Decoding therapeutic roles of adipose tissue-derived stromal cells and their extracellular
vesicles in liver disease
Afsharzadeh, Danial
DOI:10.33612/diss.121499227
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date: 2020
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Afsharzadeh, D. (2020). Decoding therapeutic roles of adipose tissue-derived stromal cells and their extracellular vesicles in liver disease. University of Groningen. https://doi.org/10.33612/diss.121499227
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
CHAPTER
1
Introduction and
aim of the thesis
Danial Afsharzadeh1
Martin C. Harmsen2
Klaas Nico Faber1,3
Departments of 1Hepatology and Gastroenterology, 2 Pathology and Medical Biology and 3Laboratory Medicine, Center for Liver, Digestive and Metabolic Disease, University of Groningen, University Medical Center Groningen Groningen, The Netherlands.
1.1 INTRODUCTION
Liver disease is a significant health problem and accounts for over two million deaths per year worldwide1-4. In clinical practice, liver disease is divided into
acute and chronic forms, based on the (initial) presentation and persistence of liver injury. However, this classification is an oversimplification of the broad spectrum of the different types of liver diseases and chronic liver injury is, in part, the result of extended acute liver injury that has been continued over time5,6. Still,
chronic liver diseases are accompanied by additional pathological processes when compared to acute forms, which may require different therapeutic approaches. Acute liver damage is typically induced by viruses or toxins and is characterized by hepatocyte death that can lead to significant impairment of liver function and structure7. The mode of hepatocyte death can be either through necrosis,
apoptosis or mixed variants thereof, and is determined by the type of damage, etiology, duration and extent of liver injury. Acute liver injury can progress to acute liver failure (ALF), which is a critical condition with a mortality rate of more than 80%8. Drug intoxication is considered as the leading cause of ALF
and acetaminophen (APAP) overdose represents the most common cause of drug-induced ALF worldwide9. APAP-induced ALF may occur after taking a
large single oral dose of APAP (>7,500 mg) or after higher than recommended doses for several consecutive days9,10. Viral infection, with hepatitis A, B and
E, is another common cause of ALF. Autoimmune hepatitis, metabolic disease, non-viral infection and cancer are among less frequent causes of ALF9. Loss of
functional liver mass triggers the liver to regenerate, a remarkable feature of this organ. Patients may survive loss of over 50% of liver function and, if the cause of liver injury can be eradicated, the liver will regain to its original volume in several weeks. Thus, ALF only occurs when there is a misbalance between the disease-induced liver damage and the capacity of the liver to regenerate. The liver regeneration process comprises the proliferation of remaining healthy hepatocytes as well as activation, proliferation, differentiation and maturation of hepatic progenitor cells, thereby enabling restoration of the hepatic function and architecture11,12,12. Chronic liver injury is a result of continued hepatocyte injury
and death, which may slowly develop to liver failure over many years. Damaged hepatocytes release reactive oxygen species (ROS) and inflammatory mediators, such as TGF-β1, TNF-α, EGF and IGF that activate liver resident macrophages (Kupffer cells), recruit circulating macrophages and inflammatory T-cells. While this is a primary response to clear possible infections and initiate liver regeneration, persistent activation of these cells also leads to the activation of liver fibroblasts13, particularly hepatic stellate cells (HSC) and portal myofibroblasts
1
(PMF). In normal physiology, HSC are quiescent (qHSC) in the (healthy) liver,where these play a central role in controlling systemic vitamin A homeostasis. Upon liver injury, qHSC transdifferentiate to migratory and proliferative myofibroblasts (activated HSC; aHSC), a process in which the lose their vitamin A stores. The activated liver fibroblasts, both aHSC and PMF, produce and deposit extracellular matrix proteins (ECM), in particular collagen, fibronectin and laminin, in the liver, required for the healing of the damaged tissue. In a normal
Figure 1. Spectrum of non-alcoholic fatty liver disease (NAFLD). Non-alcoholic fatty
liver disease (NAFLD) is a spectrum of diseases ranging from fatty liver (simple steatosis), non-alcoholic steatohepatitis (NASH) and NASH-associated fibrosis and cirrhosis, which predisposes for the development of hepatocellular carcinoma.
