Mesenchymal Stem Cell-Derived Exosomes : New Opportunity in Cell-Free Therapy

2016 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2016, 6(3), 293-299 doi: 10.15171/apb.2016.041 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
Mesenchymal stromal/stem cells (MSCs) as nonhematopoietic stem cells resided in the stroma of the bone marrow 1,2 and comprise 0.001%-0.01% of the total nucleated bone marrow cells. 3,4Although MSCs are isolated from human adipose tissue, liver, spleen, thymus, umbilical cord blood, placenta, Wharton's jelly, brain, lung, dental pulp, palatine tonsils, peripheral blood and other sources, [5][6][7][8][9][10][11] but they are mainly present in the bone marrow. 12,13SCs don't have identical markers due to species diversity, various tissue sources and culture conditions probably. 14MSCs derived from different sources are similar in phenotype, but are different in functions. 8The international society for cellular therapy (ISCT) has suggested some criteria for characterizing human MSCs which summarized in 1) Plastic adherence property in standard culture conditions, 2) Expression of CD105, CD90, and CD73, and lack expression of CD34, CD45, CD14 or CD11b, CD79a or CD19 and HLA-DR markers and 3) Differentiation potency to adipocytes, chondroblasts, and osteoblasts in vitro. 15SCs exert their roles in the bone marrow via direct cell-to-cell cross-talk as well as secretion of broadspectrum soluble factors. 16MSCs can migrate to injured tissues and because of their differentiation capability into various cell lineages and through secretion of soluble molecules can regenerate those injured tissues. 17,18MSCs augment angiogenesis and inhibit fibrosis via angiogenic and antifibrotic factors, respectively. 196][27][28][29][30] In addition, MSCs suppress HSCs apoptosis. 29,31Major soluble mediators which produced by MSCs contain cytokines, various growth factors, microRNAs, and exosomes which may affect the differentiation capacities of MSCs and promote tissue repairs. 19,32,33he search for MSC-derived exosomes (MSC-DEs) is an attractive scope of the investigation because they are the paracrine effectors of MSCs and involved in cell-to-cell interactions.In this paper, we have summarized characteristics, properties, and isolation of the exosomes secreted by MSCs, also its applications in regenerative medicine as cell-free therapy.

Extracellular vesicles
6][47][48] Among all of them, exosomes have attracted much interest in the last decades.

Isolation and Storage of Exosomes
The basic and common method for exosome isolation and purification from cell culture supernatants and in different biological fluids is ultracentrifugation that is often combined with sucrose density gradients. 36onsecutive centrifuge forces cause to cells and larger particle's removal and exosomes precipitation by centrifugation at least 100,000 ×g force. 60Other procedures for exosome isolations include highperformance liquid chromatography (HPLC), ultrafiltration, exosome precipitation by volume excluding polymers, e.g.Polyethylene glycols (PEGs), affinity purification with specific antibodies against CD9, CD63, CD81, and CD82. 60,61In addition, today exosome isolation kits are commercially available that are based on efficient techniques and provide convenient separation.Conditions with low PH increase isolation and existence of exosomes. 62][65] After the isolation and characterization, exosomes for in vivo or in vitro applications must be frozen because they are unstable at room temperature and 37 °C.Exosomes can be stored for 6 months at -20 °C without cryopreservative agents. 66Sokolova et al. have examined the stability of exosomes during storage at -20 °C, 4 °C and 37 °C.They reported that at 4 °C and 37 °C the size of the exosomes decreased and also degradations or structural changes occurre.Several freeze and thawing cycles (up to -20 °C) and ultracentrifugation did not change the size of exosomes. 65Hence, -20 °C or lower temperatures are suitable for exosome storage without changes in the size and structure of the exosomes.

