Repair of Infarcted Myocardium Mediated by Transplanted Bone Marrow–Derived CD34+ Stem Cells in a Nonhuman Primate Model
http://www.100md.com
《干细胞学杂志》
a Center for Molecular Medicine;
b Division of Hematology, and
c Division of Cardiology, Department of Internal Medicine;
d Division of Cardiovascular Surgery, Department of Surgery; and
e Department of Anatomy, Jichi Medical School, Minamikawachi, Tochigi;
f Tsukuba Primate Center, National Institute of Infectious Diseases, Tsukuba, Ibaraki;
g Department of Organ Regeneration, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, Japan
Key Words. Nonhuman primate ? Acute myocardial infarction ? Stem cell transplantation ? Genetic marking ? Lentivirus vector ? Plasticity ? Neoangiogenesis
Correspondence: Yutaka Hanazono, M.D., Ph.D., Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Tochigi 329-0498, Japan. Telephone: 81-285-58-7450; Fax: 81-285-44-5205; e-mail: hanazono@jichi.ac.jp
ABSTRACT
Recent clinical studies have shown that the introduction of bone marrow cells can restore blood flow in ischemic myocardium and ameliorate cardiac function . Despite enthusiasm for these studies, it is unclear how transplanted bone marrow cells contribute to the clinical improvement. Because endothelial progenitor cells are identified in bone marrow cells , these cells might participate in the repair of vascular tissue. On the other hand, it has been reported that hematopoietic stem cells differentiate into endothelial cells and cardiomyocytes when transplanted into the ischemic myocardium in mice . More recently, however, it has been reported that hematopoietic stem cells do not give rise to nonhematopoietic cells in the ischemic myocardium in murine models .
In vivo tracking and plastic properties of hematopoietic stem or progenitor cells have not been examined in primate cardiac ischemia. We have transplanted genetically marked autologous CD34+ cells to the ischemic myocardium in a nonhuman primate (cynomolgus macaque) model and tracked the in vivo fate of the cells. We have used CD34+ cells because the cells are widely used as a fraction of hematopoietic stem cells in clinical and nonhuman primate studies . In addition, CD34+ cells contain vascular endothelial progenitor cells . Thus, the present study can address the question of whether transplanted CD34+ cells really give rise to endothelial cells and cardiomyocytes in ischemic myocardium in primates.
MATERIALS AND METHODS
Lentiviral Marking
The CD34+ fraction of autologous bone marrow cells was used for transplantation in our study (Table 1). Before transplantation, CD34+ cells were genetically marked with GFP using an SIV-based lentivirus vector. The ex vivo transduction results are summarized in Table 1. The transduced cells were frozen until transplantation. An aliquot of the transduced cells was examined in vitro for the endothelial differentiation ability. After the differentiation culture, a vessel-like structure was observed (Fig. 1A). The ability of cells to take up DiI-acetylated LDL and the expression of CD31, vWF, VE-cadherin, and VEGFR-2 suggested the endothelial lineage (Fig. 1B). We and others have already confirmed the ability of hematopoietic differentiation of the cells . Taken together, the SIV-mediated GFP gene transfer does not spoil the differentiation abilities of CD34+ cells. In addition, on average, 41% of cells fluoresced 48 hours after transduction, and 56% of endothelial cells still fluoresced after in vitro differentiation (Table 1), showing that the GFP expression is stable during the in vitro differentiation to endothelial cells. Thus, GFP was expected to serve as a good genetic tag after transplantation.
Table 1. Summary of ex vivo transduction and transplantation
Figure 1. In vitro endothelial differentiation of cynomolgus CD34+ cells lentivirally transduced with GFP. The transduced CD34+ cells were differentiated to endothelial cells after 7 days in culture. (A): Representative vessel-like structure derived from CD34+ cells observed under a phase-contrast microscope (left) and a fluorescent microscope (right). (B): The transduced CD34+ cells differentiated into fluorescent cells (green) positive for the cellular intake of acetylated LDL and immunostaining for von Willebrand factor (vWF) (stained in red). Bar = 100 μm. Abbreviations: GFP, green fluorescent protein; LDL, low-density lipoprotein.