repair process, any excessive ECM is degraded by specific matrix metalloproteases (MMPs) and normal liver architecture is restored. However, persistent damage causes a misbalance in ECM production and turnover, because degradation is often insufficiently induced or even decreased. The excessive ECM deposition leads to formation of interstitial scar tissue, which is called fibrosis14. Over time,
fibrosis can spread throughout most of the liver, destroying the internal structure and impair liver regeneration and thereby reducing liver function. Such severe and irreversible scarring of the liver is called cirrhosis. Cirrhosis causes increased intrahepatic resistance to blood flow and portal hypertension, as well as hepatic insufficiency15. Advanced cirrhosis is a risk factor for developing hepatocellular
carcinoma16 and most of the morbidity and mortality related to liver disease occur
after cirrhosis develops17.
In a global perspective, non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease affecting approximately 25% of the world population, in particular in Westernized countries. As the name implies, NAFLD is characterized by excessive fat accumulation in the liver, especially triglycerides and cholesterol, without a clear relationship with alcohol intake. Obesity is the main trigger for NAFLD, which is actually a spectrum of liver diseases, ranging from simple steatosis, non-alcoholic steatohepatitis (NASH) to NASH-associated fibrosis and cirrhosis that predisposes for hepatic carcinogenesis. In this spectrum, NASH distinguishes from simple steatosis by the presence hepatic inflammation, which may co-exist with different stages (F0-F4) of fibrosis. Hepatocyte damage and inflammation play leading roles in disease progression, amongst others via activation of liver fibroblasts and the resultant fibrosis18. Even though only 20%
of patients with NAFLD also meet the criteria for NASH18,19, the latter condition
is being considered the major cause of liver fibrosis and cirrhosis in patients with chronic liver disease (see Figure 1).
Treatment options for patients with end-stage liver disease are still extremely limited, and liver transplantation remains as the only life-saving therapy20,21.
However, scarce availability of donor organs limit the number of patients who can benefit from this treatment. Moreover, liver transplantation remains a risky procedure, associated with high health care expenses, risks of complications related to the surgery, and the necessity for lifelong immunosuppression, which negatively impact the patient’s quality of life22. Thus, there is an evident need for
alternative therapeutic approaches for liver disease patients that otherwise can only be helped by liver transplantation. Therapies using mesenchymal stromal cells (MSC) have recently emerged as being one of the most promising options for patients with chronic liver disease, being a less aggressive procedure and potentially equally curative as liver transplantation23. MSC encompass a population
1
of undifferentiated cells24 that reside in virtually all tissues of the body, includingbone marrow25, umbilical cord26 and adipose tissue27. As a common feature, MSC
are able to differentiate in vitro into cartilage, adipocytes and osteogenic cells. The yield of MSC isolated from different tissues varies considerably, between relatively low from bone marrow and high (around hundred million per liter) from fat tissue. The ease of obtaining subcutaneous adipose tissue by a minimally invasive method is a clear advantage of adipose tissue-derived mesenchymal stromal cells (ASC) over MSCs from other tissues29-31. Demonstration of clinical feasibility and
Figure 2. Chemokine receptors in MSC. MSC chemokine receptors are represented in
therapeutic efficacy of MSC in the past two decades have opened doors for a large number of clinical trials applying MSC in a variety of common and life-threatening diseases, such as stroke32,33, heart failure34, COPD35 and liver failure36. Some trials
have indicated that the route of administration of MSC is a determining factor for their therapeutic efficacy37,38, but to date, no Gold Standard route of MSC delivery
has been established. Several studies proposed that systemic infusion of MSC is the preferred route of administration39-41, but improving tissue-specific homing of
MSC is still one of the major challenges in MSC-based therapies. Indeed, various imaging studies show that only a small percentage of intravenously administered MSC reach the target tissue42-44. A growing body of evidence ascribes this low
efficiency to the limited expression of homing molecules, i.e. chemokine receptors, during the in vitro expansion of MSC45,46. MSC are shown to be attracted to the
chemical gradient of chemokines which are being released at the site of injury47-49.
