- Email: [email protected]
Improved Proliferation and Differentiation of Bone Marrow Mesenchymal Stem Cells Into Vascular Endothelial Cells With Sphingosine 1-Phosphate
Improved Proliferation and Differentiation of Bone Marrow Mesenchymal Stem Cells Into Vascular Endothelial Cells With Sphingosine 1-Phosphate W. Lu, X. Xiu, Y. Zhao*, and M. Gui* The First Affiliated Hospital with Nanjing Medical University, Nanjing, China
ABSTRACT The practical use of bone marrow mesenchymal stem cells (MSCs), considered to be the best candidate in the field of regenerative medicine, is limited by the low efficiency of MSC differentiation. Sphingosine 1-phosphate (S1P) could promote proliferation, survival, and differentiation of many types of cells, but its effects on MSCs remain elusive. In this study, S1P was added during primary MSCs (PR-MSCs) culture and the effects of S1P on proliferation, survival, and differentiation of PR-MSCs were evaluated. The results showed that S1P could improve PR-MSCs proliferation activity in a concentration-dependent manner, and the apoptosis of PR-MSCs cultured in hypoxia was significantly reduced in the S1P-treated group compared to the control group. After being cultured with vascular endothelial growth factor for 7 days, the specific genes of endothelial cells were highly expressed in S1P-treated PR-MSCs compared to control group, which coincided with the augumented production of hepatocyte growth factor, stromal cell-derived factor-1, and insulin-like growth factor-1. In summary, our results suggest that S1P can promote proliferation, survival, and differentiation into vascular endothelial cells of PR-MSCs. These results will promote the clinical application of PR-MSCs and deepen our understanding of the function mechanism of S1P.
B
ONE marrow mesenchymal stem cells (MSCs) are currently considered to be the best candidates in the field of regenerative medicine [1], and numerous experimental studies have shown the beneficial effects of MSC transplantation in tissue and organ repair and regeneration, including ischemic heart disease (IHD) [2,3]. However, the practical use of MSCs is limited by the low efficiency of MSC proliferation and differentiation [4,5]. Moreover, apoptosis was soon observed in most MSCs after transplantation of MSCs into the body [4,6], although some studies reduced apoptosis by improving the resistance of seed cells to apoptosis [7,8]. Therefore, further efforts are needed to improve the proliferation, survival, and differentiation of MSCs. Sphingosine 1-phosphate (S1P) is a natural potent and multifunctional phospholipid catalyzed by sphingosine kinase (SPK) from sphingosine (Sp) [9]. Many previous studies have shown that S1P could promote proliferation, differentiation, and survival of many types of cells [9e13]. But the effects of S1P on proliferation,
differentiation, and survival of MSCs remain unclear. Most of the known actions of S1P are mediated by a family of 5 specific G proteinecoupled receptors (S1P1e5) [14,15]. After binding S1P-receptors, S1P can activate the ERK/MAPK, PI3K-Akt signaling pathways, which regulate cell proliferation and differentiation [16,17]. In this study, S1P was added during the culture of primary MSCs (PR-MSCs), and the effects of S1P on proliferation, survival, and differentiation of PR-MSCs were evaluated. Further, the mechanism of S1P on PR-MSCs was also investigated. This will promote the application of MSCs in revascularization.
*Address correspondence to Ming Gui, PhD, The First Affiliated Hospital with Nanjing Medical University, Nanjing 210000, China. E-mail: [email protected]; and Yingming Zhao, PhD, The First Affiliated Hospital with Nanjing Medical University, Nanjing 210000, China. E-mail: [email protected]
ª 2015 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/15 http://dx.doi.org/10.1016/j.transproceed.2015.05.032
Transplantation Proceedings, 47, 2035e2040 (2015)
2035
2036
MATERIALS AND METHODS The reagents and media used in our study were purchased from Life Technologies (Mass, USA), R&D, Millipore (Minn, USA), and Biolnd (Kibbutz Beit-Haemek, Israel), unless otherwise stated.
Animals C57BL/6J mice for Bone marrow mesenchymal stem cells (MSCs) were purchased from Comparative Medicine Centre of Yangzhou University. All studies adhered to procedures consistent with the Nanjing Medical University of Biological Sciences Guide for the care and use of laboratory animals.
Cell Culture and Treatments MSCs were isolated from femura and tibiae of male C57BL/6J mice, following the previous procedure [18], and expanded in vitro in Dulbecco’s Modified Eagle Media: Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS). During isolation of S1P PR-MSCs, after seeding cells on wells, cells were treated with 1 mmol/L S1P. Before the detection of apoptosis, PR-MSCs were cultured in 95% N2/5% CO2 for 24 hours. To detect their differentiation capacity, PR-MSCs were cultured in endothelium differentiation medium for 7 days, which consisted of DMEM, 2% FBS, 100 units/mL penicillin/streptomycin, and 50 ng/mL vascular endothelial growth factor (VEGF).
Cell Proliferation Analysis PR-MSCs and S1P PR-MSCs were collected at 24 hours, 48 hours and 72 hours for cell proliferation analysis using CCK-8 assay (YEASEN, Shanghai, China) following the manufacturer’s instructions. Cell Counting Kit-8 (CCK-8) allows very convenient assays by utilizing Dojindo’s highly water-soluble tetrazolium salt. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt] produces a water-soluble formazandye upon reduction by dehydrogenase in mitochondria in the presence of an electron carrier. Color depth is proportional to the cell proliferation. The OD value in the 450-nm wavelengths by enzyme standard instrument reflects the number of living cells indirectly.
