The mesenchymal stem cell secretome: A new paradigm towards cell-free therapeutic mode in regenerative medicine

Mesenchymal Stem Cells (MSCs) have been shown to be a promising candidate for cell-based therapy. The therapeutic potential of MSCs, towards tissue repair and wound healing is essentially based on their paracrine effects. Numerous pre-clinical and clinical studies of MSCs have yielded encouraging results. Further, these cells have been shown to be relatively safe for clinical applications. MSCs harvested from numerous anatomical locations including the bone marrow, adipose tissue, Wharton’s jelly of the umbilical cord etc., display similar immunophenotypic profiles. However, there is a large body of evidence showing that MSCs secrete a variety of biologically active molecules such as growth factors, chemokines, and cytokines. Despite the similarity in their immunophenotype, the secretome of MSCs appears to vary significantly, depending on the age of the host and niches where the cells reside. Thus, by implication, proteomics-based profiling suggests that the therapeutic potential of the different MSC populations must also be different. Analysis of the secretome points to its influence on varied biological processes such as angiogenesis, neurogenesis, tissue repair, immunomodulation, wound healing, anti-fibrotic and anti-tumour for tissue maintenance and regeneration. Though MSC based therapy has been shown to be relatively safe, from a clinical standpoint, the use of cell-free infusions can altogether circumvent the administration of viable cells for therapy. Understanding the secretome of in vitro cultured MSC populations, by the analysis of the corresponding conditioned medium, will enable us to evaluate its utility as a new therapeutic option. This review will focus on the accumulating evidence that points to the therapeutic potential of the conditioned medium, both from pre-clinical and clinical studies. Finally, this review will emphasize the importance of profiling the conditioned medium for assessing its potential for cell-free therapy therapy.     

Secretome analysis of human bone marrow derived mesenchymal stromal cells

As an essential cellular component of the bone marrow (BM) microenvironment mesenchymal stromal cells (MSC) play a pivotal role for the physiological regulation of hematopoiesis, in particular through the secretion of cytokines and chemokines. Mass spectrometry (MS) facilitates the identification and quantification of a large amount of secreted proteins (secretome), but can be hampered by the false-positive identification of contaminating proteins released from dead cells or derived from cell medium. To reduce the likelihood of contaminations we applied an approach combining secretome and proteome analysis to characterize the physiological secretome of BM derived human MSC. Our analysis revealed a secretome consisting of 315 proteins. Pathway analyses of these proteins revealed a high abundance of proteins related to cell growth and/or maintenance, signal transduction and cell communication thereby representing key biological functions of BM derived MSC on protein level. Within the MSC secretome we identified several cytokines and growth factors such as VEGFC, TGF-β1, TGF-β2 and GDF6 which are known to be involved in the physiological regulation of hematopoiesis. By comparing the peptide patterns of secretomes and cell lysates 17 proteins were identified as candidates for proteolytic processing. Taken together, our combined MS work-flow reduced the likelihood of contaminations and enabled us to carve out a specific overview about the composition of the secretome from human BM derived MSC. This methodological approach and the specific secretome signature of BM derived MSC may serve as basis for future comparative analyses of the interplay of MSC and HSPC in patients with hematological malignancies.     

Harnessing the mesenchymal stem cell secretome for the treatment of cardiovascular disease

The broad repertoire of secreted trophic and immunomodulatory cytokines produced by mesenchymal stem cells (MSCs), generally referred to as the MSC secretome, has considerable potential for the treatment of cardiovascular disease. However, harnessing this MSC secretome for meaningful therapeutic outcomes is challenging due to the limited control of cytokine production following their transplantation. This review outlines the current understanding of the MSC secretome as a therapeutic for treatment of ischemic heart disease. We discuss ongoing investigative directions aimed at improving cellular activity and characterizing the secretome and its regulation in greater detail. Finally, we provide insights on and perspectives for future development of the MSC secretome as a therapeutic tool.     