Acute Myocardial Infarction and Autologous Transplantation
Cynomolgus acute myocardial infarction was generated by ligating the left anterior descending artery. One to two hours after the ligation, GFP-transduced, autologous CD34+ cells were injected in the peri-ischemic zone at 10 sites (total, 1.20 ± 0.73 x 106 cells; n = 4). In the control group, saline was injected in the same way (n = 4). We conducted contrast echocardiography immediately after the coronary ligation and found no significant differences in the blood flow defect size (percent blood flow defect compared with the total) between the cell-treated and saline-treated groups (13.0 ± 2.1% versus 12.3 ± 3.5%, p = .75), suggesting that the initial risk of infarction did not differ between the two groups. In addition, we tried to assess the cardiac isozyme of serum creatine kinase (CK) to evaluate the infarct size; however, either the immuno-inhibition assay or chemical luminescence immunoassay did not work well for cynomolgus monkey samples. We were at least able to show that total CK values at 24 hours after the ligation did not significantly differ between the two groups (p = .83).
One of the control monkeys (CTR01061 died of heart failure 5 days after myocardial infarction, and the other control monkeys showed a decrease in %FS at 2 weeks after infarction (Fig. 2). Thus, all four control animals showed the deteriorated cardiac function. In the cell-treated group, one monkey (*, BM01051) underwent ventricular fibrillation immediately after the ligation and survived after cardiopul-monary resuscitation but eventually developed a ventricular aneurysm. Only this animal showed a decrease in %FS despite CD34+ cell treatment; the other animals receiving CD34+ cells showed an increase in %FS (Fig. 2). CD34+ cell treatment may not be able to rescue such a heavily impaired heart but otherwise had a significant effect on cardiac function. Even an old monkey (BM90047, Table 1) showed improved %FS.
Figure 2. Improved cardiac function after CD34+ cell transplantation. Cardiac function was assessed by echocardiography in terms of percent fractional shortening (%FS) before and 2 weeks after treatment. One monkey in the saline-treated group (CTR01061 died of heart failure 5 days after myocardial infarction and is not included in the figure. One monkey in the CD34+ cell–treated group (*, BM01051) developed a left ventricular aneurysm after myocardial infarction. If this animal was excluded from the statistical analysis, the cardiac function was significantly improved in the CD34+ cell–treated compared with the saline-treated group in terms of the ratio of %FS at day 14 versus day 0 after transplant (p = .017).
The relative blood flow in the peri-infarct to nonischemic control region was also significantly ameliorated in the CD34+ cell–treated monkeys compared with the saline-treated ones, as assessed using contrast echocardiography (Fig. 3A) and colored microspheres (Fig. 3B). An excellent correlation was found between the two methods (Fig. 3C; correlation coefficient = 0.93). Two groups (CD34+ cell–treated and saline-treated) were well separated on the panel, showing an obvious positive effect of CD34+ cell injection on the blood flow in the peri-infarct zone after acute myocardial infarction. In fact, the average myocardial blood flow in the peri-infarct region in the absolute value was 0.988 ml/g per minute and 0.383 ml/g per minute for the cell-treated and saline-treated groups, respectively. Of note, the blood flow in the peri-infarct zone was ameliorated even in the animal with a ventricular aneurysm. On the other hand, the relative blood flow in the infarct to nonischemic region did not show a significant difference between the CD34+ cell–treated and saline-treated groups. The peri-infarct region was the injection site, and thus the highest degree of change would be expected there.