Flow Cytometry At passage 1, MSCs were labeled using specific FITC-conjugated antibodies against CD29, CD34, CD45, and CD90 and processed through an FACSCalibur system (BD, San Jose, Calif., USA) according to the manufacturer’s protocol. Results were compared to appropriate isotype controls. Primary antibodies are the following: anti-integrin beta 1 antibody (MEM-101A) (FITC) (ab21845), antiCD34 antibody (4H11[APG]) (FITC) (ab18227), and anti-CD45.1 antibody (A20) (FITC) (ab24917; all from Abcam, Cambridge, UK).
Real-timeePCR Total RNA was extracted from collected samples using RNeasy mini kit (Qiagen, Venlo, The Netherlands). Reverse transcription was performed using PrimeScript RT Reagent Kit (Perfect Real Time, TaKaRa). For quantitative real-time-PCR, reactions were performed using SYBR Premix ExTaqTM II (Perfect Real Time, TaKaRa) and 7500 Real-Time PCR System (Applied Biosystems). For each sample, the cycle threshold (CT) values were obtained from three replicates. Primer sequences are the following: GAPDH forward: 50 -AGGTCGG TGTGAACGGATTTG-30 , reverse: 50 -GGGGTCGTTGATGGCAA-
LU, XIU, ZHAO ET AL CA-30 ; VEGF forward: 50 -TGTCTATCAAGGGAGTGTGTGC-30 , reverse: 50 -TGGAGTATTTCCGTGACCG-30 ; angiopoietin-1 forward: 50 -CTGATGGACTGGGAAGGG-30 , reverse: 50 -AGAATGTG CCACGGCTAA-30 . The relative expression levels of target genes were analyzed using the 2DDCT method.
Western Blot Cell extracts or culture media were prepared as previously described [19]. Proteins were electrophoresed in 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and incubated overnight at 4 C in PBS buffer containing 5% BSA. Primary antibodies are the following: antieCaspase-3 antibody (ab4051), anti-Bax antibody (ab7977), antieBcl-2 antibody (ab7973), anti-CD31 antibody (ab28364), anti-VCAM1 antibody (EPR5047) (ab134047), anti-HGF (EPR12230) antibody (ab178395), anti-SDF1 antibody (ab49124), anti-IGF1 antibody (ab40657), anti-AKT1 (phosphoS473) antibody (ab66138), anti-Erk1 (pT202/pY204) þ Erk2 (pT185/pY187) antibody (ab76165; all from Abcam). b-Tubulin expression levels were determined with a monoclonal antibody (Sigma, T5201) to monitor protein loading and retention.
Statistical Analysis Each experiment was repeated at least 3 times. Differences of data (mean SEM) were analyzed by SPSS statistical software (IBM, Chicago, Ill., United States). Statistical significance was determined by Student t test. Differences were considered to be statistically significant when P < .05.
RESULTS S1P Promoted Proliferation of PR-MSCs
After seeding of PR-MSCs on wells, the cells were incubated with different concentrations of S1P for 72 hours. The effect of S1P on PR-MSC proliferation was analyzed by CCK-8 assay. The results showed that S1P concentration dependently improved PR-MSC proliferation activity, which became apparent at 1 mmol/L (Fig 1A, B). Further, MSCs were identified by analysis of the surface markers using flow cytometry. The results showed CD29 and CD90, surface markers of MSCs, were both detected in most cells of control and S1P-treated groups (Fig 2). And CD34 and CD45, surface markers of HSCs and leukocyte cells, respectively, were both undetected in the control and the S1P-treated group (Fig 2). S1P Inhibited Apoptosis of PR-MSCs Cultured in Hypoxia
Endothelialization occurred in vivo under hypoxia, which could lead cell apoptosis. So the apoptosis of PR-MSCs was analyzed by Annexin V assay after PR-MSCs were cultured in 95% N2/5% CO2 for 24 hours. The results showed that 21.96% of PR-MSCs experienced early apoptosis (Fig 3A), whereas this occurred only for 3.02% of PR-MSCs treated with S1P (P < .05) (Fig 3B). In addition, levels of apoptosisrelated proteins in S1P-treated PR-MSCs were also detected. The results showed Caspase-3 and Bax, apoptosis genes, had low expression in S1P-PR-MSCs compared to the control group, whereas antiapoptotic gene Bcl-2 was highly expressed in S1P-PR-MSCs compared to control group (Fig 3C).
IMPROVED STEM CELL PROLIFERATION WITH S1P
2037
Fig 1. S1P increased proliferation of PR-MSCs. (A) Cells were isolated and incubated with different concentrations of S1P for 72 hours. CCK-8 assay was performed for proliferation of PR-MSCs, normalized to cells incubated in control medium. Data are expressed as means SE; n ¼ 3, *P < .05 vs control. (B) Representative images of PR-MSCs on 7 days cultured with 1 mmol/L S1P and control group. Bar, 100 mm.
S1P Improved Endothelialization of PR-MSCs Under VEGF Induction
To investigate whether S1P treatment could improve the differentiation of PR-MSCs, the expression of specific genes of endothelial cell were detected in PR-MSCs under VEGF induction for 7 days. It was found that the protein of CD31 and VCAM-1 and the mRNA of VEGF and angiopoietin-1 were significantly highly expressed in the S1P-treated group than in the control group (P < .05) (Fig 4).
