Hypoxic preconditioning potentiates the trophic effects of mesenchymal stem cells on co-cultured human primary hepatocytes

Stem Cell Research & Therapy

Robin D. Hughes¹  Anil Dhawan²  Sharon Lehec¹  Song Sun¹  Céline Filippi³  Harry H. Qin¹ 

[1] Dhawan Lab, Institute of Liver Studies, King’s College London and King’s College Hospital NHS Foundation Trust, London SE5 9PJ, UK;Paediatric Liver, GI and Nutrition Centre, King’s College Hospital Denmark Hill, London SE5 9RS, UK;NIHR Biomedical Research Centre at Guy’s and St Thomas NHS Foundation Trust and King’s College London, London SE1 9RT, UK

关键词: Apoptosis;    Cytokines;    Collagen;    Reactive oxygen species;    Hypoxic preconditioning;    Co-culture;    Hepatocyte;    Mesenchymal stem cell;   

Others  :  1235090
DOI  :  10.1186/s13287-015-0218-7

 received in 2015-06-10, accepted in 2015-10-28,  发布年份 2015

【 摘 要 】

Introduction

Mesenchymal stem/stromal cells (MSCs) improve the metabolic function of co-cultured hepatocytes. The present study aimed to further enhance the trophic effects of co-culture with hepatocytes using hypoxic preconditioning (HPc) of the MSCs and also to investigate the underlying molecular mechanisms involved.

Methods

Human adipose tissue-derived MSCs were subjected to hypoxia (2 % O ₂ ; HPc) or normoxia (20 % O ₂ ) for 24 h and then co-cultured with isolated human hepatocytes. Assays of metabolic function and apoptosis were performed to investigate the hepatotrophic and anti-apoptotic effects of co-culture. Indirect co-cultures and co-culture with MSC-conditioned medium investigated the role of paracrine factors in the hepatotrophic effects of co-culture. Reactive oxygen species (ROS) activity was antagonised with N-acetylcysteine to investigate whether HPc potentiated the effects of MSCs by intracellular ROS-dependent mechanisms. Tumour necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and extracellular collagen production was determined and CASP9 and BAX/BCL-2 signalling pathways analysed to investigate the role of soluble factors, extracellular matrix deposition, and apoptosis-associated gene signalling in the effects of co-culture.

Results

HPc potentiated the hepatotrophic and anti-apoptotic effects of co-culture by ROS-dependent mechanisms. There was increased MSC TGF-β1 production, and enhanced MSC deposition of extracellular collagen, with reduced synthesis of TNF-α, as well as a downregulation of the expression of pro-apoptotic CASP9, BAX, BID and BLK genes and upregulated expression of anti-apoptotic BCL-2 in hepatocytes.

Conclusions

HPc potentiated the trophic and anti-apoptotic effects of MSCs on hepatocytes via mechanisms including intracellular ROS, autocrine TGF-β, extracellular collagen and caspase and BAX/BCL-2 signalling pathways.

