- Email: [email protected]
Injection of Cardiac Stem Cells Prolongs the Survival of Cardiac Allograft Rats
BASIC INVESTIGATION
Injection of Cardiac Stem Cells Prolongs the Survival of Cardiac Allograft Rats Xiao-Song Rong, PhD, Ming-Qiu Li, PhD, Yong Xu, PhD, Xiao-Qin Hua, PhD, Guo-qing Jiao, PhD and Liu-yan Wei, PhD
Abstract: Cardiac c-kit+ cells isolated from cardiac explant-derived cells modestly improve cardiac functions after myocardial infarction; however, their full potential has not yet been realized. The present study was undertaken to determine the isolation and culture of c-kit+ cardiac stem cells (CSCs), and the roles of myocardial injection of CSCs on the survival of rat cardiac allograft. Recipient Sprague-Dawley rats were transplanted with hearts from Wistar rats. In the in vitro experiment, c-kit+ cells were isolated from mouse heart fragment culture by magnetic cell sorting. CSCs expressed of cardiomyocyte specific protein cardiac troponin I, a smooth muscle actin and von Willebrand factor in conditioned culture. CSC injection increased graft survival of cardiac allograft rats. The effects of CSCs on increase in graft survival of cardiac allograft rats were blocked by stromal-derived factor-1 (SDF-1) knockdown. The expression of SDF-1 was increased after CSC injection into the cardiac of cardiac allograft rats. These results indicate that CSC injection into the cardiac prolongs graft survival of cardiac allograft rats. SDF-1 plays an important role in the effects of CSCs on the graft survival of cardiac allograft rats. Key Indexing Terms: Cardiac stem cells; Cardiac allograft; Stromalderived factor-1; Graft survival. [Am J Med Sci 2015;349(1):67–71.]
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ardiac transplantation has been established as an effective form of therapy for cardiac diseases.1,2 Pediatric heart transplantation has excellent outcomes with survival beyond 20 years after transplant common, especially in the infant.3 Stem cell transplantation has emerged as a promising therapeutic method, and donor-specific tolerance could be induced by injection of donor mesenchymal stem cells at the time of heart transplantation.4 For a long time, it was believed that cardiac tissue is terminally differentiated. However, cardiac stem cells (CSCs), progenitor cells in the cardiac, were isolated in rodents cardiac tissue.5,6 CSCs have been classified according to biological markers such as c-kit+, side population and Islet-1+.7,8 CSCs form cell lines that can be expanded in culture while keeping their progenitor state. Subsequently, they can differentiate in cardiovascular cell types, such as cardiomyocytes, endothelial cells, and smooth muscle cells.9–11 Transplantation of CSCs aims to use and enlarge the heart’s own regeneration capacity. After myocardial infarction (MI), CSCs and newly formed myocytes are found in the border of the infarct area, suggesting that the heart has some regeneration capacity.12 The first phase 1 clinical trials using CSCs infusion were published, intracoronary infusion of autologous CSCs is effective in improving left
From the Department of Cardiothoracic Surgery, Wuxi People’s Hospital, Wuxi, China. Submitted April 24, 2014; accepted in revised form July 15, 2014. The authors have no financial or other conflicts of interest to disclose. Correspondence: Ming-Qiu Li, PhD, Department of Cardiothoracic Surgery, Wuxi People’s Hospital, 299 Qingyang Road, Wuxi 214023, China (E-mail: [email protected]).
The American Journal of the Medical Sciences
ventricular systolic function and reducing infarct size in patients with heart failure after MI.13 It has been demonstrated that intracoronary infusion of autologous CDCs after MI is safe, warranting the expansion of such therapy to phase 2 study.14 The stromal-derived factor-1 (SDF-1) is a 68-amino acid chemokine with a broad range of functions including lymphocyte and mononuclear cells trafficking to embryonic development, inflammatory sites, angiogenesis, and bone marrow stem cell homeostasis.15,16 SDF-1 and its cellular receptor C-X-C chemokine receptor type 4 (CXCR4) are known as the most prominent stem cell chemotaxis. It has been shown that exogenously expressed vascular endothelial growth factor promoted myocardial repair at least in part through SDF-1a/CXCR4mediated migration of CSCs.17 The present study was designed to determine the isolation and culture and multiple differentiation of CSCs in vitro, and the effects of CSCs on the graft survival after cardiac transplantation.
