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Stem cells and exosomes in cardiac repair
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ScienceDirect Stem cells and exosomes in cardiac repair Dinender K Singla Cardiac diseases currently lead in the number of deaths per year, giving rise an interest in transplanting embryonic and adult stem cells as a means to improve damaged tissue from conditions such as myocardial infarction and coronary artery disease. After testing these cells as a treatment option in both animal and human models, it is believed that these cells improve the damaged tissue primarily through the release of autocrine and paracrine factors. Major concerns such as teratoma formation, immune response, difficulty harvesting cells, and limited cell proliferation and differentiation, hinder the routine use of these cells as a treatment option in the clinic. The advent of stem cell-derived exosomes circumvent those concerns, while still providing the growth factors, miRNA, and additional cell protective factors that aid in repairing and regenerating the damaged tissue. These exosomes have been found to be anti-apoptotic, anti-fibrotic, pro-angiogenic, as well as enhance cardiac differentiation, all of which are key to repairing damaged tissue. As such, stem cell derived exosomes are considered to be a potential new and novel approach in the treatment of various cardiac diseases. Address Division of Metabolic and Cardiovascular Sciences, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32816, USA Corresponding author: Singla, Dinender K ([email protected])
Current Opinion in Pharmacology 2016, 27:19–23 This review comes from a themed issue on Cardiovascular and renal Edited by Gary O Rankin and Nalini Santanam
the formation of fibrotic tissue and cardiac dysfunction [5]. Significant pharmacological interventional, changes in lifestyle, and healthy eating have contributed to improving patients’ health [9]. Unfortunately, there are still increased incidents of cardiac diseases not able to be treated with current methods, giving rise to an urgent need to identify novel therapeutic options. Cell therapy is considered to be a viable option to treat cardiac diseases. There have been published studies on various cell types examined in animal models, which include neo-natal, fetal and adult cardiomyocytes, bone marrow stem cells (BMSCs), skeletal myoblasts, human umbilical cord blood cells (UCBCs), cardiac stem cells (CSCs), cardiospheres, endothelial progenitor cells (EPCs), embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs) [1,6,7,10,11,12,13]. Many of these stem cells therapies have been examined in the clinical trials [2,14]. Recent studies suggest that there is an existence of stem cell derived exosomes as a cell-free system to regenerate the injured myocardium [15,16,17–20]. These exosomes will have significant applications compared to stem cells, which pose a threat of teratoma formation and issues of immune response [6]. In this review article, we will discuss the up-to-date experimental and clinical findings on stem cells, their potential limitations in clinical practice, and an emerging role of exosomes as a future therapeutic option.
Cell therapy http://dx.doi.org/10.1016/j.coph.2016.01.003 1471-4892/# 2016 Elsevier Ltd. All rights reserved.
Introduction Myocardial infarction and coronary artery disease are the most common cardiovascular disorders, and are leading causes of heart failure and death in the modern world [1–6]. The development and progression of cardiovascular diseases are complex, dynamic, and time dependent processes that involve a variety of pathophysiological modifications that contribute to adverse cardiac remodeling [7,8]. The complete etiology of adverse cardiac remodeling is not well characterized; however, in general the process involves left ventricular dilation, increased cardiac hypertrophy, and loss of cardiac myocytes and non-myocytes, leading to www.sciencedirect.com
The general definition of stem cells is considered to be a cell that has the potential to divide into daughter cells as well as have the ability to differentiate into specialized cell types present in the body. Various investigators have identified tissue specific stem and progenitor cells; however, as a broader classification we divide these cells into three types: firstly, ASCs, secondly, ESCs, and thirdly, iPSCs [6,21]. These three major cell types are used in the cell culture system to generate disease models for drug toxicity testing and also to examine their regenerative potential [22–24].
Adult stem cells in cardiac regeneration Cell transplantation to treat cardiac diseases has been studied in animal models and patients [13]. Animal models have demonstrated successful engraftment in the injured myocardium following transplantation [13,25–27]. The rationale to use stem cells as a therapeutic option is that regeneration of injured myocardium with new heart cell types improve mechanical properties, and increase Current Opinion in Pharmacology 2016, 27:19–23
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contractility of the infarcted portion of the left ventricle [28]. Most cell transplantation studies have shown successfully differentiated cardiac cell types and significantly improved cardiac function, despite often limited regeneration [28]. Skeletal myoblasts and BMSCs are the most commonly used cell types to regenerate infarcted myocardium in animal models [29]. Transplantation of autologous skeletal muscle cells has resulted in improved function in areas affected by myocardial infarction in sheep and rats [30,31]. Moreover, transplantation of Lin_c-kitpos BMSCs in infarcted mice hearts resulted in the formation of endothelial cells, smooth muscle cells and cardiomyocytes, all of which helped to regenerate the injured heart tissue [32]. This encouraging data generated interest in clinicians and scientists, which spurred clinical testing of the potential use of adult stem cells for cardiac therapy [2,33–35]. Transplantation of autologous skeletal muscle cells or BMSCs in patients resulted in improvement of regional and global ventricular function [2,33–35]. The majority of clinical studies performed on the transplantation of MSCs, BMSCs and CSCs into the patients successfully demonstrated that these cells are safe, feasible and efficacious to use in patients [2,33–35]. Moreover, these studies show significantly improved cardiac function in most clinical trials [2,33–35]. Other recent randomized clinical trials conducted at various centers demonstrated mixed results of significant success and concerns on the use of these stem cells [2,33–35]. The major concern is that function following transplantation of these cells was only marginally improved, at 6–12% [2,33–35]. The results following cell therapy in patients are usually combined with surgical or percutaneous revascularization, and the patients continue to take their regular medications [2,33–35]. Therefore, it remains unclear whether transplanted stem cells are the sole source to improve cardiac function, or if surgical procedures and medications have boosted the body’s natural effects in addition to the transplanted cells.
