proteomic

miRNA

exosome

mesenchymal stem cells

secretion

Mesenchymal stem cells (MSCs) were first described in 1968 as multipotent fibroblast-like cells from bone marrow [1] with a potential to differentiate into osteocytes, chondrocytes, adipocytes, and myoblasts [2,3]. MSCs have now been isolated from diverse sources of tissues such adipose tissue [4,5], liver [6], muscle [7], amniotic fluid [8], placenta [9,10], umbilical cord blood [4], dental pulp [11,12], and other sources [6,13]. Despite the difference in tissue sources, MSCs share several common characteristics: plastic adherence, expression of CD105, CD73, and CD90 but not CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules, and an in vitro differentiation potential of osteogenesis, adipogenesis, and chondrogenesis [14].

MSCs are currently the most evaluated stem cells with more than 118 registered clinical trials (http://www.clinicaltrials.gov/, January 2014). The attractiveness of MSCs could be attributed to their regenerative potential, ease of isolation from adult tissues such as bone marrow and adipose tissue, and a large ex vivo expansion capacity (reviewed in [15–17]).

The use of MSCs as therapeutics was predicated on the hypothetical potential of transplanted MSCs to home in engraft and differentiate to repair injured tissues. However, it has been estimated that <1% of transplanted cells actually reached the target tissue with most of the cells being trapped in the liver, spleen, and lung [18] and few of the appropriately homed and engrafted cells demonstrated unambiguous evidence of differentiation [19–21]. Instead, the therapeutic efficacy of MSC therapy was observed to be independent of the engraftment or differentiation of transplanted MSCs at the site of injury [22–26]. Hence, it was proposed that MSCs exert their therapeutic effects through the secretion of growth factors and cytokines [27]. This proposal provides for a more all-encompassing mechanism of action for MSC efficacy in a wide range of disease indications and injuries [28–35].

The search for soluble mediators of MSC therapy has focused initially on chemokines, cytokines, or secreted proteins. For example, MSC that was approved for treatment of pediatric graft-versus-host disease in Canada and New Zealand was postulated to modulate regulatory T cells or Tregs, a subpopulation of T cells through the secretion of soluble mediators known to enhance Treg expansion. Many candidates such as transforming growth factor beta, prostaglandin E2, human leukocyte antigen G, interleukin-10, and indoleamine-pyrrole 2,3-dioxygenase were proposed (reviewed in [36]). However, none of the soluble mediators identified to date was sufficient in mediating the MSC immune modulatory effect [37].

To conduct an unbiased search for a secreted mediator, we took a first principle approach by profiling the proteome of MSC secretion, which is essentially culture medium conditioned by MSC. At that time, proteomic analysis of MSC-conditioned culture medium was not amenable to high-throughput unbiased mass spectrometry as MSC culture was still dependent on serum, which contains high abundance serum proteins such as albumin and immunoglobulins that obscure the detection of low abundance secreted proteins. To circumvent this, MSCs were cultured for 3 days in serum-free Dulbecco’s modified eagle’s medium supplemented with five peptides, namely insulin, transferrin, selenoprotein, fibroblast growth factor 2 with bovine serum albumin carrier, and platelet-derived growth factor AB [38]. Under this culture condition, MSCs remain viable for at least a week and when returned to serum-containing medium, they regain their proliferative activity without significant loss in their differentiation potential (unpublished observation). Using this defined medium, the conditioned medium (CM) was highly amenable to sensitive unbiased high-throughput mass spectrometry analysis to elucidate MSC secretome. As mass spectrometry is relatively insensitive in detecting small peptides such as cytokines, chemokines, and growth factors, mass spectrometry analysis was complemented with commercially available antibody arrays to detect small proteins that had commercially available antibodies. Using these two different analytical approaches, we detected 201 proteins secreted by MSCs. Of the 33 proteins that were previously reported to be secreted by MSCs, 29 proteins were present in the 201 proteins leaving 172 of the 201 proteins not known to be secreted by MSCs [38]. Among the 172 proteins, a significant proportion was cytoplasmic proteins that were not known to be secreted. Based on this proteome, we hypothesized that MSC-conditioned culture medium could be cardioprotective [38]. This was confirmed in a pig and mouse model of acute myocardial ischemia/reperfusion injury from [39] and a pig model of chronic myocardial ischemia [40].

To identify the active therapeutic agent/s in the complex MSC secretion, we first estimated the size range of the agent by ultrafiltration using membranes with different pore sizes, and then testing the filtrate or retentate for cardioprotective activity. Using this method, we established the size range of the active agent as 50–200 ηm, which was then confirmed visually by electron microscopy [39,41]. Subsequent biochemical and biophysical analysis identified these agents as lipid membrane vesicles with a detergent-sensitive flotation density range of 1.10–1.18 g/mL in sucrose, enriched in exosome-associated protein markers such as CD9, CD81, and Alix, and membrane lipids such as cholesterol, sphingomyelin, and phosphatidylcholine [41].

When these membrane vesicles were isolated by size exclusion high performance liquid chromatography (HPLC), they constituted a population of homogeneously sized particles with a hydrodynamic radius of 55–65 ηm and were as efficacious as CM in reducing infarct size in a mouse model. Therefore, the cardioprotective agent in MSC secretion is an exosome. Following our report, exosomes were implicated as the agent mediating the biological activity of MSCs in attenuating type 1 diabetes and multiple myeloma progression [42,43].

The implication of exosome as the active agent underpinning therapeutic efficacy of MSC converts MSC-based...