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Identification and differentiation of magnetically labeled mesenchymal stem cells in vivo in swines with myocardial infarction
Letters to the Editor [6] Kessel Richard G, Kardon Randy H, editors. Tissues and Organs: A Text Atlas of Scanning Electron Microscopy. USA: WH Freeman and Company; 1979. 41–44pp. [7] Nichlas Wilmer W, O'Rourke Michael F Eds, 2005. McDonalds blood flows in arteries- theoretical, experimental and clinical principles, 5th Ed., Hodder Arnold Publications, London. 11 to 48, 321 to 338 pp.
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[8] Farb Andrew, Burke Allen P, Kolodgie Frank D, Virmani Renu. Pathological mechanisms of fatal late coronary stent thrombosis in humans. Circulation 2003;108:1701–6. [9] Joner Michael, Finn Aloke V, Farb Andrew, et al. Pathology of drugeluting stents in humans-delayed healing and late thrombotic risk. J Am Coll Cardiol 2006;48:193–202.
0167-5273/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2007.07.111
Identification and differentiation of magnetically labeled mesenchymal stem cells in vivo in swines with myocardial infarction Chun-mei Qi, Gen-shan Ma ⁎, Nai-feng Liu, Cheng-xing Shen, Zhong Chen, Xiao-jun Liu, Yao-peng Hu, Xiao-li Zhang, Ji-yuan Chen, Sheng-hong Ju,Yao-liang Tang Received 5 July 2007; accepted 6 July 2007 Available online 11 December 2007
Abstract We aim to track mesenchymal stem cells (MSCs) after magnetically labeling and test the ability of these cells differentiate into cardiomyocytes in vivo. Therefore, 20 swines were divided into four groups, sham-operated group (n = 3); acute myocardial infarction (AMI) transplanted with PBS (n = 3); labeled MSCs (n = 7) and unlabeled MSCs (n = 7) group. 107 labeled or unlabeled cells were intracoronary delivered after MI (4.8 ± 1.3 days), and serial cardiac MR (3.0T) imaging studies were performed at 0, 4 and 8 weeks after transplantation, then the results were confirmed by histological and western blot analysis. We demonstrated that labeled MSCs can be reliably detected and tracked in vivo using MR imaging. In particular, we provided the evidence of regeneration of labeled MSCs in vivo by histological examination and western blot analysis. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Magnetic resonance imaging; Contrast media; Mesenchymal stem cell; Differentiation; Myocardial infarction
1. Results
2. Discussion
MSCs after labeled with dual-contrast particles (magnetite as an MRI marker and Dragon Green fluorescent dye as a histological marker) [1] were accurately identified along the rim or in the region of MI on MR imaging (Fig. 1). Signal to noise ratio and contrast to noise ratio (CNR) decreased (16.07 ± 5.85 vs.10.96± 1.34, P b 0.05) from 4 weeks to 8 weeks after labeled MSCs transplantation. Labeled MSCs were further confirmed by Prussian blue and immunofluorescent staining. Immunohistochemistry confirmed that the myogenic cells differentiated from labeled MSCs were positive for desmin and negative for CD68 (Fig. 2). Western blot analysis demonstrated that there was an increase expression of cardiomyocyte markers such as myosin heavy chain and troponin T in MSCs treatment groups compared with sham-operated group and AMI group (Fig. 3).
There is growing enthusiasm for the application of stem cell-based therapies to the regeneration of damaged myocardium. Although the results of preliminary studies and clinical trials are promising, many practical issues regarding this therapy remain highly controversial [2]. To address these important issues, molecular markers capable of tracking transplanted cells and determining the fate of engrafted cells would be extremely useful. Cells labeling with MR contrast media were used as a new technology to monitor the in vivo behavior of MSCs [3]. In the current study, we use the realtime tracking technology, MR imaging, to determine whether labeled MSCs indeed migrate into injured regions and further differentiate into cardiomyocytes after transplantation. A major difficulty in the present study is how to find the labeled MSCs after intracoronary delivery. With the idea of stem cells homing to injured myocardium, bone marrow stem cells are spontaneously mobilized to the infarction after MI [4]. Thus, identification of the infarction may serve as an alternative approach to finding the location of labeled MSCs.