Effects of S1P on Paracrine of PR-MSCs
MSCs generate and release some cytokines by autocrine or paracrine signaling, which play an important role in the process of vascular endothelial cell differentiation and maturation. We therefore investigated the effects of S1P on cytokine generation in PR-MSCs. As shown in Fig 5A, the quantity of HGF, SDF-1, and IGF-1 in culture media were significantly higher in the S1P-treated group compared to the control group. Moreover, P-ERK1/2 and P-AKT also
Fig 2. Flow cytometry analysis of the surface markers of MSCs on P1. (AeD) The proportions of surface marker CD29, CD34, CD45, and CD90 in control MSCs, respectively. (EeH) The proportions of surface marker CD29, CD34, CD45, and CD90 in S1P-treated MSCs, respectively. Experiment was repeated 3 times, P > .05. The representative result was shown.
2038
LU, XIU, ZHAO ET AL
Fig 3. S1P inhibited apoptosis of PR-MSCs cultured in hypoxia. (A, B) Flow cytometry analysis of Annexin V assay on control (A) and S1P-treated (B) MSCs, respectively. Experiment was repeated 3 times, P < .05. The representative result was shown. (C) Western blot analysis of Caspase-3, Bax, and Bcl-2 in PR-MSCs (S1P) and S1P-PR-MSCs (S1Pþ).
were detected in PR-MSCs. The results showed that PERK1/2 was significantly higher in the S1P-treated group compared to the control group, whereas P-AKT showed no significant difference between the 2 groups (Fig 5B). DISCUSSION
MSCs can repair damage of the myocardium and improve the function of the heart through differentiation into the myocardial and endothelial cells [1,2]. Obtaining MSCs with high efficiency in vitro is essential for its medical application. Some studies showed that the capability of MSC differentiation reduced gradually after passaged [5,20]. It also had been found that PR-MSCs improved cardiac function in mice with myocardial infarction (MI) [6,21]. But it was difficult to obtain sufficient PR-MSCs in clinical application [22]. Here we showed that S1P could increase proliferation of PR-MSCs and can promote the clinical application of PR-MSCs. On the other hand, most MSCs die soon after being transplanted into the body, which limits their repair function [23]. Here we found that S1P could inhibit apoptosis of
PR-MSCs cultured in hypoxia. This suggests that most of S1P-PR-MSCs could likely survive after transplantation. Previous reports reduced apoptosis of transplanted cells and improved heart function though overexpression of some antiapoptosis genes in stem cells, such as Akt and Bcl-2 [7]. Similarly, we found S1P could affect expression of some apoptosis-related genes in PR-MSCs to inhibit apoptosis. S1P can promote proliferation, differentiation, and survival of many cell types. And some studies showed that S1P can significantly induce endothelial cell migration and proliferation, and can promote the activity of angiogenesis. After cultured with VEGF for 7 days, it was shown that the expression of endothelial cellerelated genes were higher in the S1P PR-MSCs group than in the control group, which suggested PR-MSCs treated with S1P were more prone to experience endothelialization. Meanwhile, S1P significantly augmented PR-MSC production of HGF, SDF-1, and IGF1, cytokines that can play an important role in the process of vascular endothelial cell differentiation and maturation [24e26]. It can be speculated that these cytokines accelerated the differentiation of PR-MSCs into endothelial cells under VEGF induction.
Fig 4. S1P-PR-MSCs were prone to endothelialization under VEGF induction. (A) Western blot analysis of CD31 and VCAM-1 in S1PPR-MSCs group (S1Pþ) and control group (S1P). (B, C) The mRNA level of VEGF and angiopoietin-1 in control group and S1P-PRMSCs group. a, b represent significant difference, P < .05.
IMPROVED STEM CELL PROLIFERATION WITH S1P
2039
Fig 5. Effect of S1P on paracrine of PR-MSCs. (A) Western blot analysis of HGF, SDF-1, and IGF-1 in culture media of the S1P-PR-MSCs group (S1Pþ) and control group (S1P). (B) Western blot analysis of P-ERK1/ 2 and P-AKT in PR-MSCs (S1P) and S1P-PR-MSCs (S1Pþ). Experiment was repeated 3 times, and the representative results were shown. a, b represent significant difference, P < .05.
S1P binding with receptors could activate the ERK/ MAPK and PI3K-Akt signaling pathways, which regulate cell proliferation, migration, and other biological effects, such as angiogenesis and inflammation processes. In the study, we found that P-ERK1/2 was significantly activated in S1P-treated PR-MSCs, but not P-AKT. The results suggest that S1P may improve proliferation and differentiation of PR-MSCs through the ERK pathway. In this study, we found that S1P could promote proliferation, survival, and differentiation into vascular endothelial cells of PR-MSCs. And S1P might affect paracrine signaling of PR-MSCs through the ERK pathway. This will promote the clinical application of PR-MSCs and deepen our understanding of the function mechanism of S1P. ACKNOWLEDGMENT Project supported by the National Science Foundation for Distinguished Young Scholars of China (Grant No. 81200081).
REFERENCES [1] Bianco P, Riminucci M, Gronthos S, Robey PG. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 2001;19:180e92.