Embryonic and induced pluripotent stem cells for cardiac regeneration ESCs are immortal, self-renewing, and undifferentiated pluripotent cells derived from the inner cell mass (ICM) of the pre-implantation blastocyst [6]. IPSCs are derived from somatic cells using transcription factors such as Oct 3/4, Sox 2, Klf 4, and c-Myc [1,21,36]. These factors genetically reprogram somatic cells into a pluripotent cell with the potential to self-renew and differentiate into any cell type in the body. These cells have very similar characteristics to ESCs, while also eliminating ethical concerns [6]. These cells are unique because they have potential to differentiate into the three cardiac cell types (cardiac myocytes, endothelial cells, and vascular smooth muscle cells). We Current Opinion in Pharmacology 2016, 27:19–23
have reported cardiac repair, regeneration and significantly improved cardiac function in the infarcted heart following mouse ESC or iPSC transplantation [37,38]. Another recent study shows purified cardiac myocytes from human ESCs can form contracting aggregates [7]. Following transplantation into the infarcted rat heart, these aggregates can engraft, and improve heart function and survival [7]. Transplanted human-ESCs into non-human primate hearts show differentiation into cardiac myocytes, and remuscularization; however, authors cautioned on the formation of arrhythmias in the small number of animals used in this study [10]. Transplanted beating aggregates isolated from smooth muscle-iPSCs and MSC-iPSCs demonstrated cardiac repair and significantly improved cardiac function [39,40]. Dai et al. generated a patch that consists of iPS-derived cardiac myocytes, endothelial cells and mouse ESCs. Following transplantation into the infarcted myocardium there was a significant reduction in cardiac fibrosis [41]. Zhang et al. have generated sheets of cardiac myocytes from human cardiac iPSCs, and transplanted these into infarcted mouse hearts [11]. Their data shows approximately 32% engraftment at D28, which resulted in increased vascularity, reduction of cardiac apoptosis, and improved heart function [11]. The cardiac regeneration potential of these cells is great; however, the major limitations that hinder these cells from being used in clinical applications are the formation of teratomas, and immune response post-transplantation [1]. Another major concern of the use of ESCs is the ethical issue, which has been partially resolved by the generation of iPSCs, which have also been shown to potentially regenerate the infarcted heart [1].
Stem cell-derived exosomes After extensive research of stem cells in animal models and patients, the general consensus of scientists and clinicians is that the benefit from these stem cell therapies result from autocrine and paracrine factors released by the cells [20,42]. Recent studies suggest that the autocrine and paracrine factors are not the only released factors by stem cells; however, they also release extracellular vesicles called exosomes [15,16,17,20,42]. Exosomes are relatively homogenous in size but differ from other membrane vesicles in density and composition of miRNAs, growth factors, lipids, and proteins. Multiple cell types, including stem cells, secrete these exosomes.
Isolation and purification of exosomes Stem cell-derived exosomes have been isolated based on their presence of tetraspanins, such as CD63, CD81, and CD9, and Hsp70 [19,20,42]. Recent studies suggest that exosomes are obtained from ESCs, CD34 stem cells, MSCs, and human neural cells [42]. Exosomes are isolated from the cell culture supernatant which is taken from www.sciencedirect.com
Exosomes in the heart Singla 21
growing stem cells in serum free conditions [42]. The most common method to use exosomes is standardized by individual labs with the use of ultracentrifugation methods and/or by using commercially available kits such as exoEasy (Qiagen), ExoQuick-TC (SBI, USA), etc. [42]. Isolated exosomes from these kits are further confirmed with Western blot analysis for tetraspanin markers such as CD63, combined with HSP70, and also with ELISA methods or electron microscopy [19]. Derived exosomes are 30–100 nm, compared to macrovesicles which are 100 nm to 1 mm, and apoptotic bodies are 1–5 mm in size [15,19]. Importantly, the composition of exosomes is distinct from other vesicles with a lipid bilayer, proteins, and unique set of microRNAs, as these are secreted by multivesicular bodies of the cell [19].