Figure 3. Improved regional blood flow after CD34+ cell transplantation. Myocardial contrast echocardiography (A) and colored microspheres (B) showed a significantly ameliorated blood flow ratio (the peri-infarct to nonischemic control region) in the CD34+ cell–treated monkeys (n = 3) compared with the saline-treated monkeys (n = 3) at 2 weeks after treatment. (C): An excellent correlation was found between the two methods. A CD34+ cell–treated monkey (?, BM97080) that was examined at 12 weeks after transplant is included in the panel (C) but excluded from the statistical analysis in (A) and (B).
All monkeys except one CD34+ cell–treated monkey (BM97080) were examined for cardiac function and blood flow at 2 weeks after transplantation, and their tissue sections were finally prepared at this time point (see below). BM97080 was examined at 12 weeks, at which time the cardiac function was still improved compared with immediately after infarction (data not shown) and the blood flow data were in a position similar to the cell-treated group at 2 weeks (Fig. 3C).
In Vivo Tracking of Transplanted Cells
Two weeks after the transplantation, tissue sections were prepared from the infarct, peri-infarct, and nonischemic regions. Immunostaining of an endothelial marker CD31 demonstrated more vessels in the peri-infarct region of the CD34+ cell-treated than saline-treated myocardium (Fig. 4A). In fact, the capillary density of the peri-infarct region was significantly better preserved in the cell-treated than saline-treated group, although there was no significant difference in the capillary density of the nonischemic control regions between the two groups (Fig. 4B).
Figure 4. Neoangiogenesis in the ischemic myocardium. Tissue sections were prepared at 2 weeks after the treatment. (A): Representative results of immunostaining with anti-CD31 (stained in brown) in the peri-infarct region of the saline-treated and CD34+ cell–treated myocardium. Bar = 50 μm. (B): The density of CD31-positive capillaries in the peri-infarct and control nonischemic regions in the saline-treated and CD34+ cell–treated groups. Five fields for each section were randomly selected (n = 3 for the saline injection, n = 3 for the CD34+ cell injection), and the number of CD31-positive capillaries was counted (average ± standard deviation).
Double immunostaining with anti-CD31 and anti-GFP showed that some cells in vessels were positive for both CD31 and GFP in the peri-infarct region (Fig. 5A). The result clearly indicates that at least some transplanted CD34+ cells gave rise to endothelial cells. However, we found that the transplanted cell progeny accounted for only a small fraction of endothelial cells after examining more than 100 sections of the peri-infarct region. In situ PCR for proviral GFP sequences also showed that few CD31-positive endothelial cells contained the GFP-provirus (Fig. 5B). There were no GFP-positive cardiomyocytes in more than 100 sections. Most of the transplanted cell progeny were found not incorporated in vessels (Fig. 5C). Hematoxylineosin staining did not show any noncardiac tissue regeneration in the myocardium.
Figure 5. In vivo fate of transplanted cells. Cardiac sections were prepared at 2 weeks after transplantation. (A): Double immunohistochemistry (IHC) with anti-CD31 and anti–green fluorescent protein (GFP) in the peri-infarct region of the CD34+ cell–treated myocardium. Some cells (arrow) were positive for both CD31 (stained in brown) and GFP (stained in black), but such cells were rare. (B, C): Serial sections from the peri-infarct region of the CD34+ cell–treated myocardium. One section (left) was stained with anti-CD31 (stained in brown), and the other (right) was assessed by in situ poly-merase chain reaction (PCR) for proviral GFP sequences (stained in black). (B): Some CD31-positive endothelial cells contained the GFP-provirus (arrow, right panel), but such cells were rare. (C): Transplanted cell progeny (cells positive for GFP-provirus in the right panel) were not incorporated in vessels (cells positive for CD31 in the left panel). Bar = 50 μm.
On the other hand, we found that in vitro conditioned medium of CD34+ cell culture for endothelial differentiation contained high levels of VEGF, whereas unconditioned medium did not contain detectable VEGF, as assessed by ELISA (Fig. 6A). In addition, in vivo VEGF levels in the peri-infarct tissue were significantly higher in the CD34+ cell–treated than saline-treated group (Fig. 6B, left), although in vivo levels of bFGF differed little between the two groups (Fig. 6B, right).