The effect of Trimetazidine and Diazoxide on immunomodulatory activity of human embryonic stem cell-derived mesenchymal stem cell secretome

Comprehensive proteome profiling of the factors secreted by mesenchymal stem cells (MSCs), referred to as secretome, revealed that it consists of cytokines, chemokines, growth factors, extracellular matrix proteins, and components of regeneration, vascularization, and hematopoiesis pathways. Harnessing this MSC secretome for therapeutic applications requires the optimization of production of secretary molecules. A variety of preconditioning methods have been introduced, which subject cells to stimulatory molecules to create the preferred response and stimulate persistent effects. Pharmacological preconditioning uses small molecules and drugs to increase survival of MSCs after transplantation or prolong release of effective secretary factors such as cytokines that improve immune system responses. In this study, we investigated the effect of secretome of human embryonic-derived mesenchymal stem cells (hESC-MSCs) preconditioned with Trimetazidine (TMZ) and Diazoxide (DZ) on immunomodulatory efficiency of these cells in LPS-induced peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from human peripheral blood and treated with concentrated hESC-MSC-derived conditioned medium and then, the secreted levels of IL-10, TNFα and IL-1β were assessed by ELISA after induction with LPS. The results showed that TMZ and DZ-conditioned medium significantly enhanced immunomodulatory potential of hESC-MSCs by increasing the secretion of IL-10, TNFα and IL-1β from LPS- induced PBMCs. We also found that hESC-MSCs did not secrete mentioned cytokines prior to or after the preconditioning with TMZ and DZ. In conclusion, our results implied that TMZ and DZ can be used to promote the immunomodulatory effects of hESC-MSC secretome. It is obvious that for applying of these findings in clinical demands, the potency of different pre-conditioned MSCs secretome on immune response needs to be more clarified.     

Proteomic techniques for characterisation of mesenchymal stem cell secretome

Mesenchymal stem cells (MSCs) are multipotent cells with a substantial potential in human regenerative medicine due to their ability to migrate to sites of injury, capability to suppress immune response and accessibility in large amount from patient's own bone marrow or fat tissue. It has been increasingly observed that the transplanted MSCs did not necessarily engraft and differentiate at the site of injury but might exert their therapeutic effects through secreted trophic signals. The MSCs secrete a variety of autocrine/paracrine factors, called secretome, that support regenerative processes in the damaged tissue, induce angiogenesis, protect cells from apoptotic cell death and modulate immune system. The cell culture medium conditioned by MSCs or osteogenic, chondrogenic as well as adipogenic precursors derived from MSCs has become a subject of intensive proteomic profiling in the search for and identification of released factors and microvesicles that might be applicable in regenerative medicine. Jointly with the methods for MSC isolation, expansion and differentiation, proteomic analysis of MSC secretome was enabled recently mainly due to the extensive development in protein separation techniques, mass spectrometry, immunological methods and bioinformatics. This review describes proteomic techniques currently applied or prospectively applicable in MSC secretomics, with a particular focus on preparation of the secretome sample, protein/peptide separation, mass spectrometry and protein quantification techniques, analysis of posttranslational modifications, immunological techniques, isolation and characterisation of secreted vesicles and exosomes, analysis of cytokine-encoding mRNAs and bioinformatics.     

In vitro augmentation of mesenchymal stem cells viability in stressful microenvironments: In vitro augmentation of mesenchymal stem cells viability

Mesenchymal stem cells (MSCs) are under intensive investigation for use in cell-based therapies because their differentiation abilities, immunomodulatory effects, and homing properties offer potential for significantly augmenting regenerative capacity of many tissues. Nevertheless, major impediments to their therapeutic application, such as low proliferation and survival rates remain as obstacles to broad clinical use of MSCs. Another major challenge to evolution of MSC-based therapies is functional degradation of these cells as a result of their exposure to oxidative stressors during isolation. Indeed, oxidative stress-mediated MSC depletion occurs due to inflammatory processes associated with chemotherapy, radiotherapy, and expression of pro-apoptotic factors, and the microenvironment of damaged tissue in patients receiving MSC therapy is typically therapeutic not favorable to their survival. For this reason, any strategies that enhance the viability and proliferative capacity of MSCs associated with their therapeutic use are of great value. Here, recent strategies used by various researchers to improve MSC allograft function are reviewed, with particular focus on in vitro conditioning of MSCs in preparation for clinical application. Preconditioning, genetic manipulation, and optimization of MSC culture conditions are some examples of the methodologies described in the present article, along with novel strategies such as treatment of MSCs with secretome and MSC-derived microvesicles. This topic material is likely to find value as a guide for both research and clinical use of MSC allografts and for improvement of the value that use of these cells brings to health care.     