Chemokines are small secreted proteins ranging in size from 7 to 13 kDa. The arrangement of four amino terminal cysteines is used to group chemokines into structurally related families. Accordingly, four families of chemokines have been defined: the CCL family, the CXCL family, the CL family and the CX3CL family50.
Similarly, chemokine receptors are being categorized based on their interaction with specific chemokine families. Thus, CXCR receptors bind CXC chemokines, and CCR receptors bind CC chemokines. However, as there are fewer chemokine receptors than chemokines, a single chemokine receptor may bind several different chemokines, and a single chemokine also may bind more than one receptor47,50
(see Figure 2).
MSC administration has been shown to be beneficial to treat patients with end-stage liver disease in several studies, as evidenced by improved liver function, reduced hepatocyte apoptosis, enhanced hepatocyte proliferation, and most importantly, increased survival of patients with severe liver disease23,51,53,54.
Nevertheless, the clinical application of MSC holds multiple safety and ethical concerns that call for more in-depth investigation to define the factors that actually determine their therapeutic effect55,. A growing body of evidence indicates that
MSC-derived extracellular vesicles (EVs) are main carriers of the therapeutic factors and largely mirror the phenotype of their parent MSC56,57. Such
MSC-derived EVs could be the basis of a novel cell-free therapy for end-stage liver disease. EVs are released from a variety of cells (including MSC) and are classified into microvesicles (MVs), exosomes, and apoptotic bodies, according to various morphological (size and shape) and biochemical parameters58,59. EVs contain
proteins, lipids and nucleic acids, which they can transport to specific tissues, as they can be captured by other cells via variety of ways, such as direct membrane
1
Figure 3. Mesenchymal stromal cell derived-extracellular vesicles (MSC-derived EVs) rescue the liver damage and ameliorate liver fibrosis. Chronic liver damage mediates by
progressive hepatocyte injury which spans for a long time. Damaged hepatocytes release reactive oxygen species (ROS) and inflammatory mediators, such as TGF-β1, TNF-α, EGF and IGF, inducing the recruitment of inflammatory cells and resulting in accumulation of activated liver fibroblast, particularly hepatic stellate cells (HSC) and portal myofibroblasts (PMF). Activated liver fibroblasts facilitate synthesizing large amounts of extracellular matrix proteins (ECM) which causes tissue fibrosis. MSC derived-EVs are able to rescue the liver damage and ameliorate liver fibrosis through alleviating hepatocyte damage and regression of activation in liver fibroblasts.
fusion, receptor mediated fusion or endocytosis or a combination60-62. So far,
MSC‐derived EVs have shown multiple therapeutic effects, including in tissue regeneration, wound healing and immunomodulation63. Importantly, MSC‐
derived EVs have been shown to be less immunogenic and, compared to MSC, show a lower risk of allogenic immune rejection by the host63,64. Small non-coding
microRNA molecules (miRNAs, mostly 22 nucleotides long) have recently gained extra attention amongst EV-cargo, and a rapidly increasing number of reports attribute the therapeutic potential of MSC-derived EVs to miRNA transfer to affected cells of injured organs65-67. The therapeutic benefits of MSC-derived EVs
are being examined in a range of acute and chronic liver disease68,69. As it appears,
MSC-derived EVs are capable of alleviating hepatocyte damage and suppress the activation of liver fibroblasts (see Figure 3). Still, the therapeutic mechanisms of MSC-derived EVs are very much unexplored and need further investigation.
