 2. Summary of the potential therapeutic roles attributed to the mesenchymal stem cell secretome.

Please cite this article in press as: J.R. Lavoie, M. Rosu-Myles, Uncovering the secretes of mesenchymal stem cells, Biochimie (2013), http:// dx.doi.org/10.1016/j.biochi.2013.06.017

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Species/MSC origin

MSC characteristics

Disease model (or shotgun approaches)

Proteomic technique

CM/EV

Proteins responsible for therapeutic effect

Functions of proteins responsible for therapeutic effect

References

Human/bone marrow Human/NA

Plastic-adherent

Nerve regeneration

ELISA

CM

ND

ND

Brohlin M. et al. Plos One 2012

Plastic-adherent

Myocardial infarction

ELISA

CM

VEGF, IL-6, HGF, PlGF, adrenomedullin

ND

Plastic-adherent, in vitro differentiation (adipocytes, osteoblasts, chondrocytes), Sca-1þ, CD29þ, CD44þ, CD105þ, CD90þ, CD34, CD45, CD14, CD3, CD19 Plastic-adherent, CD44þ, CD90þ, CD105þ, CD34 Plastic-adherent, CD44þ, CD105þ, CD166þ, CD34, CD45 Plastic-adherent, in vitro differentiation (adipocytes and osteoblasts) Plastic-adherent, in vitro differentiation (osteoblasts), CD29þ, CD44þ, CD90þ, CD105þ, CD146þ, HLA-ABCþ, CD14, CD34, CD106 Plastic-adherent, in vitro differentiation (osteoblasts) Plastic-adherent, in vitro differentiation (osteoblasts) Plastic-adherent, MHC-1þ, Sca-1þ, CD90þ, CD45, HLA-DR, CD105 Plastic-adherent, MHC-1þ, Sca-1þ, CD90þ, CD45, HLA-DR, CD105 Plastic-adherent

Wound healing

IB

CM

VEGF, angiopoietin 1,

ND

Iso Y. et al. Biochemical and Biophysical Research Communications 2007 Wu Y. et al. Stem Cells 2007

Hindlimb ischemia

ELISA

CM

VEGF, bFGF, MCP-1, PlGF

Hypoxia

ELISA

CM

VEGF, IL-6, FGF-2

Mitogenic effect (VEGF and bFGF) Mitogenic effect (VEGF and bFGF)

Kinnard T. et al. Circulation 2004 Kinnard T. et al. Circulation Research 2004

Osteogenic and adipogenic differentiation Osteogenic differentiation

1D SDS-PAGE and LCeMS/MS, IB validation 1D SDS-PAGE and LCeMS/MS, IB validation

CM

PAI-1, GRP78, PTX3, BIGH3

Chiellini C. et al. BMC Molecular Biology 2008

CM

SMOC1, CHI3L1, FGFB1. EFEMP1, TIMP3, POSTN, THBS2, CTGF

Role in adipocyte and osteoblast balance (PAI-1) Role in osteoblast differentiation (SMOC1)

Osteogenic differentiation Osteogenic differentiation Wound healing

LCMS/MS, IB validation SILAC and LCe MS/MS, IB validation LCeMS/MS, IB validation

CM

Annexin A2, LTBP1 and LTBP2 STC2

ND

Kim J.-M. et al. Cellular Physiology 2013 Kristensen L.P. et al. Molecular & Cellular Proteomics 2012 Sarojini H. et al. Journal of Cellular Biochemistry 2008

Wound healing

Shotgun

Murine/bone marrow

Murine/bone marrow Human/bone marrow Human/adipose

Human/bone marrow

Human/bone marrow Human/ND Murine/bone marrow Murine/bone marrow Human/ESC (line) Human/adipose Murine/bone marrow

Human/bone marrow

Human/adipose

Plastic-adherent, CD29þ, CD44þ, CD90þ, CD105þ FGF-selected MSCs: plasticadherent, in vitro and in vivo differentiation (osteoblasts) CD146þ, CD140aþ, CD44þ, CD90þ, CD106þ, MHC-1þ, MHC-2, CD45, CD31 Plastic-adherent, in vitro differentiation (osteoblasts, adipocytes), CD105þ, CD106þ, CD44þ, CD14, CD34, CD45 Plastic-adherent

