Beneficial Effects of Hypoxic Preconditioning on Human Umbilical Cord Mesenchymal Stem Cells

10.4077/CJP.2015.BAE369

Li Zhang；Jing Yang；Yan-Ming Tian；Hui Guo；Yi Zhang

chemical hypoxia ； differentiation ； human umbilical cord mesenchymal stem cells ； hypoxic preconditioning ； proliferation

The Chinese Journal of Physiology

58卷5期（2015 / 10 / 31）

343 - 353

英文

As human umbilical cord mesenchymal stem cells (hUC-MSCs) transplanation may be promising in heart failure treatment, it is important to know whether hypoxic preconditioning (HP) promote hUC-MSCs proliferation and differentiation and protect them against chemical hypoxic damages. This study aimed to investigate the effects of HP on proliferation and differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs). The study also aimed to confirm our hypothesis that HP could promote hUC-MSCs proliferation and differentiation to cardiomyocyte-like cells as well as effectively protecting hUC-MSCs and cardiomyocyte-like cells against chemical hypoxic damages. Isolated hUC-MSCs were cultured in hypoxia at 1%, 3% and 5% O_2 for 72 hours. 5-azacytidine (5-AZA) induced differentiation of hUC-MSCs to cardiomyocyte-like cells was determined by streptavidin-perosidase (SP) immunohistochemical staining and the content of troponin (TnI). Flow cytometry was used to measure cell cycle in hUC-MSCs and cardiomyocyte-like cells. The mitochondrial membrane potential (ΔΨ_m) and mitochondrial Ca^(2+) concentration ([Ca^(2+)]_m), were measured in hUC-MSCs and cardiomyocyte-like cells during chemical hypoxia induced by cobalt chloride (100 μmol/L). HP optimally promoted the proliferation of hUC-MSCs at 3% O_2 and enhanced the differentiation of hUC-MSCs to cardiomyocytelike cells by 5-AZA in a concentration-dependent manner. The cell cycle distribution of cardiomyocytelike cells, but not hUC-MSCs, was clearly changed by HP. Chemical hypoxic damage, decreased ΔΨ_m and increased [Ca^(2+)]_m, were alleviated significantly in HP-treated cells compared with the normaxiatreated cells. The results demonstrate that HP promoted hUC-MSCs proliferation and differentiation to cardiomyocyte-like cells, and protected both cell types against chemical hypoxic damage.