1
1.2. THE AIM AND OUTLINE OF THE THESIS
The overall aim of this thesis is to investigate the therapeutic potential of extracellular vesicles from human adipose-derived stromal cells (hASC-derived EVs) in the treatment of acute and chronic liver disease. In Chapter 2, we focused on the trafficking of hASC migration to injured and early-fibrotic liver tissue, ex vivo. We tested the migration potential of hASC to human precision-cut liver slices (PCLS). In order to distinguish the migratory signal in the fibrotic tissue, we analyzed the hASC migration towards hepatocytes, quiescent hepatic stellate cells (qHSC) and activated hepatic stellate cells (aHSC), separately. Moreover, using specific inhibitors of the chemokine receptors CXCR2, CXCR3 and CXCR4, we investigated the mechanisms controlling the migration of hASC to the fibrotic liver. In Chapter 3, we focused on the therapeutic capacity of hASC-derived EVs in liver fibrosis. Activation of hepatic fibroblasts is the hallmark for the onset of liver fibrosis. We first evaluated whether hASC suppress activation and proliferation of liver fibroblasts in a paracrine manner. As this was the case, we next evaluated the potential of hASC-derived EVs and EV-free conditioned medium to suppress activation and proliferation in liver fibroblasts in vitro, as well as in vivo mouse model of liver fibrosis and in a human ex vivo using human precision-cut liver slices. In addition, we analyzed the miRNA content of hASC-derived EVs with a focus on miRNAs with anti-fibrotic activities. In Chapter 4, we investigated the potential of hASC-derived EVs in alleviating acute liver damage in mice exposed to a single high dose of paracetamol (APAP) or CCl4. Both the prophylactic and the therapeutic potential of hASC-derived EVs to prevent liver injury were evaluated. We analyzed liver histology and serum markers of liver damage, as well as markers of hepatic inflammation and early markers of fibrosis. In Chapter 5, we studied the potential of hASC-derived EVs to ameliorate simple steatosis in mice, being the first stage in the development of NAFLD. Mice were given a high fat-high cholesterol diet for 6 weeks and treated with hASC-derived EVs in the final 3 weeks. We evaluated body and liver weights of the mice and analyzed liver histology and serum markers of the liver damage. Moreover, we determined whether hASC-derived EVs affect Western-diet induced cholesterol and triglyceride levels in liver and in serum. Finally, transcript analysis was performed to reveal potential miRNAs involved in the hASC-EV-induced effects. In Chapter 6, we summarize the results obtained in the experimental studies of this thesis and provide an outlook for future directions of hASC-derived EV therapy for patients with liver disease.
REFERENCES
1. Alejandra MR, Juan AB, Ana SR. Cell therapy for liver diseases: Current medicine and future promises. Expert Rev Gastroenterol Hepatol. 2015;9(6):837-850.
2. Wang FS, Fan JG, Zhang Z, Gao B, Wang HY. The global burden of liver disease: The major impact of china. Hepatology. 2014;60(6):2099-2108.
3. Mokdad AA, Lopez AD, Shahraz S, et al. Liver cirrhosis mortality in 187 countries between 1980 and 2010: A systematic analysis. BMC Med. 2014;12:145-014-0145-y.
4. Rowe IA. Lessons from epidemiology: The burden of liver disease. Dig Dis. 2017;35(4):304-309.
5. Zhang CY, Yuan WG, He P, Lei JH, Wang CX. Liver fibrosis and hepatic stellate cells: Etiology, pathological hallmarks and therapeutic targets. World J Gastroenterol. 2016;22(48):10512-10522.
6. Hernandez-Gea V, Friedman SL. Pathogenesis of liver fibrosis. Annu Rev Pathol. 2011;6:425-456.
7. Koch DG, Speiser JL, Durkalski V, et al. The natural history of severe acute liver injury. Am J
Gastroenterol. 2017;112(9):1389-1396.