Inflammation Bone regeneration

CM

Choi Y.-A. et al. Proteome Research 2009

CM

PEDF, collagen-alpha (I), cystatin C

Role in osteoblast differentiation Chemotactic activity (PEDF)

2D LCeMS/MS, IB validation

CM

Cyr61

Angiogenesis

Estrada R. et al. Journal of Cellular Physiology 2009

Cytokine array and LCeMS/MS LCeMS/MS, IB and ELISA validation SILAC and LCeMS/ MS, IB validation

CM

ND

ND

CM CM

Cathepsin L, PTX3, IL-6, IL-8, MCP-1, CXCL6 ND

Chemotactic activity (IL-6, IL-8, MCP-1) ND

Sze S.K. et al. Molecular & Cellular Proteomics 2007 Lee M.J. et al. Journal of Proteome Research 2010 Tasso R. et al. Biomaterials 2012

Organ failure (fulminant hepatic failure)

Antibody array

CM

Chemokines (ND)

Increased survival of rat model of fulminant hepatic failure

Parekkadan B. et al. PLoS One 2007

Alzheimer

IB and enzymatic assays

EV

NEP

Decreased beta-amyloid levels in neuroblastoma cells

Katsuda T. et al. Scientific Reports 2013

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Please cite this article in press as: J.R. Lavoie, M. Rosu-Myles, Uncovering the secretes of mesenchymal stem cells, Biochimie (2013), http:// dx.doi.org/10.1016/j.biochi.2013.06.017

Table 1 Reports on therapeutic potential of MSC-derived CM and EV that have used proteomic-based approaches.

Human/bone marrow

Human/ESC (line)

BIGH3 indicates transforming growth factor-beta-induced protein ig-h3; CHI3L1, chitinase-3-like protein 1; CM, conditioned medium; CTGF, connective tissue growth factor; CXCL6, chemokine (C-X-C motif) ligand 6; EFEMP1, EGF containing fibulin-like extracellular matrix protein 1; ESC, embryonic stem cells; EV, extracellular vesicules; FGF, fibroblast growth factor; FGFB1, fibroblast growth factor beta 1; GRP78, 78-kDa glucose-regulated protein; HGF, hepatocyte growth factor; IB, immunoblotting; IL, interleukin; LCeMS/MS, liquid chromatographyemass spectrometry; LTBP, latent TGF-beta binding protein; MCP-1, monocyte chemoattractant protein-1; MI, myocardial infarction; NA, not available; ND, not determined; NEP, neprilysin; PAI-1, plasminogen activator inhibitor-1; PEDF, pigment epithelium-derived factor; PlGF, placenta growth factor; POSTN, periostin; PTX3, pentraxin 3; SILAC, stable isotope labelling by amino acids in cell culture; SMOC1, SPARC related modular calcium binding 1; STC2, stanniocalcin; TIMP3, tissue inhibitor of metalloproteinases-3; THBS2, thrombospondin 2; VEGF, vascular endothelial growth factor.

Roccaro A.M. et al. Journal of Clinical Investigation 2013 Multiple myeloma cell growth (IL-6) IB and ELISA assays Multiple myeloma

EV

IL-6, junction plakoglobin, fibronectin

Lai R.C. et al. International Journal of Proteomics 2012 Cardioprotective effect in mouse model of MI PMSA 1e7 EV Cytokine array and LCeMS/MS, IB and enzymatic assays for validation Myocardial infarction

ND 1D SDS-PAGE and LCeMS/MS Human/bone marrow

Plastic-adherent, in vitro differentiation (adipocytes, osteoblasts, chondrocytes), CD29þ, CD44þ, CD73þ, CD90þ, CD105þ, CD31, CD34, CD45, CD106 Plastic-adherent, in vitro differentiation (adipocytes, osteoblasts, chondrocytes), CD29þ, CD44þ, CD49a and CD49eþ, CD105þ, CD166þ, and CD34, CD45 Plastic-adherent, CD73þ, CD90þ, CD105þ, CD106þ, CD14, CD34, CD45, CD138, CD19