☆ This work was supported by NSFC (National Nature Science Foundation of China,30570743), NSFC (30670853). ⁎ Corresponding author. E-mail address: [email protected] (G. Ma).
418
Letters to the Editor
Fig. 1. Serial tracking of labeled MSCs in vivo and ex vivo MR imaging. There were no local hypointense signal lesions within hyperenhanced infarct tissue before labeled MSCs transplantation (A). Local hypointense signal lesions (arrows) were detected at 4 weeks (B) and 8 weeks (C) after labeled MSCs transplantation. Corresponding views of explanted heart in short-axis show areas of hypointense signal (D).
Moreover, because of occlusion of the distal of LAD, all of the infarct lesions appear in the left ventricle short-axis view adjacent to the apex in the current study. The magnetic labeling cells also typically generates a significant T2⁎ contrast in vivo with the use of iron oxide particles, which is notable as signal loss or negative contrast on T2⁎ imaging. This method of signal amplification with internalized contrast, producing a much greater signal loss area than the size of the cell itself on MR imaging [5], increases the ability of MR imaging to detect small numbers of labeled MSCs. On the basis of these reports, all of the infarctions in the present study were observed and the sites of hypointense signals where the labeled MSCs accumulated were appreciated along the rim or in the MI region. These findings suggested that labeled MSCs are attracted to and retained in the ischemic tissue [6]. Labeled MSCs appeared as a gradual loss of the signal intensity with cell division from 4 weeks to 8 weeks after transplantation, the reasons for the decrease in the dephasing signal over time are multifactorial [7]. However, some data raise concerns on the effect of iron oxide particles on stem cell differentiation. We tested in the
current study that the differentiated function and proliferative capacity of MSCs were not affected by labeling with iron particles in vivo. H&E staining demonstrated the growth of new tissue along the rim of the MI, which appeared larger with bigger ellipse nuclei, cells’ profile were consistent with that from other research [8]. These morphological mature cells positively stained with Prussian blue and desmin staining and dragon green fluorescent dye in the same section suggested that this occurred due to labeled MSCs differentiation not due to hypertrophy of the cardiomyocytes. Furthermore, with these cells negative for CD68, we excluded the possibility of macrophages took up iron particles. Western blot analysis demonstrated the increase in expression of cardiomyocyte markers such as myosin heavy chain and troponin T in MSCs treatment groups than AMI group, also suggesting the growth of new cardiomyocytes. There was no statistical significance in the changes of the expression of cardiomyocyte markers between labeled and unlabeled group, which demonstrated the differentiation of labeled MSCs was not affected by labeling with iron particles in vivo.
Letters to the Editor
419
Fig. 2. Differentiation of labeled MSCs at 8 weeks post-MI. A, H&E staining showed some cells (arrows, magnification ×200) with bigger ellipse nuclei at the infarct area and positively stained with Prussian blue (B), desmin staining (C) and Dragon Green fluorescent dye (D) in the same section.
References
Fig. 3. Cardiomyocyte markers in four groups at 8 weeks post-MI. ⁎P b 0.05 versus AMI group. Each experiment was performed twice. There was no statistical significance between labeled and unlabeled group. 0167-5273/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2007.07.164
[1] Thompson RB, Yu ZX, Hinds KA, Pessanha BS, GUttman MA, Varney TR, et al. Serial cardiac magnetic resonance imaging of injected mesenchymal stem cells. Circulation 2003;108:1009–14. [2] Laflamme MA, Murry CE. Regenerating the heart. Nat Biotechnol 2005;23:845–56. [3] Jacquier A, Higgins CB, Saeed M. MR imaging in assessing cardiovascular interventions and myocardial injury. Contrast Media MolImaging 2007;2:1–15. [4] Nelson M, Patil S, Hardy W, Sturgeon C, Hewett T, Chung T, et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol 2001;29:244–55. [5] Shapiro EM, Sharer K, Skrtic S, Koretsky AP. In vivo detection of single cells by MRI. Magn Reson Med 2006;55:242–9. [6] Orlic D, Hill JM, Arai AE. Stem cells for myocardial regeneration. Circ Res 2002;91:1092–102. [7] Lewis BK, Zywicke HA, Bulte JW, Bryant LHJ, Frank JA. Combination of transfection agents and magnetic resonance contrast agents for cellular imaging: relationship between relaxivities, electrostatic forces, and chemical composition. Magn Reson Med 2003;50:275–82. [8] Mickle DAG, Kim EJ, Sakai T, Jia ZQ. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 1999;100:II247–56.