Cellular expansion of MSCs: Shifting the regenerative potential

Mesenchymal-derived stromal or progenitor cells, commonly called “MSCs,” have attracted significant clinical interest for their remarkable abilities to promote tissue regeneration and reduce inflammation. Recent studies have shown that MSCs' therapeutic effects, originally attributed to the cells' direct differentiation capacity into the tissue of interest, are largely driven by the biomolecules the cells secrete, including cytokines, chemokines, growth factors, and extracellular vesicles containing miRNA. This secretome coordinates upregulation of endogenous repair and immunomodulation in the local microenvironment through crosstalk of MSCs with host tissue cells. Therapeutic applications for MSCs and their secretome-derived products often involve in vitro monolayer expansion. However, consecutive passaging of MSCs significantly alters their therapeutic potential, inducing a broad shift from a pro-regenerative to a pro-inflammatory phenotype. A consistent by-product of in vitro expansion of MSCs is the onset of replicative senescence, a state of cell arrest characterized by an increased release of proinflammatory cytokines and growth factors. However, little is known about changes in the secretome profile at different stages of in vitro expansion. Some culture conditions and bioprocessing techniques have shown promise in more effectively retaining the pro-regenerative and anti-inflammatory MSC phenotype throughout expansion. Understanding how in vitro expansion conditions influence the nature and function of MSCs, and their associated secretome, may provide key insights into the underlying mechanisms driving these alterations. Elucidating the dynamic and diverse changes in the MSC secretome at each stage of in vitro expansion is a critical next step in the development of standardized, safe, and effective MSC-based therapies.     

Mesenchymal stem cells in regenerative medicine applied to rheumatic diseases: Role of secretome and exosomes

Over the last decades, mesenchymal stem cells (MSCs) have been extensively studied with regard to their potential applications in regenerative medicine. In rheumatic diseases, MSC-based therapy is the subject of great expectations for patients who are refractory to proposed treatments such as rheumatoid arthritis (RA), or display degenerative injuries without possible curative treatment, such as osteoarthritis (OA). The therapeutic potential of MSCs has been demonstrated in several pre-clinical models of OA or RA and both the safety and efficacy of MSC-based therapy is being evaluated in humans. The predominant mechanism by which MSCs participate to tissue repair is through a paracrine activity. Via the production of a multitude of trophic factors with various properties, MSCs can reduce tissue injury, protect tissue from further degradation and/or enhance tissue repair. However, a thorough in vivo examination of MSC-derived secretome and strategies to modulate it are still lacking. The present review discusses the current understanding of the MSC secretome as a therapeutic for treatment of inflammatory or degenerative pathologies focusing on rheumatic diseases. We provide insights on and perspectives for future development of the MSC secretome with respect to the release of extracellular vesicles that would have certain advantages over injection of living MSCs or administration of a single therapeutic factor or a combination of factors.     

Generation of mesenchymal stem-like cells for producing extracellular vesicles

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with therapeutic potential against autoimmune diseases, inflammation, ischemia, and metabolic disorders. Contrary to the previous conceptions, recent studies have revealed that the tissue repair and immunomodulatory functions of MSCs are largely attributed to their secretome, rather than their potential to differentiate into desired cell types. The composition of MSC secretome encompasses cytokines and growth factors, in addition to the cell-derived structures known as extracellular vesicles (EVs). EVs are membrane-enclosed nanoparticles that are capable of delivering biomolecules, and it is now believed that MSC-derived EVs are the major players that induce biological changes in the target tissues. Based on these EVs’ characteristics, the potential of EVs derived from MSC (MSC-EV) in terms of tissue regeneration and immune modulation has grown during the last decade. However, the use of MSCs for producing sufficient amount of EVs has not been satisfactory due to limitations in the cell growth and large variations among the donor cell types. In this regard, pluripotent stem cells (PSCs)-derived MSC-like cells, which can be robustly induced and expanded in vitro, have emerged as more accessible cell source that can overcome current limitations of using MSCs for EV production. In this review, we have highlighted the methods of generating MSC-like cells from PSCs and their therapeutic outcome in preclinical studies. Finally, we have also discussed future requirements for making this cell-free therapy clinically feasible.     