Therapeutic effects of MSC-Derived Exosomes
Mesenchymal stem cells improve repair of injured tissues, also modulation of immune responses.These effects of mesenchymal stem cells are widely mediated by differentiations of MSCs, paracrine signals, and several secreted molecules such as microvesicles. 67,68SC-DEs investigated largely in many activities of these cells and its effects on other cells.These exosomes probably to participate in many physiological and pathological processes because they carry trophic factors, which can be delivered to recipient cells. 35,69Therefore, the isolation and identification of exosomes from MSCs cultured media have made them a popular choice for cell-free therapy in research and clinical trials that could have clinical applications in the near future.The intravenous injection of exosomes secreted from the human umbilical cord-MSC (huc-MSC) is tolerable in animal models because they had supportive effects on weight loss and had no harmful effects on renal or liver function. 70MSC-DEs through recovery, repair, and regeneration of the tissue play an important role in maintaining tissue homeostasis 71 and have cardioprotective effects through exciting proliferation, apoptosis prevention, angiogenesis induction, and oxidative stress suppression. 4These exosomes (MSC-DEs) also have anti-apoptosis and anti-inflammatory effects, anti-cardiac remodeling, cardiac regeneration, Advanced Pharmaceutical Bulletin, 2016, 6(3), 293-299 neovascularization and anti-vascular remodeling effects in cardiovascular system. 72MSC-EVs keep cardiac tissue from ischemic injury through angiogenesis-promoting effects. 73In addition, MSC-derived exosomes decrease myocardial ischemia/reperfusion (MI/R) injury in mouse models. 74,75SC-derived exosomes by activation of PI3K/Akt pathway, increase in ATP levels, reduce oxidative stress promote the myocardial viability and cardiac function MI/R injury; therefore, MSC-DEs can be a potential adjuvant for reperfusion.76 The exosomes derived from BM-MSC keeps kidney against ischemia reperfusion damages with diminished inflammatory responses and apoptosis in rats.51 In addition, exosomes increase renal epithelial cell proliferation in vitro.77 It has proved that in the mouse models BM-MSC-derived exosomes keep the intestines from necrotizing enterocolitis (NEC).78 MicroRNAs are a type of small non-coding RNAs (~18-24 nucleotides) which regulate proliferation, differentiation, development, and cell death.79 Due to exosomes enriched with microRNAs, 53,80 probably play a crucial role in cellular functions such as tissue homeostasis and hematopoiesis.Several miRNAs in adult MSC-derived exosomes, including miR-191, miR-222, miR-21, and let-7a adjust cell proliferation, miR-222, miR-21, and let-7f induce angiogenesis and miR-6087 causes promotion of endothelial cell differentiation.81 MSC-derived exosomes accelerate muscle regeneration via promoting myogenesis and angiogenesis, which mediated by miRNAs (e.g.miR-494) to be dependent on the effect of cytokines present in exosomes.82 One study at 2013 reported that MSC-DEs due to enriched with miR-16, suppress tumor progression and angiogenesis via down-regulation of the expression of vascular endothelial growth factor (VEGF) in tumor cells 83 and another study at 2012 was reported that MSC-DEs promoted tumor growth in in vivo through the increasing VEGF expression by activating extracellular signal-regulated kinase1/2 (ERK1/2) pathway in tumor cells. 84in et al. showed that intravenous infusion of MSCderived exosomes after stroke improves neurogenesis, neurite remodeling and angiogenesis.85 Exosomes have multimodal neuroprotective effects because they can pass over the blood-brain barrier in spite of most drugs.86 In addition, MSC-exosomes induce axonal development, 87 so this can make a new window in treatment of the neurodegenerative disorders. Exsomes derived from huc-MSC have the immunomodulatory effects through an increase in the percentage of T-regulatory cells (CD4+ CD25+ FoxP3+) and dissuasion the proliferation of T CD4+ and T CD8+ cells.88 MSC-DEs improve the survival of allogenic skin graft in mice and delay the occurrence of GVHD for two days by the shift of activated T CD4+ cells to Tregulatory cells.24

Conclusions and Future Directions
There have been some attracting therapeutic effects of MSC-derived exosomes in various animal models.Exosomes are ideal vehicles for drug or gene delivery because they enriched with trophic factors.Over the past decades, some studies have been conducted on MSC-DEs showed that exosomes have the capacity in repairing tissue damages, suppressing inflammatory responses, and modulating the immune system but their effects on tumor progression remain controversial and require further studies.Exosome secretions and also its compositions rely on types of sources and environmental conditions; therefore, optimization of the exosome collection procedures from various sources of MSCs gives promising confidence to establish cell-free therapy based exosomes in future.