[2] Haider H, Ashraf M. Bone marrow stem cell transplantation for cardiac repair. Am J Physiol Heart Circ Physiol 2005;288: H2557e67. [3] Schachinger V, Erbs S, Elsasser A, Haberbosch W, Hambrecht R, Holschermann H, et al. Improved clinical outcome after intracoronary administration of bone-marrow-derived progenitor cells in acute myocardial infarction: final 1-year results of the REPAIR-AMI trial. Eur Heart J 2006;27:2775e83. [4] Stolzing A, Scutt A. Effect of reduced culture temperature on antioxidant defences of mesenchymal stem cells. Free Radic Biol Med 2006;41:326e38. [5] Kretlow JD, Jin YQ, Liu W, Zhang WJ, Hong TH, Zhou G, et al. Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells. BMC Cell Biol 2008;9:60. [6] Li RK, Mickle DA, Weisel RD, Rao V, Jia ZQ. Optimal time for cardiomyocyte transplantation to maximize myocardial function after left ventricular injury. Ann Thorac Surg 2001;72:1957e63. [7] Gnecchi M, He H, Noiseux N, Liang OD, Zhang L, Morello F, et al. Evidence supporting paracrine hypothesis for Aktmodified mesenchymal stem cell-mediated cardiac protection and functional improvement. Faseb J 2006;20:661e9. [8] Li W, Ma N, Ong LL, Nesselmann C, Klopsch C, Ladilov Y, et al. Bcl-2 engineered MSCs inhibited apoptosis and improved heart function. Stem Cells 2007;25:2118e27. [9] Hait NC, Oskeritzian CA, Paugh W, Milstien S, Spiegel S. Sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases. Biochim Biophys Acta 2006;1758:2016e26. [10] Li QF, Wu CT, Guo Q, Wang H, Wang LS. Sphingosine 1-phosphate induces Mcl-1 upregulation and protects multiple myeloma cells against apoptosis. Biochem Biophys Res Commun 2008;371:159e62.
2040 [11] Jin ZQ, Goetzl EJ, Karliner JS. Sphingosine kinase activation mediates ischemic preconditioning in murine heart. Circulation 2004;110:1980e9. [12] Pebay A, Wong RC, Pitson SM, Wolvetang EJ, Peh GS, Filipczyk A, et al. Essential roles of sphingosine-1-phosphate and platelet-derived growth factor in the maintenance of human embryonic stem cells. Stem Cells 2005;23:1541e8. [13] Inniss K, Moore H. Mediation of apoptosis and proliferation of human embryonic stem cells by sphingosine-1-phosphate. Stem Cells Dev 2006;15:78996. [14] Theilmeier G, Schmidt C, Herrmann J, Keul P, Schafers M, Herrgott I, et al. High-density lipoproteins and their constituent, sphingosine-1-phosphate, directly protect the heart against ischemia/reperfusion injury in vivo via the S1P3 lysophospholipid receptor. Circulation 2006;114:1403e9. [15] Kong Y, Wang H, Lin T, Wang S. Sphingosine-1-phosphate/ S1P receptors signaling modulates cell migration in human bone marrow-derived mesenchymal stem cells. Mediators Inflamm 2014;2014:e565369. [16] Meriane M, Duhamel S, Lejeune L, Galipeau J, Annabi B. Cooperation of matrix metalloproteinases with the RhoA/Rho kinase and mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase signaling pathways is required for the sphingosine-1-phosphate-induced mobilization of marrow-derived stromal cells. Stem Cells 2006;24:2557e65. [17] Yang W, Li Q, Pan Z. Sphingosine-1-phosphate promotes extravillous trophoblast cell invasion by activating MEK/ERK/ MMP-2 signaling pathways via S1P/S1PR1 axis activation. PLoS One 2014;9:e106725. [18] Sreejit P, Dilip KB, Verma RS. Generation of mesenchymal stem cell lines from murine bone marrow. Cell Tissue Res 2012;350:55e68.
LU, XIU, ZHAO ET AL [19] Hwang JH, Shim SS, Seok OS, Lee HY, Woo SK, Kim BH, et al. Comparison of cytokine expression in mesenchymal stem cells from human placenta, cord blood, and bone marrow. J Korean Med Sci 2009;24:547e54. [20] Assmus B, Rolf A, Erbs S, Elsasser A, Haberbosch W, Hambrecht R, et al. Clinical outcome 2 years after intracoronary administration of bone marrow-derived progenitor cells in acute myocardial infarction. Circ Heart Fail 2010;3:89e96. [21] Jin J, Zhao Y, Tan X, Guo C, Yang Z, Miao D. An improved transplantation strategy for mouse mesenchymal stem cells in an acute myocardial infarction model. PLoS One 2011;6: e21005. [22] Turan RG, Bozdag TI, Turan CH, Ortak J, Akin I, Kische S, et al. Enhanced mobilization of the bone marrow-derived circulating progenitor cells by intracoronary freshly isolated bone marrow cells transplantation in patients with acute myocardial infarction. J Cell Mol Med 2012;16:852e64. [23] Siminiak T, Kurpisz M. Myocardial replacement therapy. Circulation 2003;108:1167e71. [24] Yang ZJ, Ma DC, Wang W, Xu SL, Zhang YQ, Chen B, et al. Experimental study of bone marrow-derived mesenchymal stem cells combined with hepatocyte growth factor transplantation via noninfarct-relative artery in acute myocardial infarction. Gene Ther 2006;13:1564e8. [25] Van Overstraeten-Schlogel N, Beguin Y, Gothot A. Role of stromal-derived factor-1 in the hematopoietic-supporting activity of human mesenchymal stem cells. Eur J Haematol 2006;76:488e93. [26] Tao ZW, Li LG, Geng ZH, Dang T, Zhu SJ. Growth factors induce the improved cardiac remodeling in autologous mesenchymal stem cell-implanted failing rat hearts. J Zhejiang Univ Sci B 2010;11:238e48.