MATERIALS AND METHODS Animals Male Sprague-Dawley rats (recipient, grade SPF; 250– 300 g) and male Wistar rats (donor, grade SPF; 200–250 g) were purchased from Shanghai Laboratory Animal Centre (Chinese Academy of Sciences, Shanghai, China). All efforts were made to minimize the number of animals used and their suffering. Fifty-five rats were used in the experiment. The animals were housed in a temperature- and humidity-controlled room with a 12-hour on-off light cycle and given free access to food and water. Isolation of c-kit+ CSCs C-kit+ CSCs were isolated from neonatal BALB/c mice (3–5 days) according to previous methods.7,18–20 Briefly, mouse hearts were removed and minced into small pieces, washed with phosphate-buffered solution, and digested 2 hours at 37°C with a mixture of 0.25% trypsin (Invitrogen, Carlsbad, CA) and 0.1% collagenase IV (Sigma, St. Louis, MO). Cells were cultured in Iscove’s Modified Dubecco’s Medium (IMDM) (HyClone, Shanghai, China) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY), 2 mmol/L of L-glutamine (Invitrogen) and 0.1 mmol/L of 2-mercaptoethanol (Sigma), 100 Units/mL penicillin G (Gibco), 100 mg/mL streptomycin (Gibco) at 37°C and 5% CO2. After 10 to 15 days, a layer of small, phase-bright cells above the adherent explants were collected by washing with 0.5 g/L trypsin-0.53 mmol/L EDTA. The cell suspension was filtered through a 40-mm cell strainer and isolated by magnetic cell sorting using anti-c-kit-coupled magnetic beads (Miltenyi Biotec, San Diego, CA). Culture of C-kit+ CSCs The purified c-kit+ cells were seeded at 0.5 to 2 3 105/mL in multiwell plates precoated with Dulbecco’s modified Eagle’s
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medium/Ham’s F12 containing 10% FBS (Gibco), basic fibroblast growth factor (10 ng/mL; Sigma), insulin-transferrin-selenite (Sigma), epidermal growth factor (20 ng/mL; Sigma), stem cell factor (10 ng/mL; Sigma), human platelet-derived growth factorBB (10 ng/mL; R&D, Minneapolis, MN), 2-mercaptoethanol (0.1 mM; Sigma), erythropoietin (2.5 U; Sigma) at 37°C and 5% CO2.21 Heterotopic Cardiac Transplantation The rat model of heterotopic cardiac transplantation was performed according to previous methods.22,23 Briefly, rats were anesthetized with intraperitoneal injection of 2% sodium pentobarbital. To reduce the ischemic time, the recipient abdomen was opened before the donor heart was explanted. Immediately after explantation, the ascending aorta and pulmonary artery of the donor heart were side anastomosed to the recipient abdominal aorta and inferior vena cava. The donor heart was perfused with the recipient rat’s blood and resumed contraction. Heart grafts were checked daily by palpation for signs of rejection. The day at which no cardiac contractions were palpable was regarded as graft rejection time and rejection was verified by direct inspection of the allograft by laparotomy. Myocardial Injection The rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal) and 1 3 105 c-kit+ CSCs in 50 mL saline were injected into the myocardium with a 30-G needle at 4 points. The sham-operated (Sham) rats were treated the same as the CSCs injection rats except without CSCs. Adeno-associated viral (AAV) vectors encoding short-hairpin RNAs targeting SDF-1 (AAV-shRNA-SDF-1) was purchased from Invitrogen. The myocardium was infected with AAV-SDF-1 at a multiplicity of infection of 104 as above. In addition, a scrambled oligonucleotide sequence was used for specificity control. Multiple Differentiation of c-kit+ cells The purified c-kit+ CSCs were identified by immunofluorescence using a monoclonal antibody for c-kit. To induce in vitro differentiation,7 c-kit+ cells were seeded on 35-mm dishes (5 3 104 per dish) in Dulbecco’s modified Eagle’s medium supplement with 10% FBS, 1 mM dexamethasone (Sigma), 50 mg/mL ascorbic acid (Sigma), and 1 mM b-glycerophosphate (Sigma). The medium was changed twice a week. Immunofluorescence analysis was performed with primary antibodies specific for cardiac troponin I (cTn-I) (1:200; Santa Cruz, Dallas, TX), a-smooth muscle actin (1:200; Zymed Lab, Grand Island, NY) and von Willebrand factor (1:200; BD PharMingen, Sparks, MD). Goat anti-rabbit second antibody conjugated with fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate were used for immunocytochemistry staining. Staining was observed by a Leica fluorescence microscopy (Wetzlar, Germany). SDF-1 quantitative real time-PCR Total RNA was extracted from cultured cells using Trizol (Invitrogen). The cDNA was synthesized using a random primer from 1 mg total RNA with the RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions (Fermentas). The following primers were used for qRT-PCR, sense: 59-gctttgagtgactgggtt-39, antisense: 59-gtggcaagatgatggttt39, Polymerase chain reaction was performed following the manufacturer’s instructions. The cycling conditions were 3 minutes at 94°C, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 60 seconds. Amplified DNA fragments were resolved by electrophoresis on 1% agarose gels containing ethidium bromide.