【 授权许可】

   
2015 Qin et al.

【 图 表 】

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

【 参考文献 】

• [1]Dhawan A, Puppi J, Hughes RD, Mitry RR: Human hepatocyte transplantation: current experience and future challenges. Nat Rev Gastroenterol Hepatol. 2010, 7:288-298.
• [2]Mizumoto H, Ishihara K, Nakazawa K, Ijima H, Funatsu K, Kajiwara T: A new culture technique for hepatocyte organoid formation and long-term maintenance of liver-specific functions. Tissue Eng Part C Methods. 2008, 14:167-175.
• [3]Corlu A, Ilyin G, Cariou S, Lamy I, Loyer P, Guguen-Guillouzo C: The coculture: a system for studying the regulation of liver differentiation/proliferation activity and its control. Cell Biol Toxicol. 1997, 13:235-242.
• [4]Ijima H, Matsuo T, Kawakami K: The mixed coculture effect of primary rat hepatocytes and bone marrow cells is caused by soluble factors derived from bone marrow cells. J Biosci Bioeng. 2008, 105:226-231.
• [5]Gu J, Shi X, Zhang Y, Ding Y: Heterotypic interactions in the preservation of morphology and functionality of porcine hepatocytes by bone marrow mesenchymal stem cells in vitro. J Cell Physiol. 2009, 219:100-108.
• [6]Gu J, Shi X, Zhang Y, Chu X, Hang H, Ding Y: Establishment of a three-dimensional co-culture system by porcine hepatocytes and bone marrow mesenchymal stem cells in vitro. Hepatol Res. 2009, 39:398-407.
• [7]Gu J, Shi X, Chu X, Zhang Y, Ding Y: Contribution of bone marrow mesenchymal stem cells to porcine hepatocyte culture in vitro. Biochem Cell Biol. 2009, 87:595-604.
• [8]Fitzpatrick E, Wu Y, Dhadda P, Hughes RD, Mitry RR, Qin H, et al.: Co-culture with mesenchymal stem cells results in improved viability and function of human hepatocytes. Cell Transplant. 2015, 24:73-83.
• [9]Wang JA, Chen TL, Jiang J, Shi H, Gui C, Luo RH, et al.: Hypoxic preconditioning attenuates hypoxia/reoxygenation-induced apoptosis in mesenchymal stem cells. Acta Pharmacol Sin. 2008, 29:74-82.
• [10]Hu X, Yu SP, Fraser JL, Lu Z, Ogle ME, Wang JA, et al.: Transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis. J Thorac Cardiovasc Surg. 2008, 135:799-808.
• [11]Efimenko A, Starostina E, Kalinina N, Stolzing A: Angiogenic properties of aged adipose derived mesenchymal stem cells after hypoxic conditioning. J Transl Med. 2011, 9:10. BioMed Central Full Text
• [12]Mitry RR: Isolation of human hepatocytes. Methods Mol Biol. 2009, 481:17-23.
• [13]Kramer G, Erdal H, Mertens HJ, Nap M, Mauermann J, Steiner G, et al.: Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18. Cancer Res. 2004, 64:1751-1756.
• [14]Barrachina M, Secades J, Lozano R, Gómez-Santos C, Ambrosio S, Ferrer I: Citicoline increases glutathione redox ratio and reduces caspase-3 activation and cell death in staurosporine-treated SH-SY5Y human neuroblastoma cells. Brain Res. 2002, 957:84-90.
• [15]Eruslanov E, Kusmartsev S: Identification of ROS using oxidized DCFDA and flow-cytometry. Methods Mol Biol. 2010, 594:57-72.
• [16]Kohli R, Pan X, Malladi P, Wainwright MS, Whitington PF: Mitochondrial reactive oxygen species signal hepatocyte steatosis by regulating the phosphatidylinositol 3-kinase cell survival pathway. J Biol Chem. 2007, 282:21327-21336.
• [17]Freeman TL, Kharbanda KK, Tuma DJ, Mailliard ME: Inhibition of hepatic stellate cell collagen synthesis by N-(methylamino)isobutyric acid. Biochem Pharmacol. 2002, 63:697-706.
• [18]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−deltadeltaC(t)) method. Methods. 2001, 25:402-408.
• [19]Sérandour AL, Loyer P, Garnier D, Courselaud B, Théret N, Glaise D, et al.: TNFalpha-mediated extracellular matrix remodeling is required for multiple division cycles in rat hepatocytes. Hepatology. 2005, 41:478-486.
• [20]Gómez-Aristizábal A, Davies JE: Human umbilical cord perivascular cells improve rat hepatocyte function ex vivo. Tissue Eng Part A. 2012, 18:2487-2496.
• [21]Busletta C, Novo E, Valfrè Di Bonzo L, Povero D, Paternostro C, Ievolella M, et al.: Dissection of the biphasic nature of hypoxia-induced motogenic action in bone marrow-derived human mesenchymal stem cells. Stem Cells. 2011, 29:952-963.
• [22]Fan G, Wen L, Li M, Li C, Luo B, Wang F, et al.: Isolation of mouse mesenchymal stem cells with normal ploidy from bone marrows by reducing oxidative stress in combination with extracellular matrix. BMC Cell Biol. 2011, 12:30. BioMed Central Full Text
• [23]Park JE, Seo YK, Yoon HH, Kim CW, Park JK, Jeon S: Electromagnetic fields induce neural differentiation of human bone marrow derived mesenchymal stem cells via ROS mediated EGFR activation. Neurochem Int. 2013, 62:418-424.