ABSTRACT The practical use of bone marrow mesenchymal stem cells (MSCs), considered to be the best candidate in the field of regenerative medicine, is limited by the low efficiency of MSC differentiation. Sphingosine 1-phosphate (S1P) could promote proliferation, survival, and differentiation of many types of cells, but its effects on MSCs remain elusive. In this study, S1P was added during primary MSCs (PR-MSCs) culture and the effects of S1P on proliferation, survival, and differentiation of PR-MSCs were evaluated. The results showed that S1P could improve PR-MSCs proliferation activity in a concentration-dependent manner, and the apoptosis of PR-MSCs cultured in hypoxia was significantly reduced in the S1P-treated group compared to the control group. After being cultured with vascular endothelial growth factor for 7 days, the specific genes of endothelial cells were highly expressed in S1P-treated PR-MSCs compared to control group, which coincided with the augumented production of hepatocyte growth factor, stromal cell-derived factor-1, and insulin-like growth factor-1. In summary, our results suggest that S1P can promote proliferation, survival, and differentiation into vascular endothelial cells of PR-MSCs. These results will promote the clinical application of PR-MSCs and deepen our understanding of the function mechanism of S1P.
B
ONE marrow mesenchymal stem cells (MSCs) are currently considered to be the best candidates in the field of regenerative medicine [1], and numerous experimental studies have shown the beneficial effects of MSC transplantation in tissue and organ repair and regeneration, including ischemic heart disease (IHD) [2,3]. However, the practical use of MSCs is limited by the low efficiency of MSC proliferation and differentiation [4,5]. Moreover, apoptosis was soon observed in most MSCs after transplantation of MSCs into the body [4,6], although some studies reduced apoptosis by improving the resistance of seed cells to apoptosis [7,8]. Therefore, further efforts are needed to improve the proliferation, survival, and differentiation of MSCs. Sphingosine 1-phosphate (S1P) is a natural potent and multifunctional phospholipid catalyzed by sphingosine kinase (SPK) from sphingosine (Sp) [9]. Many previous studies have shown that S1P could promote proliferation, differentiation, and survival of many types of cells [9e13]. But the effects of S1P on proliferation,
differentiation, and survival of MSCs remain unclear. Most of the known actions of S1P are mediated by a family of 5 specific G proteinecoupled receptors (S1P1e5) [14,15]. After binding S1P-receptors, S1P can activate the ERK/MAPK, PI3K-Akt signaling pathways, which regulate cell proliferation and differentiation [16,17]. In this study, S1P was added during the culture of primary MSCs (PR-MSCs), and the effects of S1P on proliferation, survival, and differentiation of PR-MSCs were evaluated. Further, the mechanism of S1P on PR-MSCs was also investigated. This will promote the application of MSCs in revascularization.
*Address correspondence to Ming Gui, PhD, The First Affiliated Hospital with Nanjing Medical University, Nanjing 210000, China. E-mail: [email protected]; and Yingming Zhao, PhD, The First Affiliated Hospital with Nanjing Medical University, Nanjing 210000, China. E-mail: [email protected]
ª 2015 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/15 http://dx.doi.org/10.1016/j.transproceed.2015.05.032
Transplantation Proceedings, 47, 2035e2040 (2015)
2035
2036
MATERIALS AND METHODS The reagents and media used in our study were purchased from Life Technologies (Mass, USA), R&D, Millipore (Minn, USA), and Biolnd (Kibbutz Beit-Haemek, Israel), unless otherwise stated.
Animals C57BL/6J mice for Bone marrow mesenchymal stem cells (MSCs) were purchased from Comparative Medicine Centre of Yangzhou University. All studies adhered to procedures consistent with the Nanjing Medical University of Biological Sciences Guide for the care and use of laboratory animals.
Cell Culture and Treatments MSCs were isolated from femura and tibiae of male C57BL/6J mice, following the previous procedure [18], and expanded in vitro in Dulbecco’s Modified Eagle Media: Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS). During isolation of S1P PR-MSCs, after seeding cells on wells, cells were treated with 1 mmol/L S1P. Before the detection of apoptosis, PR-MSCs were cultured in 95% N2/5% CO2 for 24 hours. To detect their differentiation capacity, PR-MSCs were cultured in endothelium differentiation medium for 7 days, which consisted of DMEM, 2% FBS, 100 units/mL penicillin/streptomycin, and 50 ng/mL vascular endothelial growth factor (VEGF).
Cell Proliferation Analysis PR-MSCs and S1P PR-MSCs were collected at 24 hours, 48 hours and 72 hours for cell proliferation analysis using CCK-8 assay (YEASEN, Shanghai, China) following the manufacturer’s instructions. Cell Counting Kit-8 (CCK-8) allows very convenient assays by utilizing Dojindo’s highly water-soluble tetrazolium salt. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium, monosodium salt] produces a water-soluble formazandye upon reduction by dehydrogenase in mitochondria in the presence of an electron carrier. Color depth is proportional to the cell proliferation. The OD value in the 450-nm wavelengths by enzyme standard instrument reflects the number of living cells indirectly.