Shotgun

EV

ND

Kim H.-S. et al. J. Proteome Research 2012

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4.1. MSC-CM e targeted proteomic studies Target-based proteomic studies explore and gather information on specific molecules, usually with known roles in the biological process(es) being investigated. Research studies characterizing the MSC secretome commonly target molecules, such as growth factors and hormones, with known roles in preventing apoptosis, inducing cell proliferation and differentiation or regulating immune and inflammatory responses. Targets are alternatively selected based on their function during embryogenesis, which is postulated by some to have overlapping mechanisms with the tissue repair process in adults. The most highly utilized method for targeted characterization of the MSC secretome is the Enzyme-Linked Immunosorbent Assay (ELISA). In an elegant study by Iso et al., ELISA was used to probe CM from human MSC for molecules that play a potential role in the treatment of acute myocardial infarction [29]. The authors demonstrated an improvement in cardiac function and scarring after human MSC injection into immunodeficient mice with acute infarct, with no evidence of engraftment at 3 weeks post injection. To examine a potential role for MSC secreted factors, they went on to further show that CM from the cultured human MSC used for the in vivo study rescued both murine cardiomyocytes and human umbilical vein endothelial cells from hypoxia-induced cell death in vitro. To identify potential targets for further proteomic validation, microarray analysis was used to compare the gene expression profiles of MSC with that of human BM cells. RNA for several secreted cardioprotective factors was found to be upregulated in MSC. ELISA-based validation of the most highly expressed genes demonstrated the presence of VEGF, hepatocyte growth factor (HGF), adrenomedullin, placental growth factor (PLGF) and IL-6 in CM derived from MSC. VEGF was also detected in MSC derived CM from at least two groups investigating the benefit of MSC based treatments for wound healing [59,61]. The original experiments, conducted by Wu et al. utilized an immunoblotting technique to quantitatively compare the expression of the angiogenic factors VEGF, angiogenin-1 and angiogenin-2 in CM from MSC and with that of fibroblast cells which were shown to have no capacity to initiate wound healing. Higher levels of VEGF-alpha and angiopoietin 1, but not angiopoietin 2, were detected in BM-MSC [61]. These findings were confirmed by Chen et al. who used antibody array techniques to further identify increased quantities of the cytokines IGF-1, EGF, EPO and keratinocyte growth factor as well as the chemokines SDF-1, MIP-1a and MIP-1b [59]. Targeted approaches

EV

Soluble factors

± Pre-conditioning

MSC-conditioned medium Proteomic-based strategies

Shotgun Label-free Isotope labelling (SILAC) Antibody array

Candidate ± SDS-PAGE

+ LC-MS/MS

Bioinformatics analysis Pathway analysis Gene ontology annotation

ELISA Immunoblotting Enzymatic assays

Functional analysis (in vitro, in vivo)

Fig. 3. Proteomic-based strategies to unravel bioactive secreted factors in the mesenchymal stem cell-conditioned medium.