Cardiac repair and regeneration with exosomes The use of ESC-derived exosomes is still evolving in the field of cardiac repair and regeneration. We can divide exosomes into three types: firstly, exosomes derived from stem cells (embryonic stem cells, mesenchymal stem cells and cardiac stem cells, etc.); secondly, exosomes derived from mature cells present in the heart such as cardiomyocytes and fibroblasts; finally, exosomes derived from cells exposed to pathological conditions. Additionally, it also needs to be determined whether these exosomes, irrespective of cell type, have similar characteristics and function, or if the derived exosomes are cell type specific (Figure 1). Recent studies suggest that secreted factors/miRNA from exosomes are stem cell specific, and have a specific role in the inhibition of cardiac apoptosis, regeneration, and fibrosis [43,44,45]. For example, MSC-isolated exosomes have increased presence of miR-19a, which has been shown to be anti-apoptotic [43]. Another ESCsexosome study shows presence of miR-294, which plays a significant role in the activation and differentiation of endogenous CSCs in the infarcted heart [45]. Moreover, miR-126 and miR-130 are significantly increased in exosomes isolated from CD34 hematopoietic stem cells, which are pro-angiogenic in the infarcted heart [44]. A recent study showed that glucose deprived cardiomyocytes secrete exosomes, which are then taken up by endothelial cells (ECs) and have been shown to demonstrate an increase in glucose uptake, glycolytic activity, and pyruvate production [16]. This data shows that exosomes enhance metabolic activity, which could potentially have implications in ischemic injury when apoptotic or necrotic cells secrete exosomes in a glucose-deprived environment [16]. Further in vivo studies are required to determine whether these secreted exosomes enhance glucose metabolism in ECs, and determine their specific effects on cardiac protection. These exosomes are enriched with different types of www.sciencedirect.com
Figure 1
Stem cells
Exosomes
Growth Factors, Cell protective components, and miRNAs
Anti-apoptotic Anti-fibrotic Pro-angiogenic CSCs Differentiation
Cardiac Repair, Regeneration and Protection Current Opinion in Pharmacology
Schematic representation of stem cell derived exosome-mediated cardiac protection.
miRNA, growth factors, and cell protection components, which play an important role in cells protection. These exosomes are anti-apoptotic, pro-angiogenic, a glucose transporter and also activate CSCs for their differentiation into cardiomyocytes in the heart [16,43,44,45]. Exosomes have been shown to have some detrimental effects; however, this depends on the conditions (such as oxidatively stressed cells, or type 2 diabetes exposed cells) of the cells where these exosomes were isolated [46]. Type 2 diabetic Goto-Kakizaki (GK) cardiomyocytes, when co-cultured with ECs, demonstrated significant inhibition in the ECs migration and proliferation [46]. In contrast, this was not the case when cardiomyocytes were isolated from healthy rats [16]. This data suggests that there was a release of exosomes or paracrine factors from GK cardiomyocytes that are different from healthy cardiomyocytes, and are detrimental in nature [46]. These authors then inhibited the release of GK cardiomyocytes exosomes by inhibitor GK4869, and the detrimental effects were eliminated [46]. This data suggests that released exosomes are toxic to the cells by inhibiting cell proliferation and migration [46]. Lyu et al. suggest that Angiotensin II treated cardiac fibroblast-derived exosomes added in cultured cardiac myocytes increased the expression of Current Opinion in Pharmacology 2016, 27:19–23
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renin, angiotensinogen, AT1R and AT2R, and the production of Angiotensin II that induces pathological hypertrophy in the cell culture system [15]. However, it remains unclear whether transplantation of exosomes derived from cardiac fibroblasts in the heart can induce pathological hypertrophy, and this also requires further testing. Exosomes derived from stem cells have been tested in cell culture and in vivo models of cardiac diseases such as myocardial infarction, hypertension, metabolic disorders, and heart failure; however, there is still more research that needs to be done on exosomes derived from mature cells. These current findings suggest that exosome isolation depends on the cell source and cellular conditions.
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Conclusions and future directions Both human and mouse ESCs can form complex teratomas following transplantation, a characteristic of ESCs resulting from their pluripotential capacity [6]. Teratoma formation has been considered to be a major limitation in the therapeutic use of ESCs. ESC derived exosomes, which are cell-free, do not pose a threat of teratoma formation, thus circumventing a major challenge associated with stem cells, and providing a future alternative to current cell therapy [42]. Additionally, for adult stem cell therapy, there is a need to grow these cells in large quantities for cell transplantation to regenerate the infracted heart; however, it is considered be a tedious process due to limited adult stem cell proliferation and differentiation in cell culture. There is a need to establish if growing adult stem cells can continuously secrete a large quantity of exosomes, of excellent quality, in the cell culture system for longer periods of time before routine use is considered. Further understanding on the source of exosomes, their beneficial cellular proteins and microRNAs, mechanisms of exosome-mediated cellular protection and differentiation, and their role in various acute and chronic cardiac disease conditions is still needed.
Conflict of interest Nothing declared.