Figure 6. VEGF is implicated in the neoangiogenesis. (A): Unconditioned and conditioned media of in vitro CD34+ cell cultures for endothelial differentiation were examined for VEGF by ELISA. The average ± standard error of six culture dishes is shown. (B): Lysates (three samples per monkey) from the peri-infarct region of the CD34+ cell–treated (monkey, n = 3) and saline-treated (monkey, n = 3) myocardium were prepared and examined for VEGF and basic fibroblast growth factor (bFGF) by ELISA. Data are shown as the average ± standard error. Abbreviation: VEGF, vascular endothelial growth factor.
DISCUSSION
The SIV vector was supplied by DNAVEC Corporation (Ibaraki, Japan), and thrombopoietin was supplied by Kirin Brewery Co. Ltd. (Tokyo). We thank Masahiro Shakudo (Sumiyoshi Hospital, Osaka) for analyzing the contrast echocardiography and Yasuhiro Ochiai (Jichi Medical School) for preparing tissue sections.
REFERENCES
Assmus B, Schachinger V, Teupe C et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation 2002;106:3009–3017.
Strauer BE, Brehm M, Zeus T et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 2002;106:1913–1918.
Tse HF, Kwong YL, Chan JK et al. Angiogenesis in ischaemic myocardium by intramyocardial autologous bone marrow mononuclear cell implantation. Lancet 2003;361:47–49.
Perin EC, Dohmann HF, Borojevic R et al. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation 2003;107:2294–2302.
Stamm C, Westphal B, Kleine HD et al. Autologous bone-marrow stem-cell transplantation for myocardial regeneration. Lancet 2003;361:45–46.
Wollert KC, Meyer GP, Lotz J et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 2004;364:141–148.
Asahara T, Murohara T, Sullivan A et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–967.
Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705.
Murry CE, Soonpaa MH, Reinecke H et al. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 2004;428:664–668.
Balsam LB, Wagers AJ, Christensen JL et al. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature 2004;428:668–673.
Nygren JM, Jovinge S, Breitbach M et al. Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation. Nat Med 2004;10:494–501.
Hanazono Y, Terao K, Ozawa K. Gene transfer into non-human primate hematopoietic stem cells: implications for gene therapy. STEM CELLS 2001;19:12–23.
Shibata H, Hanazono Y, Ageyama N et al. Collection and analysis of hematopoietic progenitor cells from cynomolgus macaques (Macaca fascicularis): assessment of cross-reacting monoclonal antibodies. Am J Primatol 2003;61:3–12.
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b Division of Hematology, and
c Division of Cardiology, Department of Internal Medicine;
d Division of Cardiovascular Surgery, Department of Surgery; and
e Department of Anatomy, Jichi Medical School, Minamikawachi, Tochigi;
f Tsukuba Primate Center, National Institute of Infectious Diseases, Tsukuba, Ibaraki;
g Department of Organ Regeneration, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, Japan
Key Words. Nonhuman primate ? Acute myocardial infarction ? Stem cell transplantation ? Genetic marking ? Lentivirus vector ? Plasticity ? Neoangiogenesis
Correspondence: Yutaka Hanazono, M.D., Ph.D., Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Tochigi 329-0498, Japan. Telephone: 81-285-58-7450; Fax: 81-285-44-5205; e-mail: hanazono@jichi.ac.jp
ABSTRACT
Recent clinical studies have shown that the introduction of bone marrow cells can restore blood flow in ischemic myocardium and ameliorate cardiac function . Despite enthusiasm for these studies, it is unclear how transplanted bone marrow cells contribute to the clinical improvement. Because endothelial progenitor cells are identified in bone marrow cells , these cells might participate in the repair of vascular tissue. On the other hand, it has been reported that hematopoietic stem cells differentiate into endothelial cells and cardiomyocytes when transplanted into the ischemic myocardium in mice . More recently, however, it has been reported that hematopoietic stem cells do not give rise to nonhematopoietic cells in the ischemic myocardium in murine models .