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have been similarly used to investigate potential paracrine and anti-inflammatory mechanisms for MSC-based treatment of tissue damage due to ischemia, such as that occurring in stroke or peripheral artery disease. These studies have confirmed the presence of several different soluble factors in MSC-CM including VEGF-A, IL-6 and FGF-2 [62e64]. However, it should be noted that, while the targeted studies described here provide insight into the potential content of the MSC secretome, further validation is required to determine the functional importance of these findings. Demonstration of loss of function in the absence of the identified molecules will be critical to understanding the therapeutic relevance of these findings. It is interesting that not all targeted proteomic studies agree on the exact content of the MSC secretome. There are several factors that could account for the discrepancies between studies. These include, but are not limited to; differences assay sensitivity; the tissue of origin for MSC; and the type of media used for cell growth and collection of CM. There are certainly several studies demonstrating that MSC are highly sensitive to their environment and that even slight changes in media formulations can have a dramatic effect on the cells and what they secrete. Despite discrepancies, it is interesting that VEGF-A was consistently detected in almost all targeted proteomic studies. This may speak to the importance of VEGF-A to the function of MSC in that it is one of the few factors that are expressed by these cells regardless of their current or past microenvironment. While targeted proteomic approaches to characterizing the MSC secretome have uncovered important information for further study, they are largely confirmatory in nature rather than exploratory. As such, the level of information that can be gathered from targeted studies provides only a small snap shot of the soluble factors produced by MSC. Considering the vast functional capacity attributed to MSC, more broad based approaches may be necessary to fully understand the potential complexity of their secretome. The following section will discuss some of the knowledge that has been generated on the MSC secretome using such approaches. 4.2. MSC-CM e shotgun-based proteomic approach One of the most reliable and powerful tools for the identification of proteins in complex mixtures, such as serum, CM and whole cell lysates, is tandem mass spectrometry (MS/MS) [65]. The identification and quantification of proteins present at less than picomolar concentrations is possible by coupling MS/MS with upstream protein purification and labelling methods and downstream data processing and database comparison methods. Quantitative (differentially expressed) and qualitative shot-gun proteomic studies of MSC-CM have been reported using both 1-dimensional and 2-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis purification, label-free and stable isotype labelling by amino acids in cell culture (SILAC) approaches combined with Liquid Chromatography (LC)eMS/MS analysis. Downstream processing of data obtained from these experiments employ a global proteomics-based approach which makes comparisons to various public databases to find proteins with biological and clinical relevance. By using 2D LCeMS/MS on murine MSC-CM, Sarojini et al. have identified 19 known secreted proteins, including extracellular matrix structural proteins, collagen processing enzymes, pigment epithelium-derived factor (PEDF) and cystatin C [64]. In addition, by immunodepletion and reconstitution experiments, the authors have shown that PEDF was the protein responsible for the observed fibroblast chemotactic activity obtained from the MSC-CM. In a subsequent study, the same group profiled the proteome of murine MSC to identify other possible secreted proteins that are functionally important in regulating the wound healing response and

identified 258 proteins, among which 54 were known secreted polypeptides [66]. The authors validated by immunoblotting the presence of Cysteine-rich protein 61 (Cyr61) (regulator in the development and maintenance of vasculature) in the proteome and secretome of murine MSC. The authors also showed that the secretome of MSC promoted morphogenesis of endothelial cells by using in vitro and in vivo angiogenic assays and that this protein contributes to the angiogenic-inducing capability of the secretome MSC by immunodepletion and reconstitution experiments aimed at Cyr61. Consistent with the report published by the Wang group, Sze et al. have also identified Cyr61 in their secretome analysis of MSC [67]. Sze et al. used the HuES9.E1 MSC line, derived from a human embryonic stem cell line, and performed a cytokine antibody array and shot-gun LCeMS/MS analysis to profile the MSC-CM. A total of 201 unique proteins were found through these two approaches (132 by LCeMS/MS and 72 by antibody array, 3 of which were commonly found in both) and computational analysis predicted that these gene products significantly drive three major groups of biological processes: the metabolism, defense response and tissue differentiation (e.g., vascularization, hematopoiesis and skeletal development). Pathway analysis also predicted that the 201 identified proteins are involved in signalling pathways related to cardiovascular biology, bone development and hematopoiesis, including Jak-STAT, MAPK, Toll-like receptor, transforming growth factor-beta and mTOR signalling pathways. While the majority of shot-gun proteomic studies have been completed using BM-derived MSC, MS/MS methods have also been utilized to characterize the secretome of MSC derived from adipose and umbilical cord blood. Specifically, Lee et al. identified proteins secreted by human adipose tissue-derived MSC exposed to an inflammatory stimulus (i.e., tumour necrosis factor-alpha (TNF-a) treatment) [68]. The CM from treated MSC was collected and analyzed by LCeMS/MS to identify secreted proteins during the inflammatory process. The authors identified a total of 187 proteins, among which 118 were secreted at higher levels under TNF-a treatment when compared to non-treated. For instance, the TNF-a treatment induced the secretion of various cytokines and chemokines, such as IL-6, IL-8, chemokine (C-X-C motif) ligand 6 (CXCL6) and monocyte chemotactic protein-1 (MCP-1), proteases, including cathepsin L, matrix metalloproteases, as well as long pentraxin 3, a key inflammatory mediator implicated in innate immunity. Interestingly, the TNF-a treatment time-dependently increased the secreted levels of cathepsin L, long pentraxin 3, IL-6 and IL-8 and CXCL6, and it increased dose-dependently the levels of IL-6 and IL-8 in the MSC-CM. Moreover, the TNF-a-induced secretome stimulated the migration of monocytes via Il-6, IL-8 and MCP-1, as pretreatment with neutralizing antibodies against these molecules abrogated the migration. Tasso et al. demonstrated that the presence of bFGF in the culture medium during mouse MSC expansion in vitro is the key factor for the selection of subpopulations inducing host regenerative responses [69]. Using an ectopic model of bone regeneration, the authors demonstrated that implementation of bFGF-selected MSC led to a bone tissue formation which originated from host cells through endochondral ossification, whereas the MSC not cultured in presence of bFGF directly mediated an intramembranous ossification. The authors argued that this result indicated that the selection of less committed cells was essential to activate pathways involved in the generation of the host response. Using SILAC-LCeMS/MS quantitative proteomics on CM of MSC cultured with or without bFGF, the authors aimed to unravel the effects of MSC secretome on the microenvironment induced by bFGF treatment. A total of 804 and 818 proteins were found in two independent experiments, among which 35 were found to be upregulated (including monocyte chemoattractant protein 2, stromal cell-derived factor 1, ceruloplasmin and small