8. Rovegno M, Vera M, Ruiz A, Benitez C. Current concepts in acute liver failure. Ann Hepatol. 2019;18(4):543-552.
9. Fontana RJ. Acute liver failure including acetaminophen overdose. Med Clin North Am. 2008;92(4):761-94, viii.
10. Bernal W. Acute liver failure: Review and update. Int Anesthesiol Clin. 2017;55(2):92-106. 11. Best J, Dolle L, Manka P, Coombes J, van Grunsven LA, Syn WK. Role of liver progenitors in
acute liver injury. Front Physiol. 2013;4:258.
12. Rutherford A, Chung RT. Acute liver failure: Mechanisms of hepatocyte injury and regeneration. Semin Liver Dis. 2008;28(2):167-174.
13. Canbay A, Friedman S, Gores GJ. Apoptosis: The nexus of liver injury and fibrosis. Hepatology. 2004;39(2):273-278.
14. Vinas O, Bataller R, Sancho-Bru P, et al. Human hepatic stellate cells show features of antigen-presenting cells and stimulate lymphocyte proliferation. Hepatology. 2003;38(4):919-929. 15. Gines P, Cardenas A, Arroyo V, Rodes J. Management of cirrhosis and ascites. N Engl J Med.
2004;350(16):1646-1654.
16. Berenguer M, Prieto M, Palau A, et al. Severe recurrent hepatitis C after liver retransplantation for hepatitis C virus-related graft cirrhosis. Liver Transpl. 2003;9(3):228-235.
17. Poynard T, Bedossa P, Opolon P. Natural history of liver fibrosis progression in patients with chronic hepatitis C. the OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet. 1997;349(9055):825-832.
18. Torres DM, Harrison SA. Nonalcoholic steatohepatitis and noncirrhotic hepatocellular carcinoma: Fertile soil. Semin Liver Dis. 2012;32(1):30-38.
19. Hashimoto E, Tokushige K, Ludwig J. Diagnosis and classification of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis: Current concepts and remaining challenges. Hepatol
Res. 2015;45(1):20-28.
20. Bernal W, Wendon J. Acute liver failure. N Engl J Med. 2013;369(26):2525-2534.
21. Manns MP. Liver cirrhosis, transplantation and organ shortage. Dtsch Arztebl Int. 2013;110(6):83-84.
22. Souza BS, Nogueira RC, de Oliveira SA, et al. Current status of stem cell therapy for liver diseases. Cell Transplant. 2009;18(12):1261-1279.
23. Wu DB, Chen EQ, Tang H. Stem cell transplantation for the treatment of end-stage liver disease. World J Hepatol. 2018;10(12):907-910.
1
25. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derivedfrom adult marrow. Nature. 2002;418(6893):41-49.
26. Hass R, Kasper C, Bohm S, Jacobs R. Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. Cell Commun
Signal. 2011;9:12-811X-9-12.
27. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells.
Mol Biol Cell. 2002;13(12):4279-4295.
28. O’Donoghue K, Chan J. Human fetal mesenchymal stem cells. Curr Stem Cell Res Ther. 2006;1(3):371-386.
29. Jung WY, Kang JH, Kim KG, et al. Human adipose-derived stem cells attenuate inflammatory bowel disease in IL-10 knockout mice. Tissue Cell. 2015;47(1):86-93.
30. Haniffa MA, Wang XN, Holtick U, et al. Adult human fibroblasts are potent immunoregulatory cells and functionally equivalent to mesenchymal stem cells. J Immunol. 2007;179(3):1595-1604.
31. Liu WH, Song FQ, Ren LN, et al. The multiple functional roles of mesenchymal stem cells in participating in treating liver diseases. J Cell Mol Med. 2015;19(3):511-520.
