Xenotransplant Cardiac Chimera: Immune Tolerance of Adult Stem Cells

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1 Xenotransplant Cardiac Chimera: Immune Tolerance of Adult Stem Cells Takayuki Saito, MD, PhD, Jin-Qiang Kuang, MD, Bindu Bittira, MD, Abdulaziz Al-Khaldi, MD, and Ray C.-J. Chiu, MD, PhD Division of Cardiac Surgery, McGill University Health Center, Montreal, Quebec, Canada, and Division of Cardiovascular Surgery, Nagoya City University Medical School, Nagoya, Japan Background. Bone marrow stromal cells have been shown to engraft into xenogeneic fetal recipients. In view of the potential clinical utility as an alternative source for cellular and gene therapies, we studied the fate of xenogeneic marrow stromal cells after their systemic transplantation into fully immunocompetent adult recipients without immunosuppression. Methods. Bone marrow stromal cells were isolated from C57B1/6 mice and retrovirally transduced with LacZ reporter gene for cell labeling. We then injected labeled cells into immunocompetent adult Lewis rats. One week later, the recipient animals underwent coronary artery ligation and were sacrificed at various time points ranging from 1 day to 12 weeks after ligation. Hearts, blood, and bone marrow samples were collected for histologic and immunohistochemical studies. Results. Labeled mice cells engrafted into the bone marrow cavities of the recipient rats for at least 13 weeks after transplantation without any immunosuppression. On the other hand, circulating mice cells were positive only for the animals with 1-day-old myocardial infarction. At various time points, numerous mice cells could be found in the infarcted myocardium that were not seen before coronary ligation. Some of these cells subsequently showed positive staining for cardiomyocyte specific proteins, while other labeled cells participated in angiogenesis in the infarcted area. Conclusions. The marrow stromal cells are adult stem cells with unique immunologic tolerance allowing their engraftment into a xenogeneic environment, while preserving their ability to be recruited to an injured myocardium by way of the bloodstream and to undergo differentiation to form a stable cardiac chimera. (Ann Thorac Surg 2002;74:19 24) 2002 by The Society of Thoracic Surgeons Bone marrow stromal cells (MSCs) are adult stem cells capable of differentiating into cells of both mesenchymal and nonmesenchymal lineages [1 6]. When transfused intravenously, MSCs have been reported to engraft into the bone marrow compartment of allogeneic recipients without significant toxicity [7]. Liechty and colleagues [8] recently reported that human MSCs could engraft into the fetus of sheep after transplantation intraperitoneally, and persisted in the host organs for as long as 13 months without immunosuppression. Thus although precise mechanisms are still unknown, MSCs seem to have unique immunologic characteristics that allow their persistence in a xenogeneic fetal environment. However, whether or not these xenogeneic MSCs can engraft into fully immunocompetent adult animals after systemic administration and whether or not these cells, if they survive, still possess their abilities to migrate and to differentiate have not been demonstrated. In this study, we intravenously injected mice MSCs into rats and found their engraftment in the bone marrow of the recipients. Furthermore, we confirmed that these cells Accepted for publication March 5, Address reprint requests to Dr Chiu, Division of Cardiac Surgery, The Montreal General Hospital, MUHC, 1650 Cedar Ave, Suite C9-169, Montreal, Quebec H3G 1A4, Canada; rchiu@po-box.mcgill.ca. were capable of being recruited to an injured myocardium and undergo differentiation into several phenotypes, thus participating in the repair process. Material and Methods Animals Female C57Bl/6 mice and male Lewis rats were used in this study as the donors and recipients, respectively. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, revised 1996, and the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. Experimental Design Figure 1 shows an experimental design of this study. Isolated mice MSCs ( ) were injected into the penile vein of the recipients for 2 consecutive days. One week after the second injection, either left coronary artery ligation (MSC group) or sham operation (control group) was performed on the recipients. The animals were then sacrificed at various intervals after the procedure ranging from 1 day to 12 weeks by The Society of Thoracic Surgeons /02/$22.00 Published by Elsevier Science Inc PII S (02)

