Mesenchymal stem cells versus their conditioned medium in the treatment of ischemia/reperfusion injury: Evaluation of efficacy and hepatic specific gene expression in mice

Document Type : Original Article


1 Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran

2 Department of Anatomical Sciences, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran

3 Burn and Regenerative Medicine Research Center, Guilan University of Medical Sciences, Rasht, Iran


Objective(s): The mechanisms underlying the beneficial effects of MSCs on hepatic I/R injury are still poorly described, especially the changes in hepatocyte gene expression. In this study, the effect of bone marrow-derived mesenchymal stem cells (BMSCs) and adipose tissue-derived mesenchymal stem cells (AMSCs) and their conditioned medium on hepatocyte gene expression resulted by I/R shock were investigated.
Materials and Methods: Liver ischemia models were induced by clamping in experimental groups. Experimental groups received MSCs or conditioned medium treatments and the control group received Dulbecco’s Modified Eagle Medium (DMEM). During 1, 24 hr, and 1 week after treatment, the serum levels of alanine aminotransferase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) enzymes and tissue catalase activity (CAT) were measured. Gene expression of a number of hepatocyte-specific genes (Alb, Afp, and Ck8) and Icam-1 which is upregulated under inflammatory conditions were also evaluated in 5, 24 hr, and 1-week intervals after I/R insult.
Results: In this study, liver enzymes showed a much more shift in the control group than treated groups and it was more noticeable 5 hr post-treatment. Moreover, gene expression pattern of the control group underwent changes after I/R injury. However, treated groups gene expression analysis met a steady trend after I/R insult.
Conclusion: Our finding shows that stem cell treatment has better curative effects than conditioned medium. BMSCs, AMSCs or BMSC and AMSC-derived bioactive molecules injection have potential to be considered as a therapeutic approach for treating acute liver injury.