Mesenchymal stromal cells in wound healing applications: role of the secretome,targeted delivery and impact on recessive dystrophic epidermolysis bullosa treatment

Mesenchymal stromal cells (MSCs) are multi-potent stromal-derived cells capable of self-renewal that possess several advantageous properties for wound healing, making them of interest to the field of dermatology. Research has focused on characterizing the unique properties of MSCs, which broadly revolve around their regenerative and more recently discovered immunomodulatory capacities. Because of ease of harvesting and expansion, differentiation potential and low immunogenicity, MSCs have been leading candidates for tissue engineering and regenerative medicine applications for wound healing, yet results from clinical studies have been variable, and promising pre-clinical work has been difficult to reproduce. Therefore, the specific mechanisms of how MSCs influence the local microenvironment in distinct wound etiologies warrant further research. Of specific interest in MSC-mediated healing is harnessing the secretome, which is composed of components known to positively influence wound healing. Molecules released by the MSC secretome can promote re-epithelialization and angiogenesis while inhibiting fibrosis and microbial invasion. This review focuses on the therapeutic interest in MSCs with regard to wound healing applications, including burns and diabetic ulcers, with specific attention to the genetic skin disease recessive dystrophic epidermolysis bullosa. This review also compares various delivery methods to support skin regeneration in the hopes of combating the poor engraftment of MSCs after delivery, which is one of the major pitfalls in clinical studies utilizing MSCs.     

Secretome from mesenchymal stem cells induces angiogenesis via Cyr61

It is well known that bone marrow‐derived mesenchymal stem cells (MSCs) are involved in wound healing and regeneration responses. In this study, we globally profiled the proteome of MSCs to investigate critical factor(s) that may promote wound healing. Cysteine‐rich protein 61 (Cyr61) was found to be abundantly present in MSCs. The presence of Cyr61 was confirmed by immunofluorescence staining and immunoblot analysis. Moreover, we showed that Cyr61 is present in the culture medium (secretome) of MSCs. The secretome of MSCs stimulates angiogenic response in vitro, and neovascularization in vivo. Depletion of Cyr61 completely abrogates the angiogenic‐inducing capability of the MSC secretome. Importantly, addition of recombinant Cyr61 polypeptides restores the angiogenic activity of Cyr61‐depleted secretome. Collectively, these data demonstrate that Cyr61 polypeptide in MSC secretome contributes to the angiogenesis‐promoting activity, a key event needed for regeneration and repair of injured tissues. J. Cell. Physiol. 219: 563–571, 2009. © 2009 Wiley‐Liss, Inc.     

Role of hypoxia preconditioning in therapeutic potential of mesenchymal stem-cell-derived extracellular vesicles

The use of mesenchymal stem-cells (MSC) in cell therapy has received considerable attention because of their properties. These properties include high expansion and differentiation in vitro, low immunogenicity, and modulation of biological processes, such as inflammation, angiogenesis and hematopoiesis. Curiously, the regenerative effect of MSC is partly due to their paracrine activity. This has prompted numerous studies, to investigate the therapeutic potential of their secretome in general, and specifically their extracellular vesicles (EV). The latter contain proteins, lipids, nucleic acids, and other metabolites, which can cause physiological changes when released into recipient cells. Interestingly, contents of EV can be modulated by preconditioning MSC under different culture conditions. Among them, exposure to hypoxia stands out; these cells respond by activating hypoxia-inducible factor (HIF) at low O₂ concentrations. HIF has direct and indirect pleiotropic effects, modulating expression of hundreds of genes involved in processes such as inflammation, migration, proliferation, differentiation, angiogenesis, metabolism, and cell apoptosis. Expression of these genes is reflected in the contents of secreted EV. Interestingly, numerous studies show that MSC-derived EV conditioned under hypoxia have a higher regenerative capacity than those obtained under normoxia. In this review, we show the implications of hypoxia responses in relation to tissue regeneration. In addition, hypoxia preconditioning of MSC is being evaluated as a very attractive strategy for isolation of EV, with a high potential for clinical use in regenerative medicine that can be applied to different pathologies.     

Mesenchymal stem cell secretome and regenerative therapy after cancer

Cancer treatment generally relies on tumor ablative techniques that can lead to major functional or disfiguring defects. These post-therapy impairments require the development of safe regenerative therapy strategies during cancer remission. Many current tissue repair approaches exploit paracrine (immunomodulatory, pro-angiogenic, anti-apoptotic and pro-survival effects) or restoring (functional or structural tissue repair) properties of mesenchymal stem/stromal cells (MSC). Yet, a major concern in the application of regenerative therapies during cancer remission remains the possible triggering of cancer recurrence. Tumor relapse implies the persistence of rare subsets of tumor-initiating cancer cells which can escape anti-cancer therapies and lie dormant in specific niches awaiting reactivation via unknown stimuli. Many of the components required for successful regenerative therapy (revascularization, immunosuppression, cellular homing, tissue growth promotion) are also critical for tumor progression and metastasis. While bi-directional crosstalk between tumorigenic cells (especially aggressive cancer cell lines) and MSC (including tumor stroma-resident populations) has been demonstrated in a variety of cancers, the effects of local or systemic MSC delivery for regenerative purposes on persisting cancer cells during remission remain controversial. Both pro- and anti-tumorigenic effects of MSC have been reported in the literature. Our own data using breast cancer clinical isolates have suggested that dormant-like tumor-initiating cells do not respond to MSC signals, unlike actively dividing cancer cells which benefited from the presence of supportive MSC. The secretome of MSC isolated from various tissues may partially diverge, but it includes a core of cytokines (i.e. CCL2, CCL5, IL-6, TGFβ, VEGF), which have been implicated in tumor growth and/or metastasis. This article reviews published models for studying interactions between MSC and cancer cells with a focus on the impact of MSC secretome on cancer cell activity, and discusses the implications for regenerative therapy after cancer.     