Acknowledgements The author is thankful for Jessica Hellein for assistance in preparation and proof reading of the manuscript. The author is also very thankful for the support provided by The Estate of Rebecca Gurecki.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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10. Chong JJ, Yang X, Don CW, Minami E, Liu YW, Weyers JJ, Mahoney WM, Van BB, Cook SM, Palpant NJ, Gantz JA, Fugate JA, Muskheli V, Gough GM, Vogel KW, Astley CA, Hotchkiss CE, Baldessari A, Pabon L, Reinecke H, Gill EA, Nelson V, Kiem HP, Laflamme MA, Murry CE: Human embryonicstem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature 2014, 510:273-277. 11. Zhang L, Guo J, Zhang P, Xiong Q, Wu SC, Xia L, Roy SS, Tolar J, O’Connell TD, Kyba M, Liao K, Zhang J: Derivation and high engraftment of patient-specific cardiomyocyte sheet using induced pluripotent stem cells generated from adult cardiac fibroblast. Circ Heart Fail 2015, 8:156-166. This report is an important example of how using sheet technology improves engraftment of cardiomyocytes significantly, compared to simple injection of the cells. 12. Hartman ME, Dai DF, Laflamme MA: Human pluripotent stem cells: prospects and challenges as a source of cardiomyocytes for in vitro modeling and cell-based cardiac repair. Adv Drug Deliv Rev 2015. 13. Williams AR, Hatzistergos KE, Addicott B, McCall F, Carvalho D, Suncion V, Morales AR, Da SJ, Sussman MA, Heldman AW, Hare JM: Enhanced effect of combining human cardiac stem cells and bone marrow mesenchymal stem cells to reduce infarct size and to restore cardiac function after myocardial infarction. Circulation 2013, 127:213-223. This article extends on previous findings regarding improvement of heart function post-myocardial infarction. 14. Prowse AB, Timmins NE, Yau TM, Li RK, Weisel RD, Keller G, Zandstra PW: Transforming the promise of pluripotent stem cell-derived cardiomyocytes to a therapy: challenges and solutions for clinical trials. Can J Cardiol 2014, 30:1335-1349. 15. Lyu L, Wang H, Li B, Qin Q, Qi L, Nagarkatti M, Nagarkatti P, Janicki JS, Wang XL, Cui T: A critical role of cardiac fibroblastderived exosomes in activating renin angiotensin system in cardiomyocytes. J Mol Cell Cardiol 2015. 16. Garcia NA, Moncayo-Arlandi J, Sepulveda P, Diez-Juan A: Cardiomyocyte exosomes regulate glycolytic flux in endothelium by direct transfer of GLUT transporters and glycolytic enzymes. Cardiovasc Res 2015. This article provides strong evidence that exosomes can be used to aid in certain conditions, such as a glucose-deprived enviornment in the heart. This is very important to understanding the therapeutic potential of exosomes. www.sciencedirect.com
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17. Cervio E, Barile L, Moccetti T, Vassalli G: Exosomes for intramyocardial intercellular communication. Stem Cells Int 2015:482171. 18. Das S, Halushka MK: Extracellular vesicle microRNA transfer in cardiovascular disease. Cardiovasc Pathol 2015, 24:199-206. 19. Sluijter JP, Verhage V, Deddens JC, van den Akker F, Doevendans PA: Microvesicles and exosomes for intracardiac communication. Cardiovasc Res 2014, 102:302-311. 20. Ailawadi S, Wang X, Gu H, Fan GC: Pathologic function and therapeutic potential of exosomes in cardiovascular disease. Biochim Biophys Acta 2015, 1852:1-11. 21. Zhang Y, Mignone J, MacLellan WR: Cardiac regeneration and stem cells. Physiol Rev 2015, 95:1189-1204. 22. Chepeleva EV, Pavlova SV, Malakhova AA, Milevskaya EA, Rusakova YL, Podkhvatilina NA, Sergeevichev DS, Pokushalov EA, Karaskov AM, Sukhikh GT, Zakiyan SM: Therapy of chronic cardiosclerosis in WAG rats using cultures of cardiovascular cells enriched with cardiac stem cell. Bull Exp Biol Med 2015. 23. Lee YK, Lau YM, Ng KM, Lai WH, Ho SL, Tse HF, Siu CW, Ho PW: Efficient attenuation of Friedreich’s ataxia (FRDA) cardiomyopathy by modulation of iron homeostasis-human induced pluripotent stem cell (hiPSC) as a drug screening platform for FRDA. Int J Cardiol 2015, 203:964-971. 24. Breckwoldt K, Weinberger F, Eschenhagen T: Heart regeneration. Biochim Biophys Acta 2015. 25. Fanton Y, Robic B, Rummens JL, Daniels A, Windmolders S, Willems L, Jamaer L, Dubois J, Bijnens E, Heuts N, Notelaers K, Paesen R, Ameloot M, Mees U, Bito V, Declercq J, Hensen K, Koninckx R, Hendrikx M: Cardiac atrial appendage stem cells engraft and differentiate into cardiomyocytes in vivo: a new tool for cardiac repair after MI. Int J Cardiol 2015, 201:10-19. 26. Yang CJ, Yang J, Yang J, Fan ZX: Cardiac atrial appendage stem cells therapy: a novel and promising approach for myocardial reparation after MI. Int J Cardiol 2015. 27. Fanton Y, Robic B, Rummens JL, Daniels A, Windmolders S, Willems L, Jamaer L, Dubois J, Bijnens E, Heuts N, Notelaers K, Paesen R, Ameloot M, Mees U, Bito V, Declercq J, Hensen K, Koninckx R, Hendrikx M: Possibilities and limitations for cotransplantation of cardiac atrial appendage stem cells and mesenchymal stem cells for myocardial repair. Int J Cardiol 2015. 28. Fernandes S, Chong JJ, Paige SL, Iwata M, Torok-Storb B, Keller G, Reinecke H, Murry CE: Comparison of human embryonic stem cell-derived cardiomyocytes, cardiovascular progenitors, and bone marrow mononuclear cells for cardiac repair. Stem Cell Rep 2015. 29. Antanaviciute I, Ereminiene E, Vysockas V, Rackauskas M, Skipskis V, Rysevaite K, Treinys R, Benetis R, Jurevicius J, Skeberdis VA: Exogenous connexin43-expressing autologous skeletal myoblasts ameliorate mechanical function and electrical activity of the rabbit heart after experimental infarction. Int J Exp Pathol 2015, 96:42-53. 30. Ghostine S, Carrion C, Souza LC, Richard P, Bruneval P, Vilquin JT, Pouzet B, Schwartz K, Menasche P, Hagege AA: Longterm efficacy of myoblast transplantation on regional structure and function after myocardial infarction. Circulation 2002, 106:I131-I136. 31. Farahmand P, Lai TY, Weisel RD, Fazel S, Yau T, Menasche P, Li RK: Skeletal myoblasts preserve remote matrix architecture and global function when implanted early or late after coronary ligation into infarcted or remote myocardium. Circulation 2008, 118:S130-S137. 32. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, Anversa P: Bone marrow cells regenerate infarcted myocardium. Nature 2001, 410:701-705. 33. Menasche P: Cardiac cell therapy: lessons from clinical trials. J Mol Cell Cardiol 2011, 50:258-265.