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Immunohistochemistry was performed according to the standard protocol using rabbit polyclonal SDF-1 antibodies (Abcam, Cambridge, United Kingdom). The endogenous peroxidase activity was blocked with peroxidase block for 10 minutes, and the nonspecific antibody binding was blocked by bovine serum albumin in phosphate-buffered saline. The cardiac sections were incubated with SDF-1 antibodies at 4°C overnight. After washing, sections were incubated with biotinylated goat anti-rabbit immunoglobulin G for 10 minutes and then stained with diaminobenzidine according to the manufacturer’s instructions. Sections were covered with mounting medium and SDF-1 immunoreactivity was observed under a light microscope (DP70; Olympus, Tokyo, Japan). Western Blotting SDF-1 protein expression was determined by Western blotting. Briefly, after process of electrophoresis and transmembrane, proteins on nitrocellulose membrane were probed with the primary antibody (1:500; Cell Signaling Technology, Inc, Beverly, MA) followed by incubation with the secondary antibodies (1:5000; Immunology Consultants Lab). The bands were visualized by enhanced chemiluminescence using electrogenerated chemiluminescent (Pierce Chemical, Milwaukee, WI) and captured on x-ray films. GAPDH (Bioworld Technology Inc, Louis Park, MN) protein was used as a loading control. The total SDF-1 protein level was normalized to the GAPDH protein level. Statistical Analysis Data were analyzed using SPSS 18.0 (IBM, Armonk, NY). Comparisons between 2 observations were assessed by Student’s paired t test. One-way or 2-way analysis of variance was used followed by the Bonferroni test for post hoc analysis when multiple comparisons were made. All the data were expressed as mean 6 standard error. A value of P , 0.05 was considered statistically significant.
RESULTS Culture of Explants and c-kit+ CSCs Five days after the heart tissues culture, fibroblasts migrated from the adherent explants. Then, some small and round cells appeared (Figure 1A). After c-kit+ magnetic cell sorting isolation, these c-kit+ positive cells (green, Figure 1B) were seeded in CGM and gradually formed 3-dimensional spheres in 2 weeks (Figure 1C). Multipotent Differentiation of c-kit+ CSCs C-kit+ cells changed their shape in the conditioned culture. Confocal immunofluorescence analysis showed expression of smooth muscle-specific marker a-smooth muscle actin (red, Figure 2A), the endothelial specific marker von Willebrand factor (red, Figure 2B), and cardiomyocyte-specific protein cTn-I (green, Figure 2C). Graft Survival The mean graft survival in saline group was 4.6 days, CSCs treatment increased the graft survival to12.3 days. However, SDF-1 protein knockdown blocked the increase in the graft survival of CSCs injection (Figure 3). SDF-1 Expression CSCs injection increased the expression of SDF-1 mRNA, SDF-1 protein expression was also increased after CSCs injection in the heterotopic cardiac transplantation rats (Figure 4). Volume 349, Number 1, January 2015
Cardiac Stem Cells in Cardiac Allograft Rats
FIGURE 1. Culture and characterization of c-kit+. (A) Small, round and phase-bright of c-kit+ cells. (B) Positive for c-kit staining of c-kit+ cells (green). (C) Three-dimensional spheres of c-kit+ cells. DAPI stained nuclei (blue).
FIGURE 2. Multipotent differentiation of c-kit+ cardiac stem cells. Multiple differentiation of c-kit+ cells into smooth muscle (A, red), endothelial cells (B, red) and cardiomyocyte (C, green). DAPI stained nuclei (blue).
DISCUSSION Stem cell transplantation is an effective treatment option for patients with severe systemic sclerosis.24 Intravenous administration of mesenchymal stem cells prolonged the survival of transplanted hearts, possibly by induction of allograft tolerance.25,26 Short-term priming of human CSCs with human
peptide connective tissue growth factor peptide (CTGF-D4) and insulin may improve graft success and cardiac regeneration in patients with MI.27 Overexpression of SDF-1a enhanced CSCs migration in vitro and engraftment of transplanted CSCs.28 In the present study, we demonstrated that CSCs
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FIGURE 3. Effects of CSCs injection on the graft survival after heart transplantation. CSCs injection increased the graft survival, SDF-1 knockdown blocked the effects of CSCs injection on the graft survival. Values are mean 6 SE. *P , 0.05 versus saline; †P , 0.05 versus CSCs; and #P , 0.05 versus Control-shRNA + CSCs; n 5 6 for each group. CSC, cardiac stem cells.
injection into cardiac prolongs graft survival of cardiac allograft rats. SDF-1 protein and mRNA expression were increased after CSCs injection in the heterotopic cardiac transplantation rats. SDF-1 protein knockdown blocked the increase in the graft survival of CSCs injection in the cardiac transplantation rats. SDF-1 plays an important role in the effects of CSCs on the graft survival of cardiac allograft rats. Several types of CSCs have been reported and classified according to biological markers such as c-kit+, side population, Sca-1, and Islet-1+.7,8 C-kit+ cells were isolated from neonatal mouse heart fragment culture by magnetic cell sorting.5 In vitro data have suggested that c-kit+ CSCs have a stronger growth potential than other types, although all these CSCs categories can give rise to all cardiac cell lineages.29 It has been demonstrated that transplantation of Sca-1+ cells significantly reduces apoptosis, attenuates fibrosis, and improves global heart functions in ischemic myocardium.30 We showed that c-kit+ cells kept their capacity for self-renewal and clonogenic in vitro, and could differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells. CSCs injection into cardiac prolongs graft survival of cardiac allograft rats. SDF-1 is a member of the CXC or a chemokine subfamily and the only known ligand for the chemokine receptor CXCR4.31 SDF-1 is constitutively expressed in a large number of tissues and plays an important role in multiple stem cells migration by binding CXCR4.17,31–34 SDF-1a overexpression in vivo could promote the engraftment of the locally transplanted CSCs into the infarcted region and cardiac repair.28 However, the role of SDF-1 in CSCs migration in the cardiac transplantation is still needed to be further clarified. In the present study, we find that SDF-1 protein knockdown blocked the increase in the graft survival of CSCs injection into the cardiac transplantation rats, CSCs injection increased the expression of SDF-1 mRNA and protein expression in the heterotopic cardiac transplantation rats. These results demonstrated that SDF-1 plays an important role in the effects of CSCs on the graft survival of cardiac allograft rats.