2 20 SAITO ET AL Ann Thorac Surg XENOGENEIC MSCs REGENERATE HOST MYOCARDIUM 2002;74:19 24 Fig 1. Experimental design. (LCA left coronary artery; MSCs marrow stromal cells.) Marrow Stromal Cell Isolation and Labeling With LacZ Bone marrow cells were harvested from female C57Bl/6 mice by flushing the femurs and tibias with Dulbecco s Modified Eagle s Medium supplemented with 10% fetal bovine serum and 50 U/mL penicillin-streptomycin according to the method of Wakitani and associates [4]. Whole marrow was plated in tissue culture dishes, and 5 to 7 days later, the nonadherent hematopoietic cells were discarded and the adherent bone MSCs were cultured at 37 C with 5% CO 2. The LacZ reporter gene was transfected into MSCs with pmfg-lacz retrovirus-mediated gene transfection as described previously [9]. The resulting LacZ MSCs were expanded for 4 weeks before transplantation. Marrow Stromal Cells Staining With 5-bromo-4- chloro-3-indoyl- -d-galactoside for Detection of galactosidase Activity Cells were plated in 35-mm dishes and were fixed in 1% glutaraldehyde for 5 minutes at room temperature, then they were washed with phosphate-buffered saline. Staining solution at ph of 7.8 to 8.0 [10], which contains 1 mg/ml 5-bromo-4-chloro-3-indoyl- -d-galactoside (Xgal), 1 mmol/l ethyleneglycol-bis( -aminoethyl-ether)- N,N -tetraacetic acid, 5 mmol/l K 3 Fe(CN) 6, 5 mmol/l K 4 Fe(CN) 6 O 3H 2 O, 2 mmol/l magnesium chloride, and 0.01% sodium deoxycholate [9], was added. Then cells were incubated at 37 C and protected from light for 16 hours. Transplantation of Marrow Stromal Cells and Ligation of the Left Coronary Artery Male Lewis rats (225 to 275 g) were used in this study as recipient animals. The animals were anesthetized with isoflurane (MTC Pharmaceuticals, Cambridge, Ontario, Canada), intubated, and mechanically ventilated at 80 breaths/min. Mice MSCs ( suspended in 150 L of Dulbecco s Modified Eagle s Medium) were injected into the penile vein and were reinjected 24 hours later in the same manner. Total number of MSCs injected was per animal. One week after the second injection, the animals were again anesthetized as mentioned and underwent either coronary artery ligation or sham operation (left thoracotomy only) as previously described [9]. Animals were divided into two groups: the MSCs group received MSCs intravenously followed by coronary artery ligation (n 22); the control group received MSCs intravenously followed by sham operation (n 5). Mortality of the coronary artery ligation and sham procedure was 31.8% and 0%, respectively. Surviving animals (MSCs group, n 15; control group, n 5) were sacrificed at 1 day and 2, 4, 8, and 12 weeks after operation; thus 3 MSC and 1 control hearts were examined at each time point. Tissue Processing and Staining for -galactosidase Activity On sacrifice of the recipient animals, blood, bone marrow, and hearts were collected. The blood samples were diluted sevenfold with Dulbecco s Modified Eagle s Medium containing 10% fetal bovine serum, plated in 35-mm dishes, and cultured for 2 weeks. Culture medium was changed twice a week, and most hematopoietic cells were discarded during this procedure. Bone MSCs were isolated from recipients femurs and tibias and then cultured for 1 week. Adherent cells derived from both the blood samples and bone marrow specimens were stained with X-gal staining solution as mentioned above. The hearts were rinsed with phosphate-buffered saline and perfusion fixed in 2% paraformaldehyde in phosphatebuffered saline. The staining for -galactosidase activity was performed as described above, but with the addition of 0.02% Nonidet P-40 and 0.01% deoxycholate to the staining solution [9]. After X-gal staining, the hearts were cut longitudinally and embedded in paraffin. Histology and Immunohistochemistry Heart sections 5 m in thickness were processed for either hematoxylin and eosin staining or immunohistochemical staining. Immunohistochemical staining was performed for anti- smooth muscle actin (Sigma Laboratories, St. Louis, MO), troponin I-C (Santa-Cruz Biotechnology Inc, Santa Cruz, CA), and sarcomeric myosin heavy chain molecules with MF20 as described previously [6, 9]. Results Transfection Efficiency and Intravenous Injection of Mice Marrow Stromal Cells To trace the fate of mice MSCs after transplantation, we labeled them in vitro with a retrovirus carrying the LacZ gene. 5-Bromo-4-chloro-3-indoyl- -d-galactoside staining of these cells revealed nearly 100% of MSCs expressed -galactosidase activity (Fig 2A). We transplanted labeled mice MSCs intravenously into Lewis rats without immunosuppression. There was neither transplant-related mortalities nor morbidities associated with immunorejection. Bone marrow specimens of the recipient rats were collected at various intervals ranging from 1 to 13 weeks after transplantation. Labeled mice MSCs could be identified in the rat bone marrow specimens of both MSCs and control groups at all times studied (Fig 2B).