In vivo tracking and plastic properties of hematopoietic stem or progenitor cells have not been examined in primate cardiac ischemia. We have transplanted genetically marked autologous CD34+ cells to the ischemic myocardium in a nonhuman primate (cynomolgus macaque) model and tracked the in vivo fate of the cells. We have used CD34+ cells because the cells are widely used as a fraction of hematopoietic stem cells in clinical and nonhuman primate studies . In addition, CD34+ cells contain vascular endothelial progenitor cells . Thus, the present study can address the question of whether transplanted CD34+ cells really give rise to endothelial cells and cardiomyocytes in ischemic myocardium in primates.
MATERIALS AND METHODS
Lentiviral Marking
The CD34+ fraction of autologous bone marrow cells was used for transplantation in our study (Table 1). Before transplantation, CD34+ cells were genetically marked with GFP using an SIV-based lentivirus vector. The ex vivo transduction results are summarized in Table 1. The transduced cells were frozen until transplantation. An aliquot of the transduced cells was examined in vitro for the endothelial differentiation ability. After the differentiation culture, a vessel-like structure was observed (Fig. 1A). The ability of cells to take up DiI-acetylated LDL and the expression of CD31, vWF, VE-cadherin, and VEGFR-2 suggested the endothelial lineage (Fig. 1B). We and others have already confirmed the ability of hematopoietic differentiation of the cells . Taken together, the SIV-mediated GFP gene transfer does not spoil the differentiation abilities of CD34+ cells. In addition, on average, 41% of cells fluoresced 48 hours after transduction, and 56% of endothelial cells still fluoresced after in vitro differentiation (Table 1), showing that the GFP expression is stable during the in vitro differentiation to endothelial cells. Thus, GFP was expected to serve as a good genetic tag after transplantation.
Table 1. Summary of ex vivo transduction and transplantation
Figure 1. In vitro endothelial differentiation of cynomolgus CD34+ cells lentivirally transduced with GFP. The transduced CD34+ cells were differentiated to endothelial cells after 7 days in culture. (A): Representative vessel-like structure derived from CD34+ cells observed under a phase-contrast microscope (left) and a fluorescent microscope (right). (B): The transduced CD34+ cells differentiated into fluorescent cells (green) positive for the cellular intake of acetylated LDL and immunostaining for von Willebrand factor (vWF) (stained in red). Bar = 100 μm. Abbreviations: GFP, green fluorescent protein; LDL, low-density lipoprotein.
Acute Myocardial Infarction and Autologous Transplantation
Cynomolgus acute myocardial infarction was generated by ligating the left anterior descending artery. One to two hours after the ligation, GFP-transduced, autologous CD34+ cells were injected in the peri-ischemic zone at 10 sites (total, 1.20 ± 0.73 x 106 cells; n = 4). In the control group, saline was injected in the same way (n = 4). We conducted contrast echocardiography immediately after the coronary ligation and found no significant differences in the blood flow defect size (percent blood flow defect compared with the total) between the cell-treated and saline-treated groups (13.0 ± 2.1% versus 12.3 ± 3.5%, p = .75), suggesting that the initial risk of infarction did not differ between the two groups. In addition, we tried to assess the cardiac isozyme of serum creatine kinase (CK) to evaluate the infarct size; however, either the immuno-inhibition assay or chemical luminescence immunoassay did not work well for cynomolgus monkey samples. We were at least able to show that total CK values at 24 hours after the ligation did not significantly differ between the two groups (p = .83).