Please cite this article in press as: J.R. Lavoie, M. Rosu-Myles, Uncovering the secretes of mesenchymal stem cells, Biochimie (2013), http:// dx.doi.org/10.1016/j.biochi.2013.06.017

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inducible cytokine 6) and 32 downregulated (including periostin, collagen alpha-1 (III) chain, collagen alpha-2 (V) chain and thrombospondin) following bFGF treatment. Bioinformatics analysis of the differentially secreted proteins by MSC upon bFGF selection indicated that bFGF-selected MSC secreted higher amounts of proteins with gene ontology biological processes in natural immune response, chemotaxis, inflammatory response and response to wounding. In contrast, proteins found to be secreted in lower amounts in bFGF-selected MSC were classified into biological processes such as extracellular matrix organization, development of differentiated bone, skin, adipose and blood vessel systems, response to wounding via complement activation and lymphocytemediated immunity. Overall, many soluble factors have been identified in CM from MSC using a shotgun MS/MS proteomic approach. Many of these proteins were verified to be present in MSC-CM by alternate assays (such as Western Blot and ELISA) and represent candidate molecules for mediating the therapeutic effects attributed to MSC. A point of interest that arises from a collective analysis of these studies is the apparent capacity of MSC to adapt their proteomic profile in response to changing microenvironments. Further research to characterize soluble factors secreted from MSC in response to tissue and disease specific environments may be important. While such studies can be time consuming and costly, their completion is critical to understanding how these proteins cooperate to promote therapeutically relevant outcomes. It is also important to note that, while MS/MS can be a powerful tool for proteomic screening, there are limitations with the assay. The greatest difficulty arises when dealing with a mixture of proteins in which there are highly abundant species. Albumin containing media, which is used quite frequently for MSC growth, is an excellent example. In such cases, the high concentrations of albumin mask the detection of more interesting proteins present in low abundance. Unfortunately, it is often the low abundance proteins that are of the highest clinical interest as subtle changes in the quantity of these molecules can have large functional effects. Outside of difficulties with the detection of low abundance proteins, there can be issues with the accuracy of data analysis arising from MS/MS. It is critical that experiments are carefully designed and data analyzed using stringent criteria to avoid false discoveries.