3 Ann Thorac Surg SAITO ET AL 2002;74:19 24 XENOGENEIC MSCs REGENERATE HOST MYOCARDIUM 21 Fig 3. Histochemical staining for -galactosidase activity of mice marrow stromal cells in the gross heart. Gross heart specimen of the recipient rat was harvested at 12 weeks after coronary artery ligation. Mice marrow stromal cells were transplanted intravenously 1 week before the ligation. After fixation, the heart was stained for -galactosidase activity of mice marrow stromal cells. Bluish discoloration can be seen on the infarcted myocardium, whereas noninfarcted myocardium remains unstained. (A) Frontal view. (B) Lateral view. Fig 2. Histochemical staining for -galactosidase activity of mice marrow stromal cells in cultures. The transfected marrow stromal cells showed positive staining for -galactosidase activity (blue color). (A) Culture-expanded mice marrow stromal cells before implantation. (B) Bone marrow specimen of the recipient rat to which mice marrow stromal cells were transplanted intravenously 13 weeks before. LacZ mice marrow stromal cells (arrow) were identified, whereas host marrow stromal cells were unstained. (C) Blood sample obtained at 1 day after ligating the left coronary artery in rats to which mice marrow stromal cells were transplanted intravenously 1 week before the ligation. LacZ mice marrow stromal cells (arrows) were also identified, whereas host cells were unstained. (Original magnification [A through C], 100.) Migration to an Injured Myocardium Gross examination of the hearts revealed selectively localized blue discoloration of X-gal stain in the area of infarcted myocardium, whereas adjacent noninfarcted myocardium remained unstained (Fig 3). Histologic examination of serial cross sections confirmed the blue color seen on the gross heart specimens was indeed owing to the presence of labeled mice MSCs (Fig 4). Such cells were identified in the myocardium of all recipient rats. In the hearts harvested 1 day after coronary artery ligation, most labeled MSCs were found in the perivascular zone of noninfarcted myocardium (Figs 4C and 4D). However, in the hearts obtained after 2 weeks, most labeled MSCs were seen in the infarcted myocardium (Fig 4B). To confirm that these MSCs migrated from bone marrow to the heart by way of the bloodstream, we collected blood samples just before coronary artery ligation and 1 day and 2, 4, 8, and 12 weeks after coronary artery ligation. Mice MSCs could be detected only in the blood collected 1 day after ligation (Fig 2C). In the sham-operated rats that received mouse MSCs intravenously but without coronary artery ligations, X- gal stains of the gross and microscopic specimens were all negative, with no evidence of labeled cells in their myocardia. Differentiation of Mice Marrow Stromal Cells in an Infarcted Myocardium In the infarct scar area, some MSCs were seen in the fibrous layer. Morphologically, these MSCs had a myofibroblast-like appearance and may have contributed to scar formation (Fig 4E). Near the infarcted myocardium, there were areas of neoangiogenesis as could be expected after myocardial infarction. Some of this neovasculature contained labeled MSCs that expressed -smooth muscle actin in their cytoplasm, and some were integrated into

4 22 SAITO ET AL Ann Thorac Surg XENOGENEIC MSCs REGENERATE HOST MYOCARDIUM 2002;74:19 24 Fig 4. Histology and immunohistochemistry of the hearts with myocardial infarction. Mice marrow stromal cells were transplanted intravenously 1 week before coronary artery ligation. (A and B) Heart sample harvested at 8 weeks after ligation, and stained for -galactosidase activity and hematoxylin and eosin. Numerous mice marrow stromal cells were identified mostly in the infarcted myocardium. (C) Heart sample harvested at 1 day after ligation. LacZ mice marrow stromal cells (arrows) were seen near vessel (asterisk) in an intact area. (D) Heart sample harvested at 1 day after ligation. LacZ cell (arrow) was seen in the interstitium of an intact myocardium. (E) Heart sample harvested at 12 weeks after ligation. LacZ cell (arrow) was morphologically integrated into the scar formation. (F) Heart sample harvested at 12 weeks after ligation, and immunostained for -smooth muscle actin. LacZ cell (arrow) was seen in the vascular wall in the infarcted myocardium. (G) Heart sample harvested at 12 weeks after ligation, and immunostained for MF20. LacZ cells (arrows) had immunoreactivity for