417
[8] Farb Andrew, Burke Allen P, Kolodgie Frank D, Virmani Renu. Pathological mechanisms of fatal late coronary stent thrombosis in humans. Circulation 2003;108:1701–6. [9] Joner Michael, Finn Aloke V, Farb Andrew, et al. Pathology of drugeluting stents in humans-delayed healing and late thrombotic risk. J Am Coll Cardiol 2006;48:193–202.
0167-5273/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2007.07.111
Identification and differentiation of magnetically labeled mesenchymal stem cells in vivo in swines with myocardial infarction Chun-mei Qi, Gen-shan Ma ⁎, Nai-feng Liu, Cheng-xing Shen, Zhong Chen, Xiao-jun Liu, Yao-peng Hu, Xiao-li Zhang, Ji-yuan Chen, Sheng-hong Ju,Yao-liang Tang Received 5 July 2007; accepted 6 July 2007 Available online 11 December 2007
Abstract We aim to track mesenchymal stem cells (MSCs) after magnetically labeling and test the ability of these cells differentiate into cardiomyocytes in vivo. Therefore, 20 swines were divided into four groups, sham-operated group (n = 3); acute myocardial infarction (AMI) transplanted with PBS (n = 3); labeled MSCs (n = 7) and unlabeled MSCs (n = 7) group. 107 labeled or unlabeled cells were intracoronary delivered after MI (4.8 ± 1.3 days), and serial cardiac MR (3.0T) imaging studies were performed at 0, 4 and 8 weeks after transplantation, then the results were confirmed by histological and western blot analysis. We demonstrated that labeled MSCs can be reliably detected and tracked in vivo using MR imaging. In particular, we provided the evidence of regeneration of labeled MSCs in vivo by histological examination and western blot analysis. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Magnetic resonance imaging; Contrast media; Mesenchymal stem cell; Differentiation; Myocardial infarction
1. Results
2. Discussion
MSCs after labeled with dual-contrast particles (magnetite as an MRI marker and Dragon Green fluorescent dye as a histological marker) [1] were accurately identified along the rim or in the region of MI on MR imaging (Fig. 1). Signal to noise ratio and contrast to noise ratio (CNR) decreased (16.07 ± 5.85 vs.10.96± 1.34, P b 0.05) from 4 weeks to 8 weeks after labeled MSCs transplantation. Labeled MSCs were further confirmed by Prussian blue and immunofluorescent staining. Immunohistochemistry confirmed that the myogenic cells differentiated from labeled MSCs were positive for desmin and negative for CD68 (Fig. 2). Western blot analysis demonstrated that there was an increase expression of cardiomyocyte markers such as myosin heavy chain and troponin T in MSCs treatment groups compared with sham-operated group and AMI group (Fig. 3).
There is growing enthusiasm for the application of stem cell-based therapies to the regeneration of damaged myocardium. Although the results of preliminary studies and clinical trials are promising, many practical issues regarding this therapy remain highly controversial [2]. To address these important issues, molecular markers capable of tracking transplanted cells and determining the fate of engrafted cells would be extremely useful. Cells labeling with MR contrast media were used as a new technology to monitor the in vivo behavior of MSCs [3]. In the current study, we use the realtime tracking technology, MR imaging, to determine whether labeled MSCs indeed migrate into injured regions and further differentiate into cardiomyocytes after transplantation. A major difficulty in the present study is how to find the labeled MSCs after intracoronary delivery. With the idea of stem cells homing to injured myocardium, bone marrow stem cells are spontaneously mobilized to the infarction after MI [4]. Thus, identification of the infarction may serve as an alternative approach to finding the location of labeled MSCs.
☆ This work was supported by NSFC (National Nature Science Foundation of China,30570743), NSFC (30670853). ⁎ Corresponding author. E-mail address: [email protected] (G. Ma).