Flow Cytometry At passage 1, MSCs were labeled using specific FITC-conjugated antibodies against CD29, CD34, CD45, and CD90 and processed through an FACSCalibur system (BD, San Jose, Calif., USA) according to the manufacturer’s protocol. Results were compared to appropriate isotype controls. Primary antibodies are the following: anti-integrin beta 1 antibody (MEM-101A) (FITC) (ab21845), antiCD34 antibody (4H11[APG]) (FITC) (ab18227), and anti-CD45.1 antibody (A20) (FITC) (ab24917; all from Abcam, Cambridge, UK).
Real-timeePCR Total RNA was extracted from collected samples using RNeasy mini kit (Qiagen, Venlo, The Netherlands). Reverse transcription was performed using PrimeScript RT Reagent Kit (Perfect Real Time, TaKaRa). For quantitative real-time-PCR, reactions were performed using SYBR Premix ExTaqTM II (Perfect Real Time, TaKaRa) and 7500 Real-Time PCR System (Applied Biosystems). For each sample, the cycle threshold (CT) values were obtained from three replicates. Primer sequences are the following: GAPDH forward: 50 -AGGTCGG TGTGAACGGATTTG-30 , reverse: 50 -GGGGTCGTTGATGGCAA-
LU, XIU, ZHAO ET AL CA-30 ; VEGF forward: 50 -TGTCTATCAAGGGAGTGTGTGC-30 , reverse: 50 -TGGAGTATTTCCGTGACCG-30 ; angiopoietin-1 forward: 50 -CTGATGGACTGGGAAGGG-30 , reverse: 50 -AGAATGTG CCACGGCTAA-30 . The relative expression levels of target genes were analyzed using the 2DDCT method.
Western Blot Cell extracts or culture media were prepared as previously described [19]. Proteins were electrophoresed in 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and incubated overnight at 4 C in PBS buffer containing 5% BSA. Primary antibodies are the following: antieCaspase-3 antibody (ab4051), anti-Bax antibody (ab7977), antieBcl-2 antibody (ab7973), anti-CD31 antibody (ab28364), anti-VCAM1 antibody (EPR5047) (ab134047), anti-HGF (EPR12230) antibody (ab178395), anti-SDF1 antibody (ab49124), anti-IGF1 antibody (ab40657), anti-AKT1 (phosphoS473) antibody (ab66138), anti-Erk1 (pT202/pY204) þ Erk2 (pT185/pY187) antibody (ab76165; all from Abcam). b-Tubulin expression levels were determined with a monoclonal antibody (Sigma, T5201) to monitor protein loading and retention.
Statistical Analysis Each experiment was repeated at least 3 times. Differences of data (mean SEM) were analyzed by SPSS statistical software (IBM, Chicago, Ill., United States). Statistical significance was determined by Student t test. Differences were considered to be statistically significant when P < .05.
RESULTS S1P Promoted Proliferation of PR-MSCs
After seeding of PR-MSCs on wells, the cells were incubated with different concentrations of S1P for 72 hours. The effect of S1P on PR-MSC proliferation was analyzed by CCK-8 assay. The results showed that S1P concentration dependently improved PR-MSC proliferation activity, which became apparent at 1 mmol/L (Fig 1A, B). Further, MSCs were identified by analysis of the surface markers using flow cytometry. The results showed CD29 and CD90, surface markers of MSCs, were both detected in most cells of control and S1P-treated groups (Fig 2). And CD34 and CD45, surface markers of HSCs and leukocyte cells, respectively, were both undetected in the control and the S1P-treated group (Fig 2). S1P Inhibited Apoptosis of PR-MSCs Cultured in Hypoxia
Endothelialization occurred in vivo under hypoxia, which could lead cell apoptosis. So the apoptosis of PR-MSCs was analyzed by Annexin V assay after PR-MSCs were cultured in 95% N2/5% CO2 for 24 hours. The results showed that 21.96% of PR-MSCs experienced early apoptosis (Fig 3A), whereas this occurred only for 3.02% of PR-MSCs treated with S1P (P < .05) (Fig 3B). In addition, levels of apoptosisrelated proteins in S1P-treated PR-MSCs were also detected. The results showed Caspase-3 and Bax, apoptosis genes, had low expression in S1P-PR-MSCs compared to the control group, whereas antiapoptotic gene Bcl-2 was highly expressed in S1P-PR-MSCs compared to control group (Fig 3C).
IMPROVED STEM CELL PROLIFERATION WITH S1P
2037
Fig 1. S1P increased proliferation of PR-MSCs. (A) Cells were isolated and incubated with different concentrations of S1P for 72 hours. CCK-8 assay was performed for proliferation of PR-MSCs, normalized to cells incubated in control medium. Data are expressed as means SE; n ¼ 3, *P < .05 vs control. (B) Representative images of PR-MSCs on 7 days cultured with 1 mmol/L S1P and control group. Bar, 100 mm.
S1P Improved Endothelialization of PR-MSCs Under VEGF Induction
To investigate whether S1P treatment could improve the differentiation of PR-MSCs, the expression of specific genes of endothelial cell were detected in PR-MSCs under VEGF induction for 7 days. It was found that the protein of CD31 and VCAM-1 and the mRNA of VEGF and angiopoietin-1 were significantly highly expressed in the S1P-treated group than in the control group (P < .05) (Fig 4).