32. Bang OY, Lee JS, Lee PH, Lee G. Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol. 2005;57(6):874-882.
33. Kim SJ, Moon GJ, Chang WH, Kim YH, Bang OY, STARTING-2 (STem cell Application Researches and Trials In NeuroloGy-2) collaborators. Intravenous transplantation of mesenchymal stem cells preconditioned with early phase stroke serum: Current evidence and study protocol for a randomized trial. Trials. 2013;14:317-6215-14-317.
34. Bartunek J, Behfar A, Dolatabadi D, et al. Cardiopoietic stem cell therapy in heart failure: The C-CURE (cardiopoietic stem cell therapy in heart failURE) multicenter randomized trial with lineage-specified biologics. J Am Coll Cardiol 2013 Jun 11+ADs-61(23):2329-38 doi: 10 1016/j
jacc 2013 02 071 Epub. 2013;61(23):2329-2338.
35. Weiss DJ, Casaburi R, Flannery R, LeRoux-Williams M, Tashkin DP. A placebo-controlled, randomized trial of mesenchymal stem cells in COPD. Chest. 2013;143(6):1590-1598. 36. Shi M, Zhang Z, Xu R, et al. Human mesenchymal stem cell transfusion is safe and improves
liver function in acute-on-chronic liver failure patients. Stem Cells Transl Med. 2012;1(10):725-731.
37. Kurtz A. Mesenchymal stem cell delivery routes and fate. Int J Stem Cells. 2008;1(1):1-7. 38. Kanelidis AJ, Premer C, Lopez J, Balkan W, Hare JM. Route of delivery modulates the efficacy
of mesenchymal stem cell therapy for myocardial infarction: A meta-analysis of preclinical studies and clinical trials. Circ Res. 2017;120(7):1139-1150.
39. Ra JC, Shin IS, Kim SH, et al. Safety of intravenous infusion of human adipose tissue-derived mesenchymal stem cells in animals and humans. Stem Cells Dev. 2011;20(8):1297-1308. 40. Le Blanc K, Frassoni F, Ball L, et al. Mesenchymal stem cells for treatment of steroid-resistant,
severe, acute graft-versus-host disease: A phase II study. Lancet. 2008;371(9624):1579-1586. 41. Kim SU, de Vellis J. Stem cell-based cell therapy in neurological diseases: A review. J Neurosci
Res. 2009;87(10):2183-2200.
42. Devine SM, Cobbs C, Jennings M, Bartholomew A, Hoffman R. Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates.
Blood. 2003;101(8):2999-3001.
43. Barbash IM, Chouraqui P, Baron J, et al. Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: Feasibility, cell migration, and body distribution. Circulation. 2003;108(7):863-868.
44. Kraitchman DL, Tatsumi M, Gilson WD, et al. Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction. Circulation. 2005;112(10):1451-1461.
45. Wang Y, Fu W, Zhang S, et al. CXCR-7 receptor promotes SDF-1alpha-induced migration of bone marrow mesenchymal stem cells in the transient cerebral ischemia/reperfusion rat hippocampus. Brain Res. 2014;1575:78-86.
46. Du Z, Wei C, Cheng K, et al. Mesenchymal stem cell-conditioned medium reduces liver injury and enhances regeneration in reduced-size rat liver transplantation. J Surg Res. 2013;183(2):907-915.
47. Hocking AM. The role of chemokines in mesenchymal stem cell homing to wounds. Adv
Wound Care (New Rochelle). 2015;4(11):623-630.
48. da Silva Meirelles L, Caplan AI, Nardi NB. In search of the in vivo identity of mesenchymal stem cells. Stem Cells. 2008;26(9):2287-2299.
49. Cornelissen AS, Maijenburg MW, Nolte MA, Voermans C. Organ-specific migration of mesenchymal stromal cells: Who, when, where and why? Immunol Lett. 2015;168(2):159-169. 50. Martins-Green M, Petreaca M, Wang L. Chemokines and their receptors are key players in the orchestra that regulates wound healing. Adv Wound Care (New Rochelle). 2013;2(7):327-347. 51. Shi M, Zhang Z, Xu R, et al. Human mesenchymal stem cell transfusion is safe and improves
liver function in acute-on-chronic liver failure patients. Stem Cells Transl Med. 2012;1(10):725-731.