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tubular cells after injury via horizontal transfer of mRNA [75]. Lai et al. have demonstrated that MSC-exosomes reduced infarct size in a mouse model of myocardial ischemia/reperfusion injury [74]. Finally, in the murine model of hypoxic pulmonary hypertension (HPH), Lee et al. have shown that intravenous delivery of MSCexosomes inhibited vascular remodelling and HPH, whereas a MSC-exosomes-depleted media or fibroblast-derived exosomes had no effect [73]. In addition, the authors have showed that MSCderived exosomes suppressed the hypoxic pulmonary influx of macrophages and the induction of pro-inflammatory and proproliferative mediators. The advantage of using exosomes or microvesicles over CM for non-cell based therapies is that these EVs can serve as delivery vehicles containing a complex mixture of bioactive molecules. Moreover, EVs also have been shown to be capable of crossing the blood brain barrier [76], which is an important aspect in treating neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and prion disease. Extracellular vesicles (exosomes and microvesicles) have been purified from culture medium conditioned by MSC by several methods. The study of exosomes requires the use of either exosome depleted FBS (by ultracentrifugation) or serum free medium. To purify MSC derived exosomes from CM some groups first filter the media at 0.2 mm followed by ultracentrifuged [77,78]. Roccaro et al. further completed an exosome precipitation step followed by ultracentrifugation in order to further concentrate and purify the vesicles. A second method, utilized by Lai et al., first concentrated the MSC-CM (50) with a tangential flow filtration using a membrane with a 100 kDa molecular weight cut off (MWCO), then fractionated the filter eluate by high performance liquid chromatography. Using this technique, exosomes could be collected from the first peak of elution, concentrated using molecular weight and size-exclusionbased filters [74]. For the microvesicle preparation, Kim et al. ultracentrifuged MSC-CM using a Minimate TFFÔ capsule system with a 100 kDa MWCO filter. Microvesicles were further purified by stepwise sucrose cushion centrifugation and density gradient centrifugation steps [79]. While these multiple protocols have proven successful for the enrichment of EV from CM, the equivalency of isolates obtained from different methods remains unclear. 5.1. MSC-EV e candidate-based proteomic approach

5. Proteomic studies on MSC-derived EV The extracellular milieu contains solutions of metabolites, ions, proteins and polysaccharides, as well as numerous mobile extracellular vesicles containing proteins, but also microRNA and RNA, depending on the type of vesicles. EV is a term recommended by the International Society for Extracellular Vesicles to describe exosomes, microvesicles, microparticles and apoptotic bodies [70]. It has been increasingly recognized that EVs play important physiologic roles in both immunity and embryonic development as well as pathophysiologic roles in diseases such as cancer, neurodegenerative disorders and HIV/AIDS [71]. The term “exosome” refers to vesicles of nanometer-sized (50e100 nm in diameter) generated by exocytosis of multivesicular bodies, whereas the term “microvesicle” refers to the larger extracellular membrane vesicles (100e 1000 nm in diameter) generated by budding/blebbing of the plasma membrane [70]. The fact that EVs can transfer surface receptors, signalling molecules, mRNA and miRNAs and that MSC are among the many cell types which are known to secrete EVs made a good rationale for testing their therapeutic potential in different animal models [72e74]. A few studies have looked at the therapeutic potential of MSC-derived EVs. For instance, in a mouse model of acute kidney injury, Bruno et al. found that MSCmicrovesicles activated a proliferative program in surviving

The secretion of EV by MSC is a novel finding that has caused much excitement in the field. As such, several recent studies have characterized the content of MSC-derived EV [70,73e75,77,80,81]. One such study set out to determine the potential for MSC to treat Alzheimer’s disease through EV secretion-based mechanisms. A targeted approach was used to determine whether EV could effect the expression and activity of neprilysin, a protein which normally degrades the b-amyloid peptide that accumulates in the brain of Alzheimer’s patients [82]. Katsuda et al. showed that human adipose tissue-derived MSC secrete exosomes that carry enzymatically active neprilysin [78]. MSC-derived exosomes could transfer neprilysin directly to neuroblastoma cells (N2a cells) overexpressing human b-amyloid peptide. This exosome transfer led to a decrease in both secreted and intracellular b-amyloid peptide levels in the N2a cells. 5.2. MSC-EV e shotgun-based proteomic approach Kim et al. profiled the microvesicle (MVs) proteome from human MSC to understand its therapeutic role [79]. An LCeMS/MS analysis of MSC-MVs identified 730 MV proteins (697 unique annotated gene products), which included characteristic proteins of MVs, such as RAB proteins (e.g., RAB1A, RAB2A and RAB 7A),

Please cite this article in press as: J.R. Lavoie, M. Rosu-Myles, Uncovering the secretes of mesenchymal stem cells, Biochimie (2013), http:// dx.doi.org/10.1016/j.biochi.2013.06.017