Effects of S1P on Paracrine of PR-MSCs
MSCs generate and release some cytokines by autocrine or paracrine signaling, which play an important role in the process of vascular endothelial cell differentiation and maturation. We therefore investigated the effects of S1P on cytokine generation in PR-MSCs. As shown in Fig 5A, the quantity of HGF, SDF-1, and IGF-1 in culture media were significantly higher in the S1P-treated group compared to the control group. Moreover, P-ERK1/2 and P-AKT also
Fig 2. Flow cytometry analysis of the surface markers of MSCs on P1. (AeD) The proportions of surface marker CD29, CD34, CD45, and CD90 in control MSCs, respectively. (EeH) The proportions of surface marker CD29, CD34, CD45, and CD90 in S1P-treated MSCs, respectively. Experiment was repeated 3 times, P > .05. The representative result was shown.
2038
LU, XIU, ZHAO ET AL
Fig 3. S1P inhibited apoptosis of PR-MSCs cultured in hypoxia. (A, B) Flow cytometry analysis of Annexin V assay on control (A) and S1P-treated (B) MSCs, respectively. Experiment was repeated 3 times, P < .05. The representative result was shown. (C) Western blot analysis of Caspase-3, Bax, and Bcl-2 in PR-MSCs (S1P) and S1P-PR-MSCs (S1Pþ).
were detected in PR-MSCs. The results showed that PERK1/2 was significantly higher in the S1P-treated group compared to the control group, whereas P-AKT showed no significant difference between the 2 groups (Fig 5B). DISCUSSION
MSCs can repair damage of the myocardium and improve the function of the heart through differentiation into the myocardial and endothelial cells [1,2]. Obtaining MSCs with high efficiency in vitro is essential for its medical application. Some studies showed that the capability of MSC differentiation reduced gradually after passaged [5,20]. It also had been found that PR-MSCs improved cardiac function in mice with myocardial infarction (MI) [6,21]. But it was difficult to obtain sufficient PR-MSCs in clinical application [22]. Here we showed that S1P could increase proliferation of PR-MSCs and can promote the clinical application of PR-MSCs. On the other hand, most MSCs die soon after being transplanted into the body, which limits their repair function [23]. Here we found that S1P could inhibit apoptosis of
PR-MSCs cultured in hypoxia. This suggests that most of S1P-PR-MSCs could likely survive after transplantation. Previous reports reduced apoptosis of transplanted cells and improved heart function though overexpression of some antiapoptosis genes in stem cells, such as Akt and Bcl-2 [7]. Similarly, we found S1P could affect expression of some apoptosis-related genes in PR-MSCs to inhibit apoptosis. S1P can promote proliferation, differentiation, and survival of many cell types. And some studies showed that S1P can significantly induce endothelial cell migration and proliferation, and can promote the activity of angiogenesis. After cultured with VEGF for 7 days, it was shown that the expression of endothelial cellerelated genes were higher in the S1P PR-MSCs group than in the control group, which suggested PR-MSCs treated with S1P were more prone to experience endothelialization. Meanwhile, S1P significantly augmented PR-MSC production of HGF, SDF-1, and IGF1, cytokines that can play an important role in the process of vascular endothelial cell differentiation and maturation [24e26]. It can be speculated that these cytokines accelerated the differentiation of PR-MSCs into endothelial cells under VEGF induction.
Fig 4. S1P-PR-MSCs were prone to endothelialization under VEGF induction. (A) Western blot analysis of CD31 and VCAM-1 in S1PPR-MSCs group (S1Pþ) and control group (S1P). (B, C) The mRNA level of VEGF and angiopoietin-1 in control group and S1P-PRMSCs group. a, b represent significant difference, P < .05.
IMPROVED STEM CELL PROLIFERATION WITH S1P
2039
Fig 5. Effect of S1P on paracrine of PR-MSCs. (A) Western blot analysis of HGF, SDF-1, and IGF-1 in culture media of the S1P-PR-MSCs group (S1Pþ) and control group (S1P). (B) Western blot analysis of P-ERK1/ 2 and P-AKT in PR-MSCs (S1P) and S1P-PR-MSCs (S1Pþ). Experiment was repeated 3 times, and the representative results were shown. a, b represent significant difference, P < .05.
S1P binding with receptors could activate the ERK/ MAPK and PI3K-Akt signaling pathways, which regulate cell proliferation, migration, and other biological effects, such as angiogenesis and inflammation processes. In the study, we found that P-ERK1/2 was significantly activated in S1P-treated PR-MSCs, but not P-AKT. The results suggest that S1P may improve proliferation and differentiation of PR-MSCs through the ERK pathway. In this study, we found that S1P could promote proliferation, survival, and differentiation into vascular endothelial cells of PR-MSCs. And S1P might affect paracrine signaling of PR-MSCs through the ERK pathway. This will promote the clinical application of PR-MSCs and deepen our understanding of the function mechanism of S1P. ACKNOWLEDGMENT Project supported by the National Science Foundation for Distinguished Young Scholars of China (Grant No. 81200081).
REFERENCES [1] Bianco P, Riminucci M, Gronthos S, Robey PG. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 2001;19:180e92.