52. Squillaro T, Peluso G, Galderisi U. Clinical trials with mesenchymal stem cells: An update. Cell
Transplant. 2016;25(5):829-848.
53. Chen B, Wang YH, Qian JQ, Wu DB, Chen EQ, Tang H. Human mesenchymal stem cells for hepatitis B virus-related acute-on-chronic liver failure: A systematic review with meta-analysis. Eur J Gastroenterol Hepatol. 2018;30(10):1224-1229.
54. Huang B, Cheng X, Wang H, et al. Mesenchymal stem cells and their secreted molecules predominantly ameliorate fulminant hepatic failure and chronic liver fibrosis in mice respectively. J Transl Med. 2016;14:45-016-0792-1.
55. Charo RA, Sipp D. Rejuvenating regenerative medicine regulation. N Engl J Med. 2018;378(6):504-505.
56. Gnecchi M, Danieli P, Malpasso G, Ciuffreda MC. Paracrine mechanisms of mesenchymal stem cells in tissue repair. Methods Mol Biol. 2016;1416:123-146.
57. Gnecchi M, He H, Liang OD, et al. Paracrine action accounts for marked protection of ischemic heart by akt-modified mesenchymal stem cells. Nat Med. 2005;11(4):367-368.
58. Phinney DG, Di Giuseppe M, Njah J, et al. Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs. Nat Commun. 2015;6:8472.
59. Wen D, Peng Y, Liu D, Weizmann Y, Mahato RI. Mesenchymal stem cell and derived exosome as small RNA carrier and immunomodulator to improve islet transplantation. J Control
Release. 2016;238:166-175.
60. Arslan F, Lai RC, Smeets MB, et al. Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury. Stem Cell Res. 2013;10(3):301-312.
61. Lin KC, Yip HK, Shao PL, et al. Combination of adipose-derived mesenchymal stem cells (ADMSC) and ADMSC-derived exosomes for protecting kidney from acute ischemia-reperfusion injury. Int J Cardiol. 2016;216:173-185.
62. Kim DK, Nishida H, An SY, Shetty AK, Bartosh TJ, Prockop DJ. Chromatographically isolated CD63+CD81+ extracellular vesicles from mesenchymal stromal cells rescue cognitive impairments after TBI. Proc Natl Acad Sci U S A. 2016;113(1):170-175.
63. Baek G, Choi H, Kim Y, Lee HC, Choi C. Mesenchymal stem cell-derived extracellular vesicles as therapeutics and as a drug delivery platform. Stem Cells Transl Med. 2019;8(9):880-886. 64. Ankrum JA, Ong JF, Karp JM. Mesenchymal stem cells: Immune evasive, not immune
1
65. Rani S, Ryan AE, Griffin MD, Ritter T. Mesenchymal stem cell-derived extracellular vesicles:Toward cell-free therapeutic applications. Mol Ther. 2015;23(5):812-823.
66. Sork H, Corso G, Krjutskov K, et al. Heterogeneity and interplay of the extracellular vesicle small RNA transcriptome and proteome. Sci Rep. 2018;8(1):10813-018-28485-9.
67. Bronisz A, Godlewski J, Chiocca EA. Extracellular vesicles and MicroRNAs: Their role in tumorigenicity and therapy for brain tumors. Cell Mol Neurobiol. 2016;36(3):361-376. 68. Li T, Yan Y, Wang B, et al. Exosomes derived from human umbilical cord mesenchymal stem
cells alleviate liver fibrosis. Stem Cells Dev. 2013;22(6):845-854.
69. Haga H, Yan IK, Takahashi K, Matsuda A, Patel T. Extracellular vesicles from bone marrow-derived mesenchymal stem cells improve survival from lethal hepatic failure in mice. Stem