1. Harrison D. Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Invest. 1997; 100:2153-2157.
2. van Poll D, Parekkadan B, H Cho C, Berthiaume F, Nahmias Y, W Tilles A, et al. Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo. Hepatology 2008; 47:1634-1643.
3. Peralta C, Jiménez-Castro MB, Gracia-Sancho J. Hepatic ischemia and reperfusion injury: Effects on the liver sinusoidal milieu. J Hepatol 2013; 59:1094-1106.
4. Kawaratani H, Tsujimoto T, Douhara A, Takaya H, Moriya K, Namisaki T, et al. The effect of inflammatory cytokines in alcoholic liver disease. Mediators Inflamm 2013; 495156-495156.
5. Kuo TK, Hung SP, Chuang CH, Chen CT, Shih YRV, Fang SCY, et al. Stem cell therapy for liver disease: Parameters governing the success of using bone marrow mesenchymal stem cells. Gastroenterology 2008; 134:2111-2121.e2113.
6. Li S, Zheng X, Li H, Zheng J, Chen X, Liu W, et al. Mesenchymal stem cells ameliorate hepatic ischemia/reperfusion injury via inhibition of neutrophil recruitment. J Immunol Res 2018; 7283703-7283703.
7. Haga H, Yan IK, Borrelli DA, Matsuda A, Parasramka M, Shukla N, et al. Extracellular vesicles from bone marrow-derived mesenchymal stem cells protect against murine hepatic ischemia/reperfusion injury. Liver Transpl 2017; 23:791-803.
8. Nong K, Wang W, Niu X, Hu B, Ma C, Bai Y, et al. Hepatoprotective effect of exosomes from human-induced pluripotent stem cell–derived mesenchymal stromal cells against hepatic ischemia-reperfusion injury in rats. Cytotherapy 2016; 18:1548-1559.
9. Saat TC, van den Engel S, Bijman-Lachger W, Korevaar SS, Hoogduijn MJ, Ijzermans JNM, et al. Fate and effect of intravenously infused mesenchymal stem cells in a mouse model of hepatic ischemia reperfusion injury and resection. Stem Cells Int 2016; 5761487.
10. Papanikolaou IG, Katselis C, Apostolou K, Feretis T, Lymperi M, Konstadoulakis MM, et al. Mesenchymal stem cells transplantation following partial hepatectomy: A new concept to promote liver regeneration-systematic review of the literature focused on experimental studies in rodent models. Stem Cells Int 2017; 7567958.
11. Tian Y, Wang J, Wang W, Ding Y, Sun Z, Zhang Q, et al. Mesenchymal stem cells improve mouse non-heart-beating liver graft survival by inhibiting Kupffer cell apoptosis via TLR4-ERK1/2-Fas/FasL-caspase3 pathway regulation. Stem Cell Res Ther 2016; 7:157.
12. Xagorari A, Siotou E, Yiangou M, Tsolaki E, Bougiouklis D, Sakkas L, et al. Protective effect of mesenchymal stem cell-conditioned medium on hepatic cell apoptosis after acute liver injury. Int J Clin Exp Pathol 2013;15;6:831-840.
13. Huang B, Cheng X, Wang H, Huang W, la Ga hu Z, Wang D, 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; 9;14:45.
14. Parekkadan B, van Poll D, Megeed Z, Kobayashi N, W Tilles A, Berthiaume F, et al. Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells. Biochem Biophys Res Commun 2007; 16;363:247-252.
15. Fiore EJ, Domínguez LM, Bayo J, García MG, Mazzolini GD. Taking advantage of the potential of mesenchymal stromal cells in liver regeneration: Cells and extracellular vesicles as therapeutic strategies. World J Gastroenterol 2018; 24:2427-2440.
16. Keshtkar S, Azarpira N, Ghahremani MH. Mesenchymal stem cell-derived extracellular vesicles: novel frontiers in regenerative medicine. Stem Cell Res Ther 2018; 9:63.
17. Zaminy A, Shokrgozar M, Sadeghi Y, Norouzian M, Heidari M, Piryaei A. Transplantation of schwann cells differentiated from adipose stem cells improves functional recovery in rat spinal cord injury. Arch Iran Med 2013; 16:533-541.
18. Zaminy A, Shokrgozar M, Sadeghi Y, Noroozian M, Heidari M, Piryaei A. Mesenchymal stem cells as an alternative for schwann cells in rat spinal cord injury. Iran Biomed J 2013; 17: 113–122.
19. Osugi M, Katagiri W, Yoshimi R, Inukai T, Hibi H, Ueda M. Conditioned media from mesenchymal stem cells enhanced bone regeneration in rat calvarial bone defects. Tissue Eng Part A Part A 2012; 18:1479-1489.
20. Aebi H. [13] Catalase in vitro.  Methods in Enzymology. 105: Academic Press; 1984. p. 121-126.
21. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248-254.
22. Radonić A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 2004; 313:856-862.
23. Suzuki S, Koizumi T, Sakaguchi S, Baba S, Muro H, Fujise Y. The beneficial effect of a prostaglandin 12 analog on ischemic rat liver. Transplantation 1991; 52:97Y 983.
24. Masuda Y, Vaziri ND, Takasu C, Li S, Robles L, Pham C, et al. Salutary effect of pre-treatment with an Nrf2 inducer on ischemia reperfusion injury in the rat liver. Gastroenterol Hepatol 2014; 1:1-7.
25. Rowart P, Erpicum P, Detry O, Weekers L, Grégoire C, Lechanteur C, et al. Mesenchymal stromal cell therapy in ischemia/reperfusion injury. J Immunol Res 2015: 602597.
26. Lee MW, Ryu S, Kim DS, Sung KW, Koo HH, Yoo KH. Strategies to improve the immunosuppressive properties of human mesenchymal stem cells. Stem Cell Res Ther 2015; 6:179-179.
27. Monsel A, Zhu Y-G, Gudapati V, Lim H, Lee JW. Mesenchymal stem cell derived secretome and extracellular vesicles for acute lung injury and other inflammatory lung diseases. Expert Opin Biol Ther 2016; 16:859-871.
28. Havakhah S, Sankian M, Kazemzadeh GH, Sadri K, Bidkhori HR, Naderi-Meshkin H, et al. In vivo effects of allogeneic mesenchymal stem cells in a rat model of acute ischemic kidney injury. Iran J Basic Med Sci 2018; 21:824-831.
29. Cunningham CJ, Redondo-Castro E, Allan SM. The therapeutic potential of the mesenchymal stem cell secretome in ischaemic stroke. J Cereb Blood Flow Metab 2018; 38:1276-1292.
30.  Saidi R, R. Rajeshkumar B, Shariftabrizi A, Bogdanov A, Zheng S, Dresser K, et al. Human Adipose Derived Mesenchymal Stem Cells Attenuate Liver Ischemia-Reperfusion Injury and Promote Liver Regeneration. Surgery 2014; 156:1225-1231.
31. Li Y, Li T, Qi H, Yuan F. Minocycline protects against hepatic ischemia/reperfusion injury in a rat model. Biomed Rep 2015; 3:19-24.
32. Farhood A, McGuire GM, Manning AM, Miyasaka M, Smith CW, Jaeschke H. Intercellular adhesion molecule 1 (ICAM-1) expression and its role in neutrophil-induced ischemia-reperfusion injury in rat liver. J Leukoc Biol 1995; 57:368-374.
33. Nasef A, Christelle M, Bouchet S, Francois S, Chapel A, Thierry D, et al. Leukemia inhibitory factor: Role in human mesenchymal stem cells mediated immunosuppression. Cell Immunol 2008; 253:16-22.
34. Mohsin S, Shams S, Ali Nasir G, Khan M, Javaid Awan S, Khan SN, et al. Enhanced hepatic differentiation of mesenchymal stem cells after pretreatment with injured liver tissue. Differentiation 2011; 81:42-48.
35. Nikoozad Z, Ghorbanian MT, Rezaei A. Comparison of the liver function and hepatic specific genes expression in cultured mesenchymal stem cells and hepatocytes. Iran J Basic Med Sci 2014; 17:27-33.
36. Omary M, Ku N-O, Toivola D. Keratins: Guardians of the liver. Hepatology (Baltimore, Md) 2002; 35:251-257.
37. Lau A, Chiu J-F. The possible role of cytokeratin 8 in cadmium-induced adaptation and carcinogenesis. Cancer Research 2007; 67:2107-2113.
38. Z. A. Alrefaie LR. Leucocyte infiltration in experimental warm hepatic ischemia reperfusion; effect of ischemic pre and post conditioning; implications of adhesion molecules. Life Sci J 2011; 9:2290-2295.
39. Pan G-Z, Yang Y, Zhang J, Liu W, Wang G, Zhang Y-C, et al. Bone marrow mesenchymal stem cells ameliorate hepatic ischemia/reperfusion injuries via inactivation of the MEK/ERK signaling pathway in rats. J Surg Res 2012; 178:935-948.
40. Jiao Z, Liu X, Ma Y, Ge Y, Zhang Q, Liu B, et al. Adipose-derived stem cells protect ischemia-reperfusion and partial hepatectomy by attenuating endoplasmic reticulum stress. frontiers in cell and developmental biology. Front Cell Dev Biol 2020; 177-187.
41. Zheng J, Chen L, Lu T, Zhang Y, Sui X, Li Y, et al. MSCs ameliorate hepatocellular apoptosis mediated by PINK1-dependent mitophagy in liver ischemia/reperfusion injury through AMPKα activation. Cell Death Dis 2020; 11:256-275.