[2] Haider H, Ashraf M. Bone marrow stem cell transplantation for cardiac repair. Am J Physiol Heart Circ Physiol 2005;288: H2557e67. [3] Schachinger V, Erbs S, Elsasser A, Haberbosch W, Hambrecht R, Holschermann H, et al. Improved clinical outcome after intracoronary administration of bone-marrow-derived progenitor cells in acute myocardial infarction: final 1-year results of the REPAIR-AMI trial. Eur Heart J 2006;27:2775e83. [4] Stolzing A, Scutt A. Effect of reduced culture temperature on antioxidant defences of mesenchymal stem cells. Free Radic Biol Med 2006;41:326e38. [5] Kretlow JD, Jin YQ, Liu W, Zhang WJ, Hong TH, Zhou G, et al. Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells. BMC Cell Biol 2008;9:60. [6] Li RK, Mickle DA, Weisel RD, Rao V, Jia ZQ. Optimal time for cardiomyocyte transplantation to maximize myocardial function after left ventricular injury. Ann Thorac Surg 2001;72:1957e63. [7] Gnecchi M, He H, Noiseux N, Liang OD, Zhang L, Morello F, et al. Evidence supporting paracrine hypothesis for Aktmodified mesenchymal stem cell-mediated cardiac protection and functional improvement. Faseb J 2006;20:661e9. [8] Li W, Ma N, Ong LL, Nesselmann C, Klopsch C, Ladilov Y, et al. Bcl-2 engineered MSCs inhibited apoptosis and improved heart function. Stem Cells 2007;25:2118e27. [9] Hait NC, Oskeritzian CA, Paugh W, Milstien S, Spiegel S. Sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases. Biochim Biophys Acta 2006;1758:2016e26. [10] Li QF, Wu CT, Guo Q, Wang H, Wang LS. Sphingosine 1-phosphate induces Mcl-1 upregulation and protects multiple myeloma cells against apoptosis. Biochem Biophys Res Commun 2008;371:159e62.
2040 [11] Jin ZQ, Goetzl EJ, Karliner JS. Sphingosine kinase activation mediates ischemic preconditioning in murine heart. Circulation 2004;110:1980e9. [12] Pebay A, Wong RC, Pitson SM, Wolvetang EJ, Peh GS, Filipczyk A, et al. Essential roles of sphingosine-1-phosphate and platelet-derived growth factor in the maintenance of human embryonic stem cells. Stem Cells 2005;23:1541e8. [13] Inniss K, Moore H. Mediation of apoptosis and proliferation of human embryonic stem cells by sphingosine-1-phosphate. Stem Cells Dev 2006;15:78996. [14] Theilmeier G, Schmidt C, Herrmann J, Keul P, Schafers M, Herrgott I, et al. High-density lipoproteins and their constituent, sphingosine-1-phosphate, directly protect the heart against ischemia/reperfusion injury in vivo via the S1P3 lysophospholipid receptor. Circulation 2006;114:1403e9. [15] Kong Y, Wang H, Lin T, Wang S. Sphingosine-1-phosphate/ S1P receptors signaling modulates cell migration in human bone marrow-derived mesenchymal stem cells. Mediators Inflamm 2014;2014:e565369. [16] Meriane M, Duhamel S, Lejeune L, Galipeau J, Annabi B. Cooperation of matrix metalloproteinases with the RhoA/Rho kinase and mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase signaling pathways is required for the sphingosine-1-phosphate-induced mobilization of marrow-derived stromal cells. Stem Cells 2006;24:2557e65. [17] Yang W, Li Q, Pan Z. Sphingosine-1-phosphate promotes extravillous trophoblast cell invasion by activating MEK/ERK/ MMP-2 signaling pathways via S1P/S1PR1 axis activation. PLoS One 2014;9:e106725. [18] Sreejit P, Dilip KB, Verma RS. Generation of mesenchymal stem cell lines from murine bone marrow. Cell Tissue Res 2012;350:55e68.
LU, XIU, ZHAO ET AL [19] Hwang JH, Shim SS, Seok OS, Lee HY, Woo SK, Kim BH, et al. Comparison of cytokine expression in mesenchymal stem cells from human placenta, cord blood, and bone marrow. J Korean Med Sci 2009;24:547e54. [20] Assmus B, Rolf A, Erbs S, Elsasser A, Haberbosch W, Hambrecht R, et al. Clinical outcome 2 years after intracoronary administration of bone marrow-derived progenitor cells in acute myocardial infarction. Circ Heart Fail 2010;3:89e96. [21] Jin J, Zhao Y, Tan X, Guo C, Yang Z, Miao D. An improved transplantation strategy for mouse mesenchymal stem cells in an acute myocardial infarction model. PLoS One 2011;6: e21005. [22] Turan RG, Bozdag TI, Turan CH, Ortak J, Akin I, Kische S, et al. Enhanced mobilization of the bone marrow-derived circulating progenitor cells by intracoronary freshly isolated bone marrow cells transplantation in patients with acute myocardial infarction. J Cell Mol Med 2012;16:852e64. [23] Siminiak T, Kurpisz M. Myocardial replacement therapy. Circulation 2003;108:1167e71. [24] Yang ZJ, Ma DC, Wang W, Xu SL, Zhang YQ, Chen B, et al. Experimental study of bone marrow-derived mesenchymal stem cells combined with hepatocyte growth factor transplantation via noninfarct-relative artery in acute myocardial infarction. Gene Ther 2006;13:1564e8. [25] Van Overstraeten-Schlogel N, Beguin Y, Gothot A. Role of stromal-derived factor-1 in the hematopoietic-supporting activity of human mesenchymal stem cells. Eur J Haematol 2006;76:488e93. [26] Tao ZW, Li LG, Geng ZH, Dang T, Zhu SJ. Growth factors induce the improved cardiac remodeling in autologous mesenchymal stem cell-implanted failing rat hearts. J Zhejiang Univ Sci B 2010;11:238e48.