1. Akita, T.,Murohara, T.,Ikeda, H.,Sasaki, K.,Shimada, T.,Egami, K.,Imaizumi, T.(2003).Hypoxic preconditioning augments efficacy of human endothelial progenitor cells for therapeutic neovascularization.Lab. Invest.,83,65-73.
2. Basciano, L.,Nemos, C.,Foliguet, B.,de Isla, N.,de Carvalho, M.,Tran, N.,Dalloul, A.(2011).Long term culture of mesenchymal stem cells in hypoxia promotes a genetic program maintaining their undifferentiated and multipotent status.BMC Cell Biol.,12,12.
3. Bedada, F.B.,Chan, S.S.,Metzger, S.K.,Zhang, L.,Zhang, J.,Garry, D.J.,Kamp, T.J.,Kyba, M.,Metzger, J.M.(2014).Acquisition of a quantitative, stoichiometrically conserved ratiometric marker of maturation status in stem cell-derived cardiac myocytes.Stem Cell Reports,3,594-605.
4. Bertolo, A.,Hafner, S.,Taddei, A.R.,Baur, M.,Potzel, T.,Steffen, F.,Stoyanov, J.(2015).Injectable microcarriers as human mesenchymal stem cell support and their application for cartilage and degenerated intervertebral disc repair.Eur. Cell Mater.,29,70-81.
5. Boyette, L.B.,Creasey, O.A.,Guzik, L.,Lozito, T.,Tuan, R.S.(2014).Human bone marrow-derived mesenchymal stem cells display enhanced clonogenicity but impaired differentiation with hypoxic preconditioning.Stem Cells Transl. Med.,3,241-254.
6. Caplan, A.I.,Dennis, J.E.(2006).Mesenchymal stem cells as trophic mediators.J. Cell. Biochem.,98,1076-1084.
7. Ceradini, D.J.,Kulkarni, A.R.,Callaghan, M.J.,Tepper, O.M.,Bastidas, N.,Kleinman, M.E.,Capla, J.M.,Galiano, R.D.,Levine, J.P.,Gurtner, G.C.(2004).Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1.Nat. Med.,10,858-864.
8. Delalat, B.,Pourfathollah, A.A.,Soleimani, M.,Mozdarani, H.,Ghaemi, S.R.,Movassaghpour, A.A.,Kaviani, S.(2009).Isolation and ex vivo expansion of human umbilical cord blood-derived CD34+ stem cells and their cotransplantation with or without mesenchymal stem cells.Hematology,14,125-132.
9. Di Lisa, F.,Blank, P.S.,Colonna, R.,Gambassi, G.,Silverman, H.S.,Stern, M.D.,Hansford, R.G.(1995).Mitochondrial membrane potential in single living adult rat cardiac myocytes exposed to anoxia or metabolic inhibition.J. Physiol.,486,1-13.
10. Dominici, M.,Le Blanc, K.,Mueller, I.,Slaper-Cortenbach, I.,Marini, F.,Krause, D.,Deans, R.,Keating, A.,Prockop, D.J.,Horwitz, E.(2006).Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.Cytotherapy,8,315-317.
11. Estrada, J.C.,Albo, C.,Benguria, A.,Dopazo, A.,Lopez-Romero, P.,Carrera-Quintanar, L.,Roche, E.,Clemente, E.P.,Enriquez, J.A.,Bernad, A.,Samper, E.(2012).Culture of human mesenchymal stem cells at low oxygen tension improves growth and genetic stability by activating glycolysis.Cell Death Differ.,19,743-755.
12. Gavira, J.J.,Perez-Ilzarbe, M.,Abizanda, G.,Garcia-Rodriguez, A.,Orbe, J.,Paramo, J.A.,Belzunce, M.,Rabago, G.,Barba, J.,Herreros, J.,Panizo, A.,de Jalon, J.A.,Martinez-Caro, D.,Prosper, F.(2006).A comparison between percutaneous and surgical transplantation of autologous skeletal myoblasts in a swine model of chronic myocardial infarction.Cardiovasc. Res.,71,744-753.
13. Geng, Y.J.(2003).Molecular mechanisms for cardiovascular stem cell apoptosis and growth in the hearts with atherosclerotic coronary disease and ischemic heart failure.Ann. NY Acad. Sci.,1010,687-697.
14. Govindasamy, V.,Ronald, V.S.,Abdullah, A.N.,Nathan, K.R.,Ab Aziz, Z.A.,Abdullah, M.,Musa, S.,Kasim, N.H.,Bhonde, R.R.(2011).Differentiation of dental pulp stem cells into islet-like aggregates.J. Dent. Res.,90,646-652.
15. Herrmann, J.L.,Abarbanell, A.M.,Weil, B.R.,Wang, Y.,Wang, M.,Tan, J.,Meldrum, D.R.(2009).Cell-based therapy for ischemic heart disease: a clinical update.Ann. Thorac. Surg.,88,1714-1722.
16. Holzwarth, C.,Vaegler, M.,Gieseke, F.,Pfister, S.M.,Handgretinger, R.,Kerst, G.,Muller, I.(2010).Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells.BMC Cell Biol.,11,11.
17. Horwitz, E.M.,Le Blanc, K.,Dominici, M.,Mueller, I.,Slaper-Cortenbach, I.,Marini, F.C.,Deans, R.J.,Krause, D.S.,Keating, A.(2005).Clarification of the nomenclature for MSC: the International Society for Cellular Therapy position statement.Cytotherapy,7,393-395.
18. Hu, X.,Yu, S.P.,Fraser, J.L.,Lu, Z.,Ogle, M.E.,Wang, J.A.,Wei, L.(2008).Transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis.J. Thorac. Cardiovasc. Surg.,135,799-808.
19. Hung, S.P.,Ho, J.H.,Shih, Y.R.,Lo, T.,Lee, O.K.(2012).Hypoxia promotes proliferation and osteogenic differentiation potentials of human mesenchymal stem cells.J. Orthop. Res.,30,260-266.