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positive markers of MSC (e.g., CD13, CD29, CD44, CD73 and CD105), as well as signalling molecules controlling MSC self-renewal and differentiation in various important signalling pathways, such as Wnt (e.g., RAC1, PRKCB and PPP2R1A), TGF-b (e.g., COL1A2, CD105 and ENG) and MAPK (e.g., FLNA, HSPA8 and EGFR). Functional enrichment analysis showed that cellular processes represented by the MSC-MV-derived proteins included vesicle-mediated transport, cell cycle and proliferation, cell adhesion, cell migration and cell morphogenesis. In 2007, the Sai Kiang Lim group demonstrated that exosomes were the active component of the MSC-CM leading to a reduction of the infarct size by w40% in a mouse model of myocardial ischemia/reperfusion injury [83]. In a subsequent study, the same group executed the protein profiling of exosomes from human ESC-derived MSC (huES9.E1) by cytokine array and LCeMS/ MS to identify candidate proteins or protein complexes that could drive their therapeutic efficacy in ameliorating myocardial ischemia/reperfusion injury [67]. The authors found 857 unique gene products, among which all 7 alpha and 7 beta chains of the 20S proteasome and also the 3 beta subunits of immunoproteasome were detected. In addition, the authors found that the presence of a functional proteasome co-purified with MSC-derived exosomes was correlated with a modest, but significant reduction in oligomerized protein in a mouse model of MI, suggesting a cardioprotective effect by MSC-derived exosomes through proteolytic degradation of misfolded proteins. Roccaro et al. showed that whereas multiple myeloma (MM) bone marrow MSC-derived exosomes resulted in promoting tumour cell growth in vivo, BMMSC-derived exosomes from healthy individuals inhibited the growth of MM cells [77]. The authors investigated the proteome content of the exosomes derived from both groups (healthy and MM) and found that MM BM-MSC-derived exosomes contained higher expression of oncogenic proteins, cytokines and protein kinases, such as IL-5, MCP-1, junction plakoglobin and fibronectin. In addition, the authors have shown that an increased in IL-6 levels in the MM MSC-CM is only present when exposed to exosomes from MM BM-MSC and not from normal BM-MSC, suggesting that the increased levels in IL-6 is functionally important in MM cell growth. 6. Discussion and future directions Using either candidate- or shotgun-based proteomic approaches, a large number of studies have reported biologicallyactive components present in the MSC-CM as soluble factors or contained into EVs. Although the above-mentioned reports bring molecular evidence in support of an MSC-mediated paracrine effect, substantial in vivo validation will be required to confirm the biological relevance of the candidates found by proteomic approaches. Indeed, in most studies described in this review, the validation of the secreted proteins was based either on literature searches or on theoretical prediction using bioinformatics tools, such as Phobius or SecretomeP, and only a few studies actually confirmed the biological relevance of their findings. Substantial validation work will be required before being able to realize the practical application of MSC-derived CM and/or EVs. In addition, another important issue is the heterogeneity of the MSC samples used in the studies to derive the CM. Indeed, many reports have pointed out the presence of heterogeneity among MSC cell preparation in terms of differentiation [84] and expansion potential [85], as well as transcriptomic and proteomic profiles [86,87]. Variation in the cell preparation will inevitably have an influence on the secretome profile, which brings more confusion to the identification of the key bioactive factors leading to the observed therapeutic benefits. To address these issues, MSC-CM-based proteomic studies will have to be conducted with properly characterized MSC,

including surface markers expression and differentiation potential, but also with potency assays, to predict MSC efficacy in vivo. Altogether, this will ensure that the proteomic study conducted with the MSC-CM is reflective of its therapeutic efficacy rather than of its variation in cell preparation and/or donor. Furthermore, the clinically relevant product might be derived from a mixture of bioactive factors. Indeed, recent studies in the ischemic hind limb model have reported a synergistic relationship between growth factors, whereby a combination of the factors led to a superior therapeutic potential when compared with factors alone [88e90]. The proper combination of bioactive factors recapitulating the beneficial effects observed with the MSC alone represents a substantial challenge to overcome. Ultimately, the clinically relevant product will have to be derived from a well-defined complex mixture of manufactured bioactive factors without or within EVs. References [1] A.J. 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Please cite this article in press as: J.R. Lavoie, M. Rosu-Myles, Uncovering the secretes of mesenchymal stem cells, Biochimie (2013), http:// dx.doi.org/10.1016/j.biochi.2013.06.017