Reduced neuroprotective potential of the mesenchymal stromal cell secretome with ex vivo expansion,age and progressive multiple sclerosis

Background

Clinical trials using ex vivo expansion of autologous mesenchymal stromal cells (MSCs) are in progress for several neurological diseases including multiple sclerosis (MS). Given that environment alters MSC function, we examined whether in vitro expansion, increasing donor age and progressive MS affect the neuroprotective properties of the MSC secretome.

Approaches to the study of the cell secretome

The secretome, or secretomics, has recently emerged as a new term to describe the global study of proteins that are secreted by a cell, tissue or organism at any given time or under certain conditions. The secretome constitutes an important class of proteins that control and regulate a multitude of biological and physiological processes, thus making it a clinically relevant source for biomarkers and therapeutic target discoveries. There are several approaches that are being implemented to study such a class of proteins; however, each of these approaches has its advantages and limitations. While genome-wide studies using signal predictions can provide a comprehensive analysis of the secretome, the detection and quantification of the actual secreted proteins in a tissue would be more relevant. The goal of this review is to provide an overview of the methods currently used to analyze such a class of proteins, as well as the challenges encountered during the study of the secretome. The implication of studying the cell secretome together with its clinical relevance will be also covered.     

Injection of synthetic mesenchymal stem cell mitigates osteoporosis in rats after ovariectomy

Osteoporosis is a severe skeletal disorder. Patients have a low bone mineral density and bone structural deterioration. Mounting lines of evidence suggest that inappropriate apoptosis of osteoblasts/osteocytes leads to maladaptive bone remodelling in osteoporosis. It has been suggested that transplantation of stem cells, including mesenchymal stem cells, may alter the trajectory of bone remoulding and mitigate osteoporosis in animal models. However, stem cells needed to be carefully stored and characterized before usage. In addition, there is great batch‐to‐batch variation in stem cell production. Here, we fabricated therapeutic polymer microparticles from the secretome and membranes of mesenchymal stem cells (MSCs). These synthetic MSCs contain growth factors secreted by MSCs. In addition, these particles display MSC surface molecules. In vitro, co‐culture with synthetic MSCs increases the viability of osteoblast cells. In a rat model of ovariectomy‐induced osteoporosis, injection of synthetic MSCs mitigated osteoporosis by reducing cell apoptosis and systemic inflammation, but increasing osteoblast numbers. Synthetic MSC offers a promising therapy to manage osteoporosis.     

Lysis and phenotypic modulation of mesenchymal stromal cells upon blood contact triggers anti-inflammatory skewing of the peripheral innate immune repertoire