sarcomeric myosin heavy-chain molecules in their cytoplasms. (H) Heart sample harvested at 12 weeks after ligation, and immunostained for troponin I-C. LacZ cells (arrows) had immunoreactivity for troponin I-C in their cytoplasms. Original magnifications: (A) 100; (B) 200; (C through F) 400; (G and H) vessel walls (Fig 4F). Moreover, some MSCs in the infarcted area stained positively for the sarcomeric myosin heavy chain (Fig 4G) and for the cardiomyocytespecific protein, troponin I-C (Fig 4H). However, up until 2 weeks, the MSC-derived cells showed an immature appearance with a large nucleus to cytoplasm ratio and were negatively stained for these proteins. Comment In this study, we demonstrated that culture expanded xenogeneic MSCs were capable of homing into the bone marrow of immunocompetent adult recipients after systemic transplantation. The apparent lack of immune response indicated by the persistence of mice MSCs in the rats is intriguing. Although the murine MSCs have been reported to express major histocompatibility complex class I on their cell surfaces and lack class II [11], the tolerance of xenogeneic cells in a fully immunocompetent recipient is perplexing according to the classic self non-self immune response paradigm of Burnet [12] and Billingham and colleagues [13]. Nevertheless, our findings could be consistent with the prediction of the more recent danger model of immune tolerance proposed by Matzinger [14] and Anderson and Matzinger [15]. This model posits that the antigen-presenting cells require the presence of danger signals as costimulant with the antigen to induce immune activation of T cells. Antigens presented without concomitant danger signal should result in tolerance. Thus minimal tissue damage associated with injection of mice MSCs in our experiments may have allowed for their survival. Recent in vitro and in vivo studies also reveal MSCs can exert multiple effects directly on the immune system [11], although precise

5 Ann Thorac Surg SAITO ET AL 2002;74:19 24 XENOGENEIC MSCs REGENERATE HOST MYOCARDIUM 23 mechanisms are still poorly understood. Most intriguing is the finding that MSCs can suppress the function of mature T cells, either directly or by stimulating suppressor T cells, and thus are tolerogenic. They also speculated that MSCs recruited to an area of tissue injury may downregulate inflammatory response to initiate tissue repair. But even if the MSCs are immune privileged, why do they not get rejected when they have differentiated? Perhaps by the time they differentiate, which may take several weeks, the danger signals from myocardial infarction have subsided. Clearly such speculations need to be confirmed by future studies. Nevertheless, the therapeutic importance of immunologic tolerance of adult stem cells cannot be overemphasized. This study demonstrated the tolerogenic properties of xenogeneic cells, and there is some evidence that allogeneic cells behave similarly [16]. The rationale for therapeutic cloning to obtain embryonic stem cells, a highly controversial issue, is to avoid immune rejection of these cells by the recipients. The reason for using autologous myoblasts and MSCs, which is currently undergoing early clinical trials, is that autologous cells can avoid immune rejection. However, use of autologous cells faces the disadvantages of a time lag between cell harvesting and implantation as well as the logistic complexity to transport them to and from a central laboratory for quality control and for isolation and expansion of donor cells. In contrast, use of tolerogenic allogeneic or xenogeneic adult stem cells will avoid the need for cloning, and frozen donor cells can be supplied to be readily available for clinical use without delay. Another unique characteristic of MSCs is their capacity to migrate to an injured site in the body [17, 18]. To assess this capability in xenogeneic MSCs, we created myocardial infarction in rats by ligating the left coronary artery 1 week after mice MSCs had been intravenously transplanted. Although sham-operated hearts did not contain mice MSCs, the hearts with myocardial infarction had MSCs in the heart tissues. Interestingly, in the hearts harvested 1 day after coronary artery ligation, most of the labeled MSCs were found in the perivascular zone of noninfarcted myocardium, possibly because the coronary artery that supplied the infarcted area had been permanently occluded. However, in