FIGURE 4. Effects of CSCs transplantation on the expression of SDF-1. (A) Effects of CSCs transplantation on the expression of SDF-1 mRNA. (B) Effects of CSCs transplantation on the expression of SDF-1 protein (immunohistochemical). (C) Effects of CSCs transplantation on the expression of SDF-1 protein (Western blotting). Values are mean 6 SE. *P , 0.05 versus saline; n 5 6 for each group. CSC, cardiac stem cells; SDF-1, stromal-derived factor-1.
Roles of SDF-1 in the Effects of CSCs on the Graft Survival of Cardiac Allograft Rats In conclusion, c-kit+ cells kept their capacity for selfrenewal and clonogenic in vitro and could differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells. CSCs injection into cardiac prolongs graft survival of cardiac
allograft rats. SDF-1 protein knockdown blocked the increase in the graft survival of CSCs injection into the cardiac transplantation rats. SDF-1 plays an important role in the effects of CSCs on the graft survival of cardiac allograft rats.
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REFERENCES 1. Gursu HA, Varan B, Erdogan I. Use of oral budesonide in the management of protein-losing enteropathy due to restrictive cardiomyopathy. Cardiol Young 2014;24:764–6. 2. Yanagawa B, Collazo L, Burton N, et al. Combined heart transplantation and thoracic endovascular aortic repair for heart failure secondary to tricuspid atresia palliated with Potts shunt. Innovations (Phila) 2013; 8:242–4. 3. Dodd DA. Pediatric heart failure and transplantation: where are we in 2013? Curr Opin Pediatr 2013;25:553–60.
18. Messina E, De Angelis L, Frati G, et al. Isolation and expansion of adult cardiac stem cells from human and murine heart. Circ Res 2004; 95:911–21. 19. Bearzi C, Rota M, Hosoda T, et al. Human cardiac stem cells. Proc Natl Acad Sci U S A 2007;104:14068–73. 20. Tang YL, Shen L, Qian K, et al. A novel two-step procedure to expand cardiac Sca-1+ cells clonally. Biochem Biophys Res Commun 2007;359:877–83.
4. English K, French A, Wood KJ. Mesenchymal stromal cells: facilitators of successful transplantation? Cell Stem Cell 2010;7:431–42.
21. Ellison GM, Torella D, Dellegrottaglie S, et al. Endogenous cardiac stem cell activation by insulin-like growth factor-1/hepatocyte growth factor intracoronary injection fosters survival and regeneration of the infarcted pig heart. J Am Coll Cardiol 2011;58:977–86.
5. Fischer AM, Mercer JC, Iyer A, et al. Regulation of CXC chemokine receptor 4-mediated migration by the Tec family tyrosine kinase ITK. J Biol Chem 2004;279:29816–20.
22. Ruzza A, Vespignani R, Czer LS, et al. Heterotopic heart transplantation in rats: improved anesthetic and surgical technique. Transplant Proc 2010;42:3828–32.
6. Al-Lamki RS, Lu W, Wang J, et al. TNF, acting through inducibly expressed TNFR2, drives activation and cell cycle entry of c-Kit cardiac stem cells in ischemic heart disease. Stem Cells 2013;31:1881–92.