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34. Menasche P, Alfieri O, Janssens S, McKenna W, Reichenspurner H, Trinquart L, Vilquin JT, Marolleau JP, Seymour B, Larghero J, Lake S, Chatellier G, Solomon S, Desnos M, Hagege AA: The Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial: first randomized placebo-controlled study of myoblast transplantation. Circulation 2008, 117:1189-1200. 35. Afzal MR, Samanta A, Shah ZI, Jeevanantham V, Abdel-Latif A, Zuba-Surma EK, Dawn B: Adult bone marrow cell therapy for ischemic heart disease: evidence and insights from randomized controlled trials. Circ Res 2015, 117:558-575. 36. Pasha Z, Haider HK, Ashraf M: Efficient non-viral reprogramming of myoblasts to stemness with a single small molecule to generate cardiac progenitor cells. PLoS ONE 2011, 6:e23667. 37. Singla DK, Long X, Glass C, Singla RD, Yan B: Induced pluripotent stem (iPS) cells repair and regenerate infarcted myocardium. Mol Pharm 2011, 8:1573-1581. 38. Yan B, Singla RD, Abdelli LS, Singal PK, Singla DK: Regulation of PTEN/Akt pathway enhances cardiomyogenesis and attenuates adverse left ventricular remodeling following thymosin beta4 overexpressing embryonic stem cell transplantation in the infarcted heart. PLOS ONE 2013, 8:e75580. 39. Ahmed RP, Haider HK, Buccini S, Li L, Jiang S, Ashraf M: Reprogramming of skeletal myoblasts for induction of pluripotency for tumor-free cardiomyogenesis in the infarcted heart. Circ Res 2011, 109:60-70. 40. Buccini S, Haider KH, Ahmed RP, Jiang S, Ashraf M: Cardiac progenitors derived from reprogrammed mesenchymal stem cells contribute to angiomyogenic repair of the infarcted heart. Basic Res Cardiol 2012, 107:301. 41. Dai B, Huang W, Xu M, Millard RW, Gao MH, Hammond HK, Menick DR, Ashraf M, Wang Y: Reduced collagen deposition in infarcted myocardium facilitates induced pluripotent stem cell engraftment and angiomyogenesis for improvement of left ventricular function. J Am Coll Cardiol 2011, 58:2118-2127. 42. Vishnubhatla I, Corteling R, Stevanato L, Hicks C, Sinden J: The development of stem cell-derived exosomes as a cell-free regenerative medicine. J Circ Biomark 2014. 43. Yu B, Kim HW, Gong M, Wang J, Millard RW, Wang Y, Ashraf M, Xu M: Exosomes secreted from GATA-4 overexpressing mesenchymal stem cells serve as a reservoir of anti-apoptotic microRNAs for cardioprotection. Int J Cardiol 2015, 182: 349-360. This report demonstrates the anti-apoptoic nature of exosomes, further providing evidence that exosomes are a good candidate for therapeutic applications. 44. Sahoo S, Klychko E, Thorne T, Misener S, Schultz KM, Millay M, Ito A, Liu T, Kamide C, Agrawal H, Perlman H, Qin G, Kishore R, Losordo DW: Exosomes from human CD34(+) stem cells mediate their proangiogenic paracrine activity. Circ Res 2011, 109:724-728. This is an excellent example of showing the pro-angiogenic properties of exosomes derived from CD34 stem cells, and highlighting their paracrine activities. 45. Khan M, Nickoloff E, Abramova T, Johnson J, Verma SK, Krishnamurthy P, Mackie AR, Vaughan E, Garikipati VN, Benedict C, Ramirez V, Lambers E, Ito A, Gao E, Misener S, Luongo T, Elrod J, Qin G, Houser SR, Koch WJ, Kishore R: Embryonic stem cell-derived exosomes promote endogenous repair mechanisms and enhance cardiac function following myocardial infarction. Circ Res 2015, 117:52-64. This data shows that ESC-derived exosomes also have the ability to promote CSC differentiation, further providing compelling evidence that exosomes are a great choice for therapeutical treatments to improve cardiac function. 46. Wang X, Huang W, Liu G, Cai W, Millard RW, Wang Y, Chang J, Peng T, Fan GC: Cardiomyocytes mediate anti-angiogenesis in type 2 diabetic rats through the exosomal transfer of miR-320 into endothelial cells. J Mol Cell Cardiol 2014, 74:139-150. This is an excellent article on how certain pathological conditions, such as type 2 diabetes, can cause exosomes to behave differently than exosomes isolated from healthy animal models.