the hearts obtained after 2 weeks, most of the labeled MSCs were seen in the infarcted myocardium, suggesting they had migrated to the injured site. To further confirm our suggestion that these MSCs migrated from bone marrow to the heart through the bloodstream, we collected blood samples just before coronary artery ligation and 1 day and 2, 4, 8, and 12 weeks after coronary artery ligation. Mice MSCs could be detected only in the blood collected 1 day after ligation. Such findings appear to be consistent with the scenario that a signal or signals were released from the damaged tissue shortly after injury, initiating MSCs recruitment from the bone marrow and their migration to the injured area by way of the bloodstream. To examine the normal physiologic response of MSCs to tissue injury in vivo, we did not artificially purify these cells to clonal subgroups before their transplantation in this study. Thus the multiple phenotypes expressed by these cells at the infarct sites may represent the existence of progenitor cells for different lineages in this cell population or the multipotential adult stem cells responding to different in situ signals in the microenvironment [17, 19]. Although the sample size of hearts examined at each point in this study is modest, it should be noted that experimental animals sacrificed at various times after myocardial infarction (n 15) were all positive for implanted labeled cells without signs of rejection. When X-gal stain for -galactosidase is performed at a ph of 7.8 to 8.0 as described above [10], no false-positive stain of myocardial scar has been seen, not only in the 5 control animals in this study, but also in all sham-operated control animals in other series of experiments that we had previously reported [20]. In summary, our findings indicate that (1) xenogeneic MSCs can home in and survive in the bone marrow cavities of hosts without immunosuppression, (2) they are capable of being recruited to the injured myocardium through the bloodstream, and (3) they can differentiate into various phenotypes such as vascular smooth muscle cells and cardiomyocytes in the infarcted myocardium, resulting in a stable cardiac chimera without the need for immunosuppressive therapy. How such a chimera may affect cardiac function is an interesting and clinically relevant question that deserves further investigation. We deeply appreciate the technical assistance of Minh Duong, BS. References 1. Johnstone B, Hering TM, Caplan A, Goldberg VM, Yoo JU. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998;238: Ferrari G, Cusella-De Angelis G, Coletta M, et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998;279: Peterson B, Bowen W, Patrene K, et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284: Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve 1995;18: Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 1999;103: Wang JS, Shum-Tim D, Galipeau J, Chedrawy E, Eliopoulos N, Chiu RCJ. Marrow stromal cells for cellular cardiomyoplasty: feasibility and potential clinical advantages. J Thorac Cardiovasc Surg 2000;120: Devine SM, Bartholomew AM, Mahmud N, et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol 2001;29: Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymal stem cells engraft and demonstrate sitespecific differentiation after in utero transplantation in sheep. Nat Med 2000;11: Wang JS, Shum-Tim D, Chedrawy E, Chiu RCJ. The coronary delivery of marrow stromal cells for myocardial regeneration: pathophysiologic and therapeutic implications. J Thorac Cardiovasc Surg 2001;122:

6 24 SAITO ET AL Ann Thorac Surg XENOGENEIC MSCs REGENERATE HOST MYOCARDIUM 2002;74: Al-Khaldi A, Lachapelle K, Galipeau J. Endogenous -galactosidase enzyme activity in normal tissues and ischemic myocardium: a comparison study with prokaryotic -galactosidase reporter enzyme detection. Card Vasc Regeneration 2000;1: McIntosh K, Bartholomew A. Stromal cell modulation of the immune system. A potential role for mesenchymal stem cells. Graft 2000;3: Burnet FM. The clonal selection theory of acquired immunity. Nashville, TN: Vanderbilt University Press, Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature 1953;172: Matzinger P. An innate sense of danger. Semin Immunol 1998;10: Anderson CC, Matzinger P. Danger: the view from the bottom of the cliff. Semin Immunol 2000;12: Caparrelli DJ, Cattaneo S, Shake JG, et al. Cellular cardiomyoplasty with