23. Ono K, Lindsey ES. Improved technique of heart transplantation in rats. J Thorac Cardiovasc Surg 1969;57:225–9.
7. Miyamoto S, Kawaguchi N, Ellison GM, et al. Characterization of long-term cultured c-kit+ cardiac stem cells derived from adult rat hearts. Stem Cells Dev 2010;19:105–16. 8. Frati C, Savi M, Graiani G, et al. Resident cardiac stem cells. Curr Pharm Des 2011;17:3252–7. 9. Goumans MJ, de Boer TP, Smits AM, et al. TGF-beta1 induces efficient differentiation of human cardiomyocyte progenitor cells into functional cardiomyocytes in vitro. Stem Cell Res 2007;1:138–49. 10. Smits AM, van Laake LW, den Ouden K, et al. Human cardiomyocyte progenitor cell transplantation preserves long-term function of the infarcted mouse myocardium. Cardiovasc Res 2009;83:527–35. 11. Noort WA, Sluijter JP, Goumans MJ, et al. Stem cells from in- or outside of the heart: isolation, characterization, and potential for myocardial tissue regeneration. Pediatr Cardiol 2009;30:699–709. 12. Hsieh PC, Segers VF, Davis ME, et al. Evidence from a genetic fatemapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat Med 2007;13:970–4. 13. Bolli R, Chugh AR, D’Amario D, et al. Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial. Lancet 2011;378:1847–57. 14. Makkar RR, Smith RR, Cheng K, et al. Intracoronary cardiospherederived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial. Lancet 2012; 379:895–904. 15. Bleul CC, Fuhlbrigge RC, Casasnovas JM, et al. A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J Exp Med 1996;184:1101–9. 16. Luster AD. Chemokines–chemotactic cytokines that mediate inflammation. N Engl J Med 1998;338:436–45. 17. Tang J, Wang J, Kong X, et al. Vascular endothelial growth factor promotes cardiac stem cell migration via the PI3K/Akt pathway. Exp Cell Res 2009;315:3521–31.
24. van Laar JM, Sullivan K. Stem cell transplantation in systemic sclerosis. Curr Opin Rheumatol 2013;25:719–25. 25. Zhou HP, Yi DH, Yu SQ, et al. Administration of donor-derived mesenchymal stem cells can prolong the survival of rat cardiac allograft. Transpl Proc 2006;38:3046–51. 26. Ge W, Jiang J, Baroja ML, et al. Infusion of mesenchymal stem cells and rapamycin synergize to attenuate alloimmune responses and promote cardiac allograft tolerance. Am J Transplant 2009;9:1760–72. 27. Iso Y, Rao KS, Poole CN, et al. Priming with ligands secreted by human stromal progenitor cells promotes grafts of cardiac stem/progenitor cells after myocardial infarction. Stem Cells 2014;32:674–83. 28. Wang K, Zhao X, Kuang C, et al. Overexpression of SDF-1alpha enhanced migration and engraftment of cardiac stem cells and reduced infarcted size via CXCR4/PI3K pathway. PLoS One 2012;7:e43922. 29. Li M, Naqvi N, Yahiro E, et al. c-kit is required for cardiomyocyte terminal differentiation. Circ Res 2008;102:677–85. 30. Feng Y, Huang W, Meng W, et al. Heat shock improves sca-1 stem cells survival and directs ischemic cardiomyocytes towards a prosurvival phenotype via exosomal transfer: a critical role for HSF1/miR34a/HSP70 pathway. Stem Cells 2014;32:462–72. 31. Zheng H, Fu G, Dai T, et al. Migration of endothelial progenitor cells mediated by stromal cell-derived factor-1alpha/CXCR4 via PI3K/Akt/eNOS signal transduction pathway. J Cardiovasc Pharmacol 2007;50:274–80. 32. Theiss HD, Vallaster M, Rischpler C, et al. Dual stem cell therapy after myocardial infarction acts specifically by enhanced homing via the SDF-1/CXCR4 axis. Stem Cell Res 2011;7:244–55. 33. Fischer KM, Din S, Gude N, et al. Cardiac progenitor cell commitment is inhibited by nuclear Akt expression. Circ Res 2011;108:960–70. 34. Huang PH, Chen YH, Tsai HY, et al. Intake of red wine increases the number and functional capacity of circulating endothelial progenitor cells by enhancing nitric oxide bioavailability. Arterioscler Thromb Vasc Biol 2010;30:869–77.
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Injection of Cardiac Stem Cells Prolongs the Survival of Cardiac Allograft Rats Xiao-Song Rong, PhD, Ming-Qiu Li, PhD, Yong Xu, PhD, Xiao-Qin Hua, PhD, Guo-qing Jiao, PhD and Liu-yan Wei, PhD
Abstract: Cardiac c-kit+ cells isolated from cardiac explant-derived cells modestly improve cardiac functions after myocardial infarction; however, their full potential has not yet been realized. The present study was undertaken to determine the isolation and culture of c-kit+ cardiac stem cells (CSCs), and the roles of myocardial injection of CSCs on the survival of rat cardiac allograft. Recipient Sprague-Dawley rats were transplanted with hearts from Wistar rats. In the in vitro experiment, c-kit+ cells were isolated from mouse heart fragment culture by magnetic cell sorting. CSCs expressed of cardiomyocyte specific protein cardiac troponin I, a smooth muscle actin and von Willebrand factor in conditioned culture. CSC injection increased graft survival of cardiac allograft rats. The effects of CSCs on increase in graft survival of cardiac allograft rats were blocked by stromal-derived factor-1 (SDF-1) knockdown. The expression of SDF-1 was increased after CSC injection into the cardiac of cardiac allograft rats. These results indicate that CSC injection into the cardiac prolongs graft survival of cardiac allograft rats. SDF-1 plays an important role in the effects of CSCs on the graft survival of cardiac allograft rats. Key Indexing Terms: Cardiac stem cells; Cardiac allograft; Stromalderived factor-1; Graft survival. [Am J Med Sci 2015;349(1):67–71.]