20. Malliaras, K.,Makkar, R.R.,Smith, R.R.,Cheng, K.,Wu, E.,Bonow, R.O.,Marbán, L.,Mendizabal, A.,Cingolani, E.,Johnston, P.V.,Gerstenblith, G.,Schuleri, K.H.,Lardo, A.C.,Marbán, E.(2014).Intracoronary cardiosphere-derived cells after myocardial infarction: evidence of therapeutic regeneration in the final 1-year results of the CADUCEUS trial (CArdiosphere-Derived aUtologous stem Cells to reverse ventricUlar dySfunction)..J. Am. Coll. Cardiol.,63,110-122.
21. Mangi, A.A.,Noiseux, N.,Kong, D.,He, H.,Rezvani, M.,Ingwall, J.S.,Dzau, V.J.(2003).Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts.Nat. Med.,9,1195-1201.
22. Martens, T.P.,See, F.,Schuster, M.D.,Sondermeijer, H.P.,Hefti, M.M.,Zannettino, A.,Gronthos, S.,Seki, T.,Itescu, S.(2006).Mesenchymal lineage precursor cells induce vascular network formation in ischemic myocardium.Nat. Clin. Pract. Cardiovasc. Med.,3(Suppl 1),S18-S22.
23. Nauta, A.J.,Fibbe, W.E.(2007).Immunomodulatory properties of mesenchymal stromal cells.Blood,100,3499-3506.
24. Nekanti, U.,Dastidar, S.,Venugopal, P.,Totey, S.,Ta, M.(2010).Increased proliferation and analysis of differential gene expression in human Wharton's jelly-derived mesenchymal stromal cells under hypoxia.Int. J. Biol. Sci.,6,499-512.
25. Panchision, D.M.(2009).The role of oxygen in regulating neural stem cells in development and disease.J. Cell. Physiol.,220,562-568.
26. Raheja, L.F.,Genetos, D.C.,Yellowley, C.E.(2010).The effect of oxygen tension on the long-term osteogenic differentiation and MMP/TIMP expression of human mesenchymal stem cells.Cells Tissues Organs,191,175-184.
27. Saller, M.M.,Prall, W.C.,Docheva, D.,Schonitzer, V.,Popov, T.,Anz, D.,Clausen-Schaumann, H.,Mutschler, W.,Volkmer, E.,Schieker, M.,Polzer, H.(2012).Increased stemness and migration of human mesenchymal stem cells in hypoxia is associated with altered integrin expression.Biochem. Biophys. Res. Commun.,423,379-385.
28. Sasaki, M.,Abe, R.,Fujita, Y.,Ando, S.,Inokuma, D.,Shimizu, H.(2008).Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type.J. Immunol.,180,2581-2587.
29. Shimizu, T.,Sekine, H.,Yamato, M.,Okano, T.(2009).Cell sheet-based myocardial tissue engineering: new hope for damaged heart rescue.Curr. Pharm. Des.,15,2807-2814.
30. Simon, M.C.,Keith, B.(2008).The role of oxygen availability in embryonic development and stem cell function.Nat. Rev. Mol. Cell Biol.,9,285-296.
31. Steinhauser, M.L.,Lee, R.T.(2011).Regeneration of the heart.EMBO Mol. Med.,3,701-712.
32. Tang, Y.L.,Tang, Y.,Zhang, Y.C.,Qian, K.,Shen, L.,Phillips, M.I.(2005).Improved graft mesenchymal stem cell survival in ischemic heart with a hypoxia-regulated heme oxygenase-1 vector.J. Am. Coll. Cardiol.,46,1339-1350.
33. Toma, C.,Pittenger, M.F.,Cahill, K.S.,Byrne, B.J.,Kessler, P.D.(2002).Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart.Circulation,105,93-98.
34. Ubl, J.J.,Chatton, J.Y.,Chen, S.,Stucki, J.W.(1996).A critical evaluation of in situ measurement of mitochondrial electrical potentials in single hepatocytes.Biochim. Biophys. Acta,1276,124-132.
35. Valorani, M.G.,Montelatici, E.,Germani, A.,Biddle, A.,D'Alessandro, D.,Strollo, R.,Patrizi, M.P.,Lazzari, L.,Nye, E.,Otto, W.R.,Pozzilli, P.,Alison, M.R.(2012).Pre-culturing human adipose tissue mesenchymal stem cells under hypoxia increases their adipogenic and osteogenic differentiation potentials.Cell Prolif.,45,225-238.
36. Wang, X.,Jameel, M.N.,Li, Q.,Mansoor, A.,Qiang, X.,Swingen, C.,Panetta, C.,Zhang, J.(2009).Stem cells for myocardial repair with use of a transarterial catheter.Circulation,120,S238-S246.
37. Yang, D.C.,Yang, M.H.,Tsai, C.C.,Huang, T.F.,Chen, Y.H.,Hung, S.C.(2011).Hypoxia inhibits osteogenesis in human mesenchymal stem cells through direct regulation of RUNX2 by TWIST.PLoS One,6,e23965.
38. Yu, Y.S.,Shen, Z.Y.,Ye, W.X.,Huang, H.Y.,Hua, F.,Chen, Y.H.,Chen, K.,Lao, W.J.,Tao, L.(2010).AKT-modified autologous intracoronary mesenchymal stem cells prevent remodeling and repair in swine infarcted myocardium.Chinese Med. J.,123,1702-1708.
39. Zhang, M.,Mal, N.,Kiedrowski, M.,Chacko, M.,Askari, A.T.,Popovic, Z.B.,Koc, O.N.,Penn, M.S.(2007).SDF-1 expression by mesenchymal stem cells results in trophic support of cardiac myocytes after myocardial infarction.FASEB J.,21,3197-3207.
40. Zhang, Y.,Marsboom, G.,Toth, P.T.,Rehman, J.(2013).Mitochondrial respiration regulates adipogenic differentiation of human mesenchymal stem cells.PLoS One,8,e77077.
41. Zhuang, Y.,Chen, X.,Xu, M.,Zhang, L.Y.,Xiang, F.(2009).Chemokine stromal cell-derived factor 1/CXCL12 increases homing of mesenchymal stem cells to injured myocardium and neovascularization following myocardial infarction.Chinese Med. J.,122,183-187.