• [24]De Barros S, Dehez S, Arnaud E, Barreau C, Cazavet A, Perez G, et al.: Aging-related decrease of human ASC angiogenic potential is reversed by hypoxia preconditioning through ROS production. Mol Ther. 2013, 21:399-408.
• [25]Moriyama M, Moriyama H, Ueda A, Nishibata Y, Okura H, Ichinose A, et al.: Human adipose tissue-derived multilineage progenitor cells exposed to oxidative stress induce neurite outgrowth in PC12 cells through p38 MAPK signaling. BMC Cell Biol. 2012, 13:21. BioMed Central Full Text
• [26]Krawczak DA, Westendorf JJ, Carlson CS, Lewis JL: Influence of bone morphogenetic protein-2 on the extracellular matrix, material properties, and gene expression of long-term articular chondrocyte cultures: loss of chondrocyte stability. Tissue Eng Part A. 2009, 15:1247-1255.
• [27]Shi XL, Gu JY, Zhang Y, Han B, Xiao JQ, Yuan XW, et al.: Protective effects of ACLF sera on metabolic functions and proliferation of hepatocytes co-cultured with bone marrow MSCs in vitro. World J Gastroenterol. 2011, 17:2397-2406.
• [28]Chia SM, Lin PC, Yu H: TGF-beta1 regulation in hepatocyte-NIH3T3 co-culture is important for the enhanced hepatocyte function in 3D microenvironment. Biotechnol Bioeng. 2005, 89:565-573.
• [29]Hung SP, Yang MH, Tseng KF, Lee OK: Hypoxia-induced secretion of TGF-β1 in mesenchymal stem cell promotes breast cancer cell progression. Cell Transplant. 2013, 22:1869-1882.
• [30]Hansen LK, Albrecht JH: Regulation of the hepatocyte cell cycle by type I collagen matrix: role of cyclin D1. J Cell Sci. 1999, 112:2971-2981.
• [31]Moghe PV, Coger RN, Toner M, Yarmush ML: Cell–cell interactions are essential for maintenance of hepatocyte function in collagen gel but not on matrigel. Biotechnol Bioeng. 1997, 56:706-711.
• [32]Müller J, Benz K, Ahlers M, Gaissmaier C, Mollenhauer J: Hypoxic conditions during expansion culture prime human mesenchymal stromal precursor cells for chondrogenic differentiation in three-dimensional cultures. Cell Transplant. 2011, 20:1589-1602.
• [33]Rodríguez JP, Montecinos L, Ríos S, Reyes P, Martínez J: Mesenchymal stem cells from osteoporotic patients produce a type I collagen-deficient extracellular matrix favoring adipogenic differentiation. J Cell Biochem. 2000, 79:557-565.
• [34]Feng G, Kaplowitz N: Mechanism of staurosporine-induced apoptosis in murine hepatocytes. Am J Physiol Gastrointest Liver Physiol. 2002, 282:G825-G834.
• [35]Schutte B, Henfling M, Kölgen W, Bouman M, Meex S, Leers MP, et al.: Keratin 8/18 breakdown and reorganization during apoptosis. Exp Cell Res. 2004, 297:11-26.
• [36]Kang YJ, Chae SW: JNK/SAPK is required in nitric oxide-induced apoptosis in osteoblasts. Arch Pharm Res. 2003, 26:937-942.
• [37]Hikita H, Takehara T, Kodama T, Shimizu S, Shigekawa M, Hosui A, et al.: Delayed-onset caspase-dependent massive hepatocyte apoptosis upon Fas activation in Bak/Bax-deficient mice. Hepatology. 2011, 54:240-251.
• [38]Li S, Zhao Y, He X, Kim TH, Kuharsky DK, Rabinowich H, et al.: Relief of extrinsic pathway inhibition by the Bid-dependent mitochondrial release of Smac in Fas-mediated hepatocyte apoptosis. J Biol Chem. 2002, 277:26912-26920.
• [39]Park KS, Kim YS, Kim JH, Choi B, Kim SH, Tan AH, et al.: Trophic molecules derived from human mesenchymal stem cells enhance survival, function, and angiogenesis of isolated islets after transplantation. Transplantation. 2010, 89:509-517.
• [40]Sun CK, Chang CL, Lin YC, Kao YH, Chang LT, Yen CH, et al.: Systemic administration of autologous adipose-derived mesenchymal stem cells alleviates hepatic ischemia-reperfusion injury in rats. Crit Care Med. 2012, 40:1279-1290.
• [41]Krappmann D, Patke A, Heissmeyer V, Scheidereit C: B-cell receptor- and phorbol ester-induced NF-kappaB and c-Jun N-terminal kinase activation in B cells requires novel protein kinase C’s. Mol Cell Biol. 2001, 21:6640-6650.
• [42]Plümpe J, Malek NP, Bock CT, Rakemann T, Manns MP, Trautwein C: NF-kappaB determines between apoptosis and proliferation in hepatocytes during liver regeneration. Am J Physiol Gastrointest Liver Physiol. 2000, 278:G173-G183.
• [43]Bian S, Zhang L, Duan L, Wang X, Min Y, Yu H: Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model. J Mol Med (Berl) 2014, 92:387-397.
• [44]Li L, Jin S, Zhang Y: Ischemic preconditioning potentiates the protective effect of mesenchymal stem cells on endotoxin-induced acute lung injury in mice through secretion of exosome. Int J Clin Exp Med. 2015, 8:3825-3832.
• [45]Yu J, Yin S, Zhang W, Gao F, Liu Y, Chen Z, et al.: Hypoxia preconditioned bone marrow mesenchymal stem cells promote liver regeneration in a rat massive hepatectomy model. Stem Cell Res Ther. 2013, 4:83. BioMed Central Full Text