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ardiac transplantation has been established as an effective form of therapy for cardiac diseases.1,2 Pediatric heart transplantation has excellent outcomes with survival beyond 20 years after transplant common, especially in the infant.3 Stem cell transplantation has emerged as a promising therapeutic method, and donor-specific tolerance could be induced by injection of donor mesenchymal stem cells at the time of heart transplantation.4 For a long time, it was believed that cardiac tissue is terminally differentiated. However, cardiac stem cells (CSCs), progenitor cells in the cardiac, were isolated in rodents cardiac tissue.5,6 CSCs have been classified according to biological markers such as c-kit+, side population and Islet-1+.7,8 CSCs form cell lines that can be expanded in culture while keeping their progenitor state. Subsequently, they can differentiate in cardiovascular cell types, such as cardiomyocytes, endothelial cells, and smooth muscle cells.9–11 Transplantation of CSCs aims to use and enlarge the heart’s own regeneration capacity. After myocardial infarction (MI), CSCs and newly formed myocytes are found in the border of the infarct area, suggesting that the heart has some regeneration capacity.12 The first phase 1 clinical trials using CSCs infusion were published, intracoronary infusion of autologous CSCs is effective in improving left
From the Department of Cardiothoracic Surgery, Wuxi People’s Hospital, Wuxi, China. Submitted April 24, 2014; accepted in revised form July 15, 2014. The authors have no financial or other conflicts of interest to disclose. Correspondence: Ming-Qiu Li, PhD, Department of Cardiothoracic Surgery, Wuxi People’s Hospital, 299 Qingyang Road, Wuxi 214023, China (E-mail: [email protected]).
The American Journal of the Medical Sciences
ventricular systolic function and reducing infarct size in patients with heart failure after MI.13 It has been demonstrated that intracoronary infusion of autologous CDCs after MI is safe, warranting the expansion of such therapy to phase 2 study.14 The stromal-derived factor-1 (SDF-1) is a 68-amino acid chemokine with a broad range of functions including lymphocyte and mononuclear cells trafficking to embryonic development, inflammatory sites, angiogenesis, and bone marrow stem cell homeostasis.15,16 SDF-1 and its cellular receptor C-X-C chemokine receptor type 4 (CXCR4) are known as the most prominent stem cell chemotaxis. It has been shown that exogenously expressed vascular endothelial growth factor promoted myocardial repair at least in part through SDF-1a/CXCR4mediated migration of CSCs.17 The present study was designed to determine the isolation and culture and multiple differentiation of CSCs in vitro, and the effects of CSCs on the graft survival after cardiac transplantation.
MATERIALS AND METHODS Animals Male Sprague-Dawley rats (recipient, grade SPF; 250– 300 g) and male Wistar rats (donor, grade SPF; 200–250 g) were purchased from Shanghai Laboratory Animal Centre (Chinese Academy of Sciences, Shanghai, China). All efforts were made to minimize the number of animals used and their suffering. Fifty-five rats were used in the experiment. The animals were housed in a temperature- and humidity-controlled room with a 12-hour on-off light cycle and given free access to food and water. Isolation of c-kit+ CSCs C-kit+ CSCs were isolated from neonatal BALB/c mice (3–5 days) according to previous methods.7,18–20 Briefly, mouse hearts were removed and minced into small pieces, washed with phosphate-buffered solution, and digested 2 hours at 37°C with a mixture of 0.25% trypsin (Invitrogen, Carlsbad, CA) and 0.1% collagenase IV (Sigma, St. Louis, MO). Cells were cultured in Iscove’s Modified Dubecco’s Medium (IMDM) (HyClone, Shanghai, China) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY), 2 mmol/L of L-glutamine (Invitrogen) and 0.1 mmol/L of 2-mercaptoethanol (Sigma), 100 Units/mL penicillin G (Gibco), 100 mg/mL streptomycin (Gibco) at 37°C and 5% CO2. After 10 to 15 days, a layer of small, phase-bright cells above the adherent explants were collected by washing with 0.5 g/L trypsin-0.53 mmol/L EDTA. The cell suspension was filtered through a 40-mm cell strainer and isolated by magnetic cell sorting using anti-c-kit-coupled magnetic beads (Miltenyi Biotec, San Diego, CA). Culture of C-kit+ CSCs The purified c-kit+ cells were seeded at 0.5 to 2 3 105/mL in multiwell plates precoated with Dulbecco’s modified Eagle’s