418
Letters to the Editor
Fig. 1. Serial tracking of labeled MSCs in vivo and ex vivo MR imaging. There were no local hypointense signal lesions within hyperenhanced infarct tissue before labeled MSCs transplantation (A). Local hypointense signal lesions (arrows) were detected at 4 weeks (B) and 8 weeks (C) after labeled MSCs transplantation. Corresponding views of explanted heart in short-axis show areas of hypointense signal (D).
Moreover, because of occlusion of the distal of LAD, all of the infarct lesions appear in the left ventricle short-axis view adjacent to the apex in the current study. The magnetic labeling cells also typically generates a significant T2⁎ contrast in vivo with the use of iron oxide particles, which is notable as signal loss or negative contrast on T2⁎ imaging. This method of signal amplification with internalized contrast, producing a much greater signal loss area than the size of the cell itself on MR imaging [5], increases the ability of MR imaging to detect small numbers of labeled MSCs. On the basis of these reports, all of the infarctions in the present study were observed and the sites of hypointense signals where the labeled MSCs accumulated were appreciated along the rim or in the MI region. These findings suggested that labeled MSCs are attracted to and retained in the ischemic tissue [6]. Labeled MSCs appeared as a gradual loss of the signal intensity with cell division from 4 weeks to 8 weeks after transplantation, the reasons for the decrease in the dephasing signal over time are multifactorial [7]. However, some data raise concerns on the effect of iron oxide particles on stem cell differentiation. We tested in the
current study that the differentiated function and proliferative capacity of MSCs were not affected by labeling with iron particles in vivo. H&E staining demonstrated the growth of new tissue along the rim of the MI, which appeared larger with bigger ellipse nuclei, cells’ profile were consistent with that from other research [8]. These morphological mature cells positively stained with Prussian blue and desmin staining and dragon green fluorescent dye in the same section suggested that this occurred due to labeled MSCs differentiation not due to hypertrophy of the cardiomyocytes. Furthermore, with these cells negative for CD68, we excluded the possibility of macrophages took up iron particles. Western blot analysis demonstrated the increase in expression of cardiomyocyte markers such as myosin heavy chain and troponin T in MSCs treatment groups than AMI group, also suggesting the growth of new cardiomyocytes. There was no statistical significance in the changes of the expression of cardiomyocyte markers between labeled and unlabeled group, which demonstrated the differentiation of labeled MSCs was not affected by labeling with iron particles in vivo.
Letters to the Editor
419
Fig. 2. Differentiation of labeled MSCs at 8 weeks post-MI. A, H&E staining showed some cells (arrows, magnification ×200) with bigger ellipse nuclei at the infarct area and positively stained with Prussian blue (B), desmin staining (C) and Dragon Green fluorescent dye (D) in the same section.
References
Fig. 3. Cardiomyocyte markers in four groups at 8 weeks post-MI. ⁎P b 0.05 versus AMI group. Each experiment was performed twice. There was no statistical significance between labeled and unlabeled group. 0167-5273/$ - see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2007.07.164
[1] Thompson RB, Yu ZX, Hinds KA, Pessanha BS, GUttman MA, Varney TR, et al. Serial cardiac magnetic resonance imaging of injected mesenchymal stem cells. Circulation 2003;108:1009–14. [2] Laflamme MA, Murry CE. Regenerating the heart. Nat Biotechnol 2005;23:845–56. [3] Jacquier A, Higgins CB, Saeed M. MR imaging in assessing cardiovascular interventions and myocardial injury. Contrast Media MolImaging 2007;2:1–15. [4] Nelson M, Patil S, Hardy W, Sturgeon C, Hewett T, Chung T, et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol 2001;29:244–55. [5] Shapiro EM, Sharer K, Skrtic S, Koretsky AP. In vivo detection of single cells by MRI. Magn Reson Med 2006;55:242–9. [6] Orlic D, Hill JM, Arai AE. Stem cells for myocardial regeneration. Circ Res 2002;91:1092–102. [7] Lewis BK, Zywicke HA, Bulte JW, Bryant LHJ, Frank JA. Combination of transfection agents and magnetic resonance contrast agents for cellular imaging: relationship between relaxivities, electrostatic forces, and chemical composition. Magn Reson Med 2003;50:275–82. [8] Mickle DAG, Kim EJ, Sakai T, Jia ZQ. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 1999;100:II247–56.