One of the control monkeys (CTR01061 died of heart failure 5 days after myocardial infarction, and the other control monkeys showed a decrease in %FS at 2 weeks after infarction (Fig. 2). Thus, all four control animals showed the deteriorated cardiac function. In the cell-treated group, one monkey (*, BM01051) underwent ventricular fibrillation immediately after the ligation and survived after cardiopul-monary resuscitation but eventually developed a ventricular aneurysm. Only this animal showed a decrease in %FS despite CD34+ cell treatment; the other animals receiving CD34+ cells showed an increase in %FS (Fig. 2). CD34+ cell treatment may not be able to rescue such a heavily impaired heart but otherwise had a significant effect on cardiac function. Even an old monkey (BM90047, Table 1) showed improved %FS.
Figure 2. Improved cardiac function after CD34+ cell transplantation. Cardiac function was assessed by echocardiography in terms of percent fractional shortening (%FS) before and 2 weeks after treatment. One monkey in the saline-treated group (CTR01061 died of heart failure 5 days after myocardial infarction and is not included in the figure. One monkey in the CD34+ cell–treated group (*, BM01051) developed a left ventricular aneurysm after myocardial infarction. If this animal was excluded from the statistical analysis, the cardiac function was significantly improved in the CD34+ cell–treated compared with the saline-treated group in terms of the ratio of %FS at day 14 versus day 0 after transplant (p = .017).
The relative blood flow in the peri-infarct to nonischemic control region was also significantly ameliorated in the CD34+ cell–treated monkeys compared with the saline-treated ones, as assessed using contrast echocardiography (Fig. 3A) and colored microspheres (Fig. 3B). An excellent correlation was found between the two methods (Fig. 3C; correlation coefficient = 0.93). Two groups (CD34+ cell–treated and saline-treated) were well separated on the panel, showing an obvious positive effect of CD34+ cell injection on the blood flow in the peri-infarct zone after acute myocardial infarction. In fact, the average myocardial blood flow in the peri-infarct region in the absolute value was 0.988 ml/g per minute and 0.383 ml/g per minute for the cell-treated and saline-treated groups, respectively. Of note, the blood flow in the peri-infarct zone was ameliorated even in the animal with a ventricular aneurysm. On the other hand, the relative blood flow in the infarct to nonischemic region did not show a significant difference between the CD34+ cell–treated and saline-treated groups. The peri-infarct region was the injection site, and thus the highest degree of change would be expected there.
Figure 3. Improved regional blood flow after CD34+ cell transplantation. Myocardial contrast echocardiography (A) and colored microspheres (B) showed a significantly ameliorated blood flow ratio (the peri-infarct to nonischemic control region) in the CD34+ cell–treated monkeys (n = 3) compared with the saline-treated monkeys (n = 3) at 2 weeks after treatment. (C): An excellent correlation was found between the two methods. A CD34+ cell–treated monkey (?, BM97080) that was examined at 12 weeks after transplant is included in the panel (C) but excluded from the statistical analysis in (A) and (B).
All monkeys except one CD34+ cell–treated monkey (BM97080) were examined for cardiac function and blood flow at 2 weeks after transplantation, and their tissue sections were finally prepared at this time point (see below). BM97080 was examined at 12 weeks, at which time the cardiac function was still improved compared with immediately after infarction (data not shown) and the blood flow data were in a position similar to the cell-treated group at 2 weeks (Fig. 3C).
In Vivo Tracking of Transplanted Cells
Two weeks after the transplantation, tissue sections were prepared from the infarct, peri-infarct, and nonischemic regions. Immunostaining of an endothelial marker CD31 demonstrated more vessels in the peri-infarct region of the CD34+ cell-treated than saline-treated myocardium (Fig. 4A). In fact, the capillary density of the peri-infarct region was significantly better preserved in the cell-treated than saline-treated group, although there was no significant difference in the capillary density of the nonischemic control regions between the two groups (Fig. 4B).
Figure 4. Neoangiogenesis in the ischemic myocardium. Tissue sections were prepared at 2 weeks after the treatment. (A): Representative results of immunostaining with anti-CD31 (stained in brown) in the peri-infarct region of the saline-treated and CD34+ cell–treated myocardium. Bar = 50 μm. (B): The density of CD31-positive capillaries in the peri-infarct and control nonischemic regions in the saline-treated and CD34+ cell–treated groups. Five fields for each section were randomly selected (n = 3 for the saline injection, n = 3 for the CD34+ cell injection), and the number of CD31-positive capillaries was counted (average ± standard deviation).