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medium/Ham’s F12 containing 10% FBS (Gibco), basic fibroblast growth factor (10 ng/mL; Sigma), insulin-transferrin-selenite (Sigma), epidermal growth factor (20 ng/mL; Sigma), stem cell factor (10 ng/mL; Sigma), human platelet-derived growth factorBB (10 ng/mL; R&D, Minneapolis, MN), 2-mercaptoethanol (0.1 mM; Sigma), erythropoietin (2.5 U; Sigma) at 37°C and 5% CO2.21 Heterotopic Cardiac Transplantation The rat model of heterotopic cardiac transplantation was performed according to previous methods.22,23 Briefly, rats were anesthetized with intraperitoneal injection of 2% sodium pentobarbital. To reduce the ischemic time, the recipient abdomen was opened before the donor heart was explanted. Immediately after explantation, the ascending aorta and pulmonary artery of the donor heart were side anastomosed to the recipient abdominal aorta and inferior vena cava. The donor heart was perfused with the recipient rat’s blood and resumed contraction. Heart grafts were checked daily by palpation for signs of rejection. The day at which no cardiac contractions were palpable was regarded as graft rejection time and rejection was verified by direct inspection of the allograft by laparotomy. Myocardial Injection The rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal) and 1 3 105 c-kit+ CSCs in 50 mL saline were injected into the myocardium with a 30-G needle at 4 points. The sham-operated (Sham) rats were treated the same as the CSCs injection rats except without CSCs. Adeno-associated viral (AAV) vectors encoding short-hairpin RNAs targeting SDF-1 (AAV-shRNA-SDF-1) was purchased from Invitrogen. The myocardium was infected with AAV-SDF-1 at a multiplicity of infection of 104 as above. In addition, a scrambled oligonucleotide sequence was used for specificity control. Multiple Differentiation of c-kit+ cells The purified c-kit+ CSCs were identified by immunofluorescence using a monoclonal antibody for c-kit. To induce in vitro differentiation,7 c-kit+ cells were seeded on 35-mm dishes (5 3 104 per dish) in Dulbecco’s modified Eagle’s medium supplement with 10% FBS, 1 mM dexamethasone (Sigma), 50 mg/mL ascorbic acid (Sigma), and 1 mM b-glycerophosphate (Sigma). The medium was changed twice a week. Immunofluorescence analysis was performed with primary antibodies specific for cardiac troponin I (cTn-I) (1:200; Santa Cruz, Dallas, TX), a-smooth muscle actin (1:200; Zymed Lab, Grand Island, NY) and von Willebrand factor (1:200; BD PharMingen, Sparks, MD). Goat anti-rabbit second antibody conjugated with fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate were used for immunocytochemistry staining. Staining was observed by a Leica fluorescence microscopy (Wetzlar, Germany). SDF-1 quantitative real time-PCR Total RNA was extracted from cultured cells using Trizol (Invitrogen). The cDNA was synthesized using a random primer from 1 mg total RNA with the RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions (Fermentas). The following primers were used for qRT-PCR, sense: 59-gctttgagtgactgggtt-39, antisense: 59-gtggcaagatgatggttt39, Polymerase chain reaction was performed following the manufacturer’s instructions. The cycling conditions were 3 minutes at 94°C, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 60 seconds. Amplified DNA fragments were resolved by electrophoresis on 1% agarose gels containing ethidium bromide.
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Immunohistochemistry was performed according to the standard protocol using rabbit polyclonal SDF-1 antibodies (Abcam, Cambridge, United Kingdom). The endogenous peroxidase activity was blocked with peroxidase block for 10 minutes, and the nonspecific antibody binding was blocked by bovine serum albumin in phosphate-buffered saline. The cardiac sections were incubated with SDF-1 antibodies at 4°C overnight. After washing, sections were incubated with biotinylated goat anti-rabbit immunoglobulin G for 10 minutes and then stained with diaminobenzidine according to the manufacturer’s instructions. Sections were covered with mounting medium and SDF-1 immunoreactivity was observed under a light microscope (DP70; Olympus, Tokyo, Japan). Western Blotting SDF-1 protein expression was determined by Western blotting. Briefly, after process of electrophoresis and transmembrane, proteins on nitrocellulose membrane were probed with the primary antibody (1:500; Cell Signaling Technology, Inc, Beverly, MA) followed by incubation with the secondary antibodies (1:5000; Immunology Consultants Lab). The bands were visualized by enhanced chemiluminescence using electrogenerated chemiluminescent (Pierce Chemical, Milwaukee, WI) and captured on x-ray films. GAPDH (Bioworld Technology Inc, Louis Park, MN) protein was used as a loading control. The total SDF-1 protein level was normalized to the GAPDH protein level. Statistical Analysis Data were analyzed using SPSS 18.0 (IBM, Armonk, NY). Comparisons between 2 observations were assessed by Student’s paired t test. One-way or 2-way analysis of variance was used followed by the Bonferroni test for post hoc analysis when multiple comparisons were made. All the data were expressed as mean 6 standard error. A value of P , 0.05 was considered statistically significant.