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ScienceDirect Stem cells and exosomes in cardiac repair Dinender K Singla Cardiac diseases currently lead in the number of deaths per year, giving rise an interest in transplanting embryonic and adult stem cells as a means to improve damaged tissue from conditions such as myocardial infarction and coronary artery disease. After testing these cells as a treatment option in both animal and human models, it is believed that these cells improve the damaged tissue primarily through the release of autocrine and paracrine factors. Major concerns such as teratoma formation, immune response, difficulty harvesting cells, and limited cell proliferation and differentiation, hinder the routine use of these cells as a treatment option in the clinic. The advent of stem cell-derived exosomes circumvent those concerns, while still providing the growth factors, miRNA, and additional cell protective factors that aid in repairing and regenerating the damaged tissue. These exosomes have been found to be anti-apoptotic, anti-fibrotic, pro-angiogenic, as well as enhance cardiac differentiation, all of which are key to repairing damaged tissue. As such, stem cell derived exosomes are considered to be a potential new and novel approach in the treatment of various cardiac diseases. Address Division of Metabolic and Cardiovascular Sciences, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32816, USA Corresponding author: Singla, Dinender K ([email protected])
Current Opinion in Pharmacology 2016, 27:19–23 This review comes from a themed issue on Cardiovascular and renal Edited by Gary O Rankin and Nalini Santanam
the formation of fibrotic tissue and cardiac dysfunction [5]. Significant pharmacological interventional, changes in lifestyle, and healthy eating have contributed to improving patients’ health [9]. Unfortunately, there are still increased incidents of cardiac diseases not able to be treated with current methods, giving rise to an urgent need to identify novel therapeutic options. Cell therapy is considered to be a viable option to treat cardiac diseases. There have been published studies on various cell types examined in animal models, which include neo-natal, fetal and adult cardiomyocytes, bone marrow stem cells (BMSCs), skeletal myoblasts, human umbilical cord blood cells (UCBCs), cardiac stem cells (CSCs), cardiospheres, endothelial progenitor cells (EPCs), embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs) [1,6,7,10,11,12,13]. Many of these stem cells therapies have been examined in the clinical trials [2,14]. Recent studies suggest that there is an existence of stem cell derived exosomes as a cell-free system to regenerate the injured myocardium [15,16,17–20]. These exosomes will have significant applications compared to stem cells, which pose a threat of teratoma formation and issues of immune response [6]. In this review article, we will discuss the up-to-date experimental and clinical findings on stem cells, their potential limitations in clinical practice, and an emerging role of exosomes as a future therapeutic option.
Cell therapy http://dx.doi.org/10.1016/j.coph.2016.01.003 1471-4892/# 2016 Elsevier Ltd. All rights reserved.
Introduction Myocardial infarction and coronary artery disease are the most common cardiovascular disorders, and are leading causes of heart failure and death in the modern world [1–6]. The development and progression of cardiovascular diseases are complex, dynamic, and time dependent processes that involve a variety of pathophysiological modifications that contribute to adverse cardiac remodeling [7,8]. The complete etiology of adverse cardiac remodeling is not well characterized; however, in general the process involves left ventricular dilation, increased cardiac hypertrophy, and loss of cardiac myocytes and non-myocytes, leading to www.sciencedirect.com
The general definition of stem cells is considered to be a cell that has the potential to divide into daughter cells as well as have the ability to differentiate into specialized cell types present in the body. Various investigators have identified tissue specific stem and progenitor cells; however, as a broader classification we divide these cells into three types: firstly, ASCs, secondly, ESCs, and thirdly, iPSCs [6,21]. These three major cell types are used in the cell culture system to generate disease models for drug toxicity testing and also to examine their regenerative potential [22–24].