Background aimsMesenchymal stromal cells (MSCs) are used to treat immune-related disorders, including graft-versus-host disease. Upon intravenous infusion, MSCs trigger the instant blood-mediated inflammatory response, resulting in activation of both complement and coagulation cascades, and are rapidly cleared from circulation. Despite no/minimal engraftment, long-term immunoregulatory properties are evident. The aim of this study was to establish the effects of blood exposure on MSC viability and immunomodulatory functions.MethodsHuman, bone marrow derived MSCs were exposed to human plasma +/– heat inactivation or whole blood. MSC number, viability and cellular damage was assessed using the JC-1 mitochondrial depolarization assay and annexin V staining. C3c binding and expression of the inhibitory receptors CD46, CD55 and CD59 and complement receptors C3aR and C5aR were evaluated by flow cytometry. MSCs pre-exposed to plasma were cultured with peripheral blood mononuclear cells (PBMCs) and monocyte subsets characterized by flow cytometry. The PBMC and MSC secretome was assessed using enzyme-linked immunosorbent assays against tumor necrosis factor alpha, interleukin (IL)-6 and IL-10. Monocyte recruitment towards the MSC secretome was evaluated using Boyden chambers and screened for chemotactic factors including monocyte chemoattractant protein (MCP)-1. MSC effects on the peripheral immune repertoire was also evaluated in whole blood by flow cytometry.ResultsPlasma induced rapid lysis of 57% of MSCs, which reduced to 1% lysis with heat inactivation plasma. Of those cells that were not lysed, C3c could be seen bound to the surface of the cells, with a significant swelling of the MSCs and induction of cell death. The MSC secretome reduced monocyte recruitment, in part due to a reduction in MCP-1, and downregulated PBMC tumor necrosis factor alpha secretion while increasing IL-6 levels in the co-culture supernatant. A significant decrease in CD14⁺ monocytes was evident after MSC addition to whole blood alongside a significant increase in IL-6 levels, with those remaining monocytes demonstrating an increase in classical and decrease in non-classical subsets. This was accompanied by a significant increase in both mononuclear and polymorphonuclear myeloid-derived suppressor cells.ConclusionsThis study demonstrates that a significant number of MSCs are rapidly lysed upon contact with blood, with those surviving demonstrating a shift in their phenotype, including a reduction in the secretion of monocyte recruitment factors and an enhanced ability to skew the phenotype of monocytes. Shifts in the innate immune repertoire, towards an immunosuppressive profile, were also evident within whole blood after MSC addition. These findings suggest that exposure to blood components can promote peripheral immunomodulation via multiple mechanisms that persists within the system long after the infused MSCs have been cleared.     

Mesenchymal stem cells promote lymphangiogenic properties of lymphatic endothelial cells

Lymphatic metastasis is one of the main prognostic factors concerning long‐term survival of cancer patients. In this regard, the molecular mechanisms of lymphangiogenesis are still rarely explored. Also, the interactions between stem cells and lymphatic endothelial cells (LEC) in humans have not been well examined. Therefore, the main objective of this study was to assess the interactions between mesenchymal stem cells (MSC) and LEC using in vitro angiogenesis assays. Juvenile LEC were stimulated with VEGF‐C, bFGF, MSC‐conditioned medium (MSC‐CM) or by co‐culture with MSC. LEC proliferation was assessed using a MTT assay. Migration of the cells was determined with a wound healing assay and a transmigration assay. To measure the formation of lymphatic sprouts, LEC spheroids were embedded in collagen or fibrin gels. The LEC's capacity to form capillary‐like structures was assessed by a tube formation assay on Matrigel^®. The proliferation, migration and tube formation of LEC could be significantly enhanced by MSC‐CM and by co‐culture with MSC. The effect of stimulation with MSC‐CM was stronger compared to stimulation with the growth factors VEGF‐C and bFGF in proliferation and transmigration assays. Sprouting was stimulated by VEGF‐C, bFGF and by MSC‐CM. With this study, we demonstrate the potent stimulating effect of the MSC secretome on proliferation, migration and tube formation of LEC. This indicates an important role of MSC in lymphangiogenesis in pathological as well as physiological processes.     

Identification of differentially regulated secretome components during skeletal myogenesis

Myogenesis is a well-characterized program of cellular differentiation that is exquisitely sensitive to the extracellular milieu. Systematic characterization of the myogenic secretome (i.e. the ensemble of secreted proteins) is, therefore, warranted for the identification of novel secretome components that regulate both the pluripotency of these progenitor mesenchymal cells, and also their commitment and passage through the differentiation program. Previously, we have successfully identified 26 secreted proteins in the mouse skeletal muscle cell line C2C12 (1). In an effort to attain a more comprehensive picture of the regulation of myogenesis by its extracellular milieu, quantitative profiling employing stable isotope labeling by amino acids in cell culture was implemented in conjunction with two parallel high throughput online reverse phase liquid chromatography-tandem mass spectrometry systems. In summary, 34 secreted proteins were quantified, 30 of which were shown to be differentially expressed during muscle development. Intriguingly, our analysis has revealed several novel up- and down-regulated secretome components that may have critical biological relevance for both the maintenance of pluripotency and the passage of cells through the differentiation program. In particular, the altered regulation of secretome components, including follistatin-like protein-1, osteoglycin, spondin-2, and cytokine-induced apoptosis inhibitor-1, along with constitutively expressed factors, such as fibulin-2, illustrate dynamic changes in the secretome that take place when differentiation to a specific lineage occurs.