RESULTS Culture of Explants and c-kit+ CSCs Five days after the heart tissues culture, fibroblasts migrated from the adherent explants. Then, some small and round cells appeared (Figure 1A). After c-kit+ magnetic cell sorting isolation, these c-kit+ positive cells (green, Figure 1B) were seeded in CGM and gradually formed 3-dimensional spheres in 2 weeks (Figure 1C). Multipotent Differentiation of c-kit+ CSCs C-kit+ cells changed their shape in the conditioned culture. Confocal immunofluorescence analysis showed expression of smooth muscle-specific marker a-smooth muscle actin (red, Figure 2A), the endothelial specific marker von Willebrand factor (red, Figure 2B), and cardiomyocyte-specific protein cTn-I (green, Figure 2C). Graft Survival The mean graft survival in saline group was 4.6 days, CSCs treatment increased the graft survival to12.3 days. However, SDF-1 protein knockdown blocked the increase in the graft survival of CSCs injection (Figure 3). SDF-1 Expression CSCs injection increased the expression of SDF-1 mRNA, SDF-1 protein expression was also increased after CSCs injection in the heterotopic cardiac transplantation rats (Figure 4). Volume 349, Number 1, January 2015
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FIGURE 1. Culture and characterization of c-kit+. (A) Small, round and phase-bright of c-kit+ cells. (B) Positive for c-kit staining of c-kit+ cells (green). (C) Three-dimensional spheres of c-kit+ cells. DAPI stained nuclei (blue).
FIGURE 2. Multipotent differentiation of c-kit+ cardiac stem cells. Multiple differentiation of c-kit+ cells into smooth muscle (A, red), endothelial cells (B, red) and cardiomyocyte (C, green). DAPI stained nuclei (blue).
DISCUSSION Stem cell transplantation is an effective treatment option for patients with severe systemic sclerosis.24 Intravenous administration of mesenchymal stem cells prolonged the survival of transplanted hearts, possibly by induction of allograft tolerance.25,26 Short-term priming of human CSCs with human
peptide connective tissue growth factor peptide (CTGF-D4) and insulin may improve graft success and cardiac regeneration in patients with MI.27 Overexpression of SDF-1a enhanced CSCs migration in vitro and engraftment of transplanted CSCs.28 In the present study, we demonstrated that CSCs
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Rong et al
FIGURE 3. Effects of CSCs injection on the graft survival after heart transplantation. CSCs injection increased the graft survival, SDF-1 knockdown blocked the effects of CSCs injection on the graft survival. Values are mean 6 SE. *P , 0.05 versus saline; †P , 0.05 versus CSCs; and #P , 0.05 versus Control-shRNA + CSCs; n 5 6 for each group. CSC, cardiac stem cells.
injection into cardiac prolongs graft survival of cardiac allograft rats. SDF-1 protein and mRNA expression were increased after CSCs injection in the heterotopic cardiac transplantation rats. SDF-1 protein knockdown blocked the increase in the graft survival of CSCs injection in the cardiac transplantation rats. SDF-1 plays an important role in the effects of CSCs on the graft survival of cardiac allograft rats. Several types of CSCs have been reported and classified according to biological markers such as c-kit+, side population, Sca-1, and Islet-1+.7,8 C-kit+ cells were isolated from neonatal mouse heart fragment culture by magnetic cell sorting.5 In vitro data have suggested that c-kit+ CSCs have a stronger growth potential than other types, although all these CSCs categories can give rise to all cardiac cell lineages.29 It has been demonstrated that transplantation of Sca-1+ cells significantly reduces apoptosis, attenuates fibrosis, and improves global heart functions in ischemic myocardium.30 We showed that c-kit+ cells kept their capacity for self-renewal and clonogenic in vitro, and could differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells. CSCs injection into cardiac prolongs graft survival of cardiac allograft rats. SDF-1 is a member of the CXC or a chemokine subfamily and the only known ligand for the chemokine receptor CXCR4.31 SDF-1 is constitutively expressed in a large number of tissues and plays an important role in multiple stem cells migration by binding CXCR4.17,31–34 SDF-1a overexpression in vivo could promote the engraftment of the locally transplanted CSCs into the infarcted region and cardiac repair.28 However, the role of SDF-1 in CSCs migration in the cardiac transplantation is still needed to be further clarified. In the present study, we find that SDF-1 protein knockdown blocked the increase in the graft survival of CSCs injection into the cardiac transplantation rats, CSCs injection increased the expression of SDF-1 mRNA and protein expression in the heterotopic cardiac transplantation rats. These results demonstrated that SDF-1 plays an important role in the effects of CSCs on the graft survival of cardiac allograft rats.
FIGURE 4. Effects of CSCs transplantation on the expression of SDF-1. (A) Effects of CSCs transplantation on the expression of SDF-1 mRNA. (B) Effects of CSCs transplantation on the expression of SDF-1 protein (immunohistochemical). (C) Effects of CSCs transplantation on the expression of SDF-1 protein (Western blotting). Values are mean 6 SE. *P , 0.05 versus saline; n 5 6 for each group. CSC, cardiac stem cells; SDF-1, stromal-derived factor-1.
Roles of SDF-1 in the Effects of CSCs on the Graft Survival of Cardiac Allograft Rats In conclusion, c-kit+ cells kept their capacity for selfrenewal and clonogenic in vitro and could differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells. CSCs injection into cardiac prolongs graft survival of cardiac
allograft rats. SDF-1 protein knockdown blocked the increase in the graft survival of CSCs injection into the cardiac transplantation rats. SDF-1 plays an important role in the effects of CSCs on the graft survival of cardiac allograft rats.
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