Adult stem cells in cardiac regeneration Cell transplantation to treat cardiac diseases has been studied in animal models and patients [13]. Animal models have demonstrated successful engraftment in the injured myocardium following transplantation [13,25–27]. The rationale to use stem cells as a therapeutic option is that regeneration of injured myocardium with new heart cell types improve mechanical properties, and increase Current Opinion in Pharmacology 2016, 27:19–23
20 Cardiovascular and renal
contractility of the infarcted portion of the left ventricle [28]. Most cell transplantation studies have shown successfully differentiated cardiac cell types and significantly improved cardiac function, despite often limited regeneration [28]. Skeletal myoblasts and BMSCs are the most commonly used cell types to regenerate infarcted myocardium in animal models [29]. Transplantation of autologous skeletal muscle cells has resulted in improved function in areas affected by myocardial infarction in sheep and rats [30,31]. Moreover, transplantation of Lin_c-kitpos BMSCs in infarcted mice hearts resulted in the formation of endothelial cells, smooth muscle cells and cardiomyocytes, all of which helped to regenerate the injured heart tissue [32]. This encouraging data generated interest in clinicians and scientists, which spurred clinical testing of the potential use of adult stem cells for cardiac therapy [2,33–35]. Transplantation of autologous skeletal muscle cells or BMSCs in patients resulted in improvement of regional and global ventricular function [2,33–35]. The majority of clinical studies performed on the transplantation of MSCs, BMSCs and CSCs into the patients successfully demonstrated that these cells are safe, feasible and efficacious to use in patients [2,33–35]. Moreover, these studies show significantly improved cardiac function in most clinical trials [2,33–35]. Other recent randomized clinical trials conducted at various centers demonstrated mixed results of significant success and concerns on the use of these stem cells [2,33–35]. The major concern is that function following transplantation of these cells was only marginally improved, at 6–12% [2,33–35]. The results following cell therapy in patients are usually combined with surgical or percutaneous revascularization, and the patients continue to take their regular medications [2,33–35]. Therefore, it remains unclear whether transplanted stem cells are the sole source to improve cardiac function, or if surgical procedures and medications have boosted the body’s natural effects in addition to the transplanted cells.
Embryonic and induced pluripotent stem cells for cardiac regeneration ESCs are immortal, self-renewing, and undifferentiated pluripotent cells derived from the inner cell mass (ICM) of the pre-implantation blastocyst [6]. IPSCs are derived from somatic cells using transcription factors such as Oct 3/4, Sox 2, Klf 4, and c-Myc [1,21,36]. These factors genetically reprogram somatic cells into a pluripotent cell with the potential to self-renew and differentiate into any cell type in the body. These cells have very similar characteristics to ESCs, while also eliminating ethical concerns [6]. These cells are unique because they have potential to differentiate into the three cardiac cell types (cardiac myocytes, endothelial cells, and vascular smooth muscle cells). We Current Opinion in Pharmacology 2016, 27:19–23
have reported cardiac repair, regeneration and significantly improved cardiac function in the infarcted heart following mouse ESC or iPSC transplantation [37,38]. Another recent study shows purified cardiac myocytes from human ESCs can form contracting aggregates [7]. Following transplantation into the infarcted rat heart, these aggregates can engraft, and improve heart function and survival [7]. Transplanted human-ESCs into non-human primate hearts show differentiation into cardiac myocytes, and remuscularization; however, authors cautioned on the formation of arrhythmias in the small number of animals used in this study [10]. Transplanted beating aggregates isolated from smooth muscle-iPSCs and MSC-iPSCs demonstrated cardiac repair and significantly improved cardiac function [39,40]. Dai et al. generated a patch that consists of iPS-derived cardiac myocytes, endothelial cells and mouse ESCs. Following transplantation into the infarcted myocardium there was a significant reduction in cardiac fibrosis [41]. Zhang et al. have generated sheets of cardiac myocytes from human cardiac iPSCs, and transplanted these into infarcted mouse hearts [11]. Their data shows approximately 32% engraftment at D28, which resulted in increased vascularity, reduction of cardiac apoptosis, and improved heart function [11]. The cardiac regeneration potential of these cells is great; however, the major limitations that hinder these cells from being used in clinical applications are the formation of teratomas, and immune response post-transplantation [1]. Another major concern of the use of ESCs is the ethical issue, which has been partially resolved by the generation of iPSCs, which have also been shown to potentially regenerate the infarcted heart [1].
Stem cell-derived exosomes After extensive research of stem cells in animal models and patients, the general consensus of scientists and clinicians is that the benefit from these stem cell therapies result from autocrine and paracrine factors released by the cells [20,42]. Recent studies suggest that the autocrine and paracrine factors are not the only released factors by stem cells; however, they also release extracellular vesicles called exosomes [15,16,17,20,42]. Exosomes are relatively homogenous in size but differ from other membrane vesicles in density and composition of miRNAs, growth factors, lipids, and proteins. Multiple cell types, including stem cells, secrete these exosomes.
Isolation and purification of exosomes Stem cell-derived exosomes have been isolated based on their presence of tetraspanins, such as CD63, CD81, and CD9, and Hsp70 [19,20,42]. Recent studies suggest that exosomes are obtained from ESCs, CD34 stem cells, MSCs, and human neural cells [42]. Exosomes are isolated from the cell culture supernatant which is taken from www.sciencedirect.com
Exosomes in the heart Singla 21
growing stem cells in serum free conditions [42]. The most common method to use exosomes is standardized by individual labs with the use of ultracentrifugation methods and/or by using commercially available kits such as exoEasy (Qiagen), ExoQuick-TC (SBI, USA), etc. [42]. Isolated exosomes from these kits are further confirmed with Western blot analysis for tetraspanin markers such as CD63, combined with HSP70, and also with ELISA methods or electron microscopy [19]. Derived exosomes are 30–100 nm, compared to macrovesicles which are 100 nm to 1 mm, and apoptotic bodies are 1–5 mm in size [15,19]. Importantly, the composition of exosomes is distinct from other vesicles with a lipid bilayer, proteins, and unique set of microRNAs, as these are secreted by multivesicular bodies of the cell [19].