Double immunostaining with anti-CD31 and anti-GFP showed that some cells in vessels were positive for both CD31 and GFP in the peri-infarct region (Fig. 5A). The result clearly indicates that at least some transplanted CD34+ cells gave rise to endothelial cells. However, we found that the transplanted cell progeny accounted for only a small fraction of endothelial cells after examining more than 100 sections of the peri-infarct region. In situ PCR for proviral GFP sequences also showed that few CD31-positive endothelial cells contained the GFP-provirus (Fig. 5B). There were no GFP-positive cardiomyocytes in more than 100 sections. Most of the transplanted cell progeny were found not incorporated in vessels (Fig. 5C). Hematoxylineosin staining did not show any noncardiac tissue regeneration in the myocardium.
Figure 5. In vivo fate of transplanted cells. Cardiac sections were prepared at 2 weeks after transplantation. (A): Double immunohistochemistry (IHC) with anti-CD31 and anti–green fluorescent protein (GFP) in the peri-infarct region of the CD34+ cell–treated myocardium. Some cells (arrow) were positive for both CD31 (stained in brown) and GFP (stained in black), but such cells were rare. (B, C): Serial sections from the peri-infarct region of the CD34+ cell–treated myocardium. One section (left) was stained with anti-CD31 (stained in brown), and the other (right) was assessed by in situ poly-merase chain reaction (PCR) for proviral GFP sequences (stained in black). (B): Some CD31-positive endothelial cells contained the GFP-provirus (arrow, right panel), but such cells were rare. (C): Transplanted cell progeny (cells positive for GFP-provirus in the right panel) were not incorporated in vessels (cells positive for CD31 in the left panel). Bar = 50 μm.
On the other hand, we found that in vitro conditioned medium of CD34+ cell culture for endothelial differentiation contained high levels of VEGF, whereas unconditioned medium did not contain detectable VEGF, as assessed by ELISA (Fig. 6A). In addition, in vivo VEGF levels in the peri-infarct tissue were significantly higher in the CD34+ cell–treated than saline-treated group (Fig. 6B, left), although in vivo levels of bFGF differed little between the two groups (Fig. 6B, right).
Figure 6. VEGF is implicated in the neoangiogenesis. (A): Unconditioned and conditioned media of in vitro CD34+ cell cultures for endothelial differentiation were examined for VEGF by ELISA. The average ± standard error of six culture dishes is shown. (B): Lysates (three samples per monkey) from the peri-infarct region of the CD34+ cell–treated (monkey, n = 3) and saline-treated (monkey, n = 3) myocardium were prepared and examined for VEGF and basic fibroblast growth factor (bFGF) by ELISA. Data are shown as the average ± standard error. Abbreviation: VEGF, vascular endothelial growth factor.
DISCUSSION
The SIV vector was supplied by DNAVEC Corporation (Ibaraki, Japan), and thrombopoietin was supplied by Kirin Brewery Co. Ltd. (Tokyo). We thank Masahiro Shakudo (Sumiyoshi Hospital, Osaka) for analyzing the contrast echocardiography and Yasuhiro Ochiai (Jichi Medical School) for preparing tissue sections.
REFERENCES
Assmus B, Schachinger V, Teupe C et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation 2002;106:3009–3017.
Strauer BE, Brehm M, Zeus T et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 2002;106:1913–1918.
Tse HF, Kwong YL, Chan JK et al. Angiogenesis in ischaemic myocardium by intramyocardial autologous bone marrow mononuclear cell implantation. Lancet 2003;361:47–49.
Perin EC, Dohmann HF, Borojevic R et al. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation 2003;107:2294–2302.
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