Cardiac repair and regeneration with exosomes The use of ESC-derived exosomes is still evolving in the field of cardiac repair and regeneration. We can divide exosomes into three types: firstly, exosomes derived from stem cells (embryonic stem cells, mesenchymal stem cells and cardiac stem cells, etc.); secondly, exosomes derived from mature cells present in the heart such as cardiomyocytes and fibroblasts; finally, exosomes derived from cells exposed to pathological conditions. Additionally, it also needs to be determined whether these exosomes, irrespective of cell type, have similar characteristics and function, or if the derived exosomes are cell type specific (Figure 1). Recent studies suggest that secreted factors/miRNA from exosomes are stem cell specific, and have a specific role in the inhibition of cardiac apoptosis, regeneration, and fibrosis [43,44,45]. For example, MSC-isolated exosomes have increased presence of miR-19a, which has been shown to be anti-apoptotic [43]. Another ESCsexosome study shows presence of miR-294, which plays a significant role in the activation and differentiation of endogenous CSCs in the infarcted heart [45]. Moreover, miR-126 and miR-130 are significantly increased in exosomes isolated from CD34 hematopoietic stem cells, which are pro-angiogenic in the infarcted heart [44]. A recent study showed that glucose deprived cardiomyocytes secrete exosomes, which are then taken up by endothelial cells (ECs) and have been shown to demonstrate an increase in glucose uptake, glycolytic activity, and pyruvate production [16]. This data shows that exosomes enhance metabolic activity, which could potentially have implications in ischemic injury when apoptotic or necrotic cells secrete exosomes in a glucose-deprived environment [16]. Further in vivo studies are required to determine whether these secreted exosomes enhance glucose metabolism in ECs, and determine their specific effects on cardiac protection. These exosomes are enriched with different types of www.sciencedirect.com
Figure 1
Stem cells
Exosomes
Growth Factors, Cell protective components, and miRNAs
Anti-apoptotic Anti-fibrotic Pro-angiogenic CSCs Differentiation
Cardiac Repair, Regeneration and Protection Current Opinion in Pharmacology
Schematic representation of stem cell derived exosome-mediated cardiac protection.
miRNA, growth factors, and cell protection components, which play an important role in cells protection. These exosomes are anti-apoptotic, pro-angiogenic, a glucose transporter and also activate CSCs for their differentiation into cardiomyocytes in the heart [16,43,44,45]. Exosomes have been shown to have some detrimental effects; however, this depends on the conditions (such as oxidatively stressed cells, or type 2 diabetes exposed cells) of the cells where these exosomes were isolated [46]. Type 2 diabetic Goto-Kakizaki (GK) cardiomyocytes, when co-cultured with ECs, demonstrated significant inhibition in the ECs migration and proliferation [46]. In contrast, this was not the case when cardiomyocytes were isolated from healthy rats [16]. This data suggests that there was a release of exosomes or paracrine factors from GK cardiomyocytes that are different from healthy cardiomyocytes, and are detrimental in nature [46]. These authors then inhibited the release of GK cardiomyocytes exosomes by inhibitor GK4869, and the detrimental effects were eliminated [46]. This data suggests that released exosomes are toxic to the cells by inhibiting cell proliferation and migration [46]. Lyu et al. suggest that Angiotensin II treated cardiac fibroblast-derived exosomes added in cultured cardiac myocytes increased the expression of Current Opinion in Pharmacology 2016, 27:19–23
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renin, angiotensinogen, AT1R and AT2R, and the production of Angiotensin II that induces pathological hypertrophy in the cell culture system [15]. However, it remains unclear whether transplantation of exosomes derived from cardiac fibroblasts in the heart can induce pathological hypertrophy, and this also requires further testing. Exosomes derived from stem cells have been tested in cell culture and in vivo models of cardiac diseases such as myocardial infarction, hypertension, metabolic disorders, and heart failure; however, there is still more research that needs to be done on exosomes derived from mature cells. These current findings suggest that exosome isolation depends on the cell source and cellular conditions.
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Conclusions and future directions Both human and mouse ESCs can form complex teratomas following transplantation, a characteristic of ESCs resulting from their pluripotential capacity [6]. Teratoma formation has been considered to be a major limitation in the therapeutic use of ESCs. ESC derived exosomes, which are cell-free, do not pose a threat of teratoma formation, thus circumventing a major challenge associated with stem cells, and providing a future alternative to current cell therapy [42]. Additionally, for adult stem cell therapy, there is a need to grow these cells in large quantities for cell transplantation to regenerate the infracted heart; however, it is considered be a tedious process due to limited adult stem cell proliferation and differentiation in cell culture. There is a need to establish if growing adult stem cells can continuously secrete a large quantity of exosomes, of excellent quality, in the cell culture system for longer periods of time before routine use is considered. Further understanding on the source of exosomes, their beneficial cellular proteins and microRNAs, mechanisms of exosome-mediated cellular protection and differentiation, and their role in various acute and chronic cardiac disease conditions is still needed.
Conflict of interest Nothing declared.
Acknowledgements The author is thankful for Jessica Hellein for assistance in preparation and proof reading of the manuscript. The author is also very thankful for the support provided by The Estate of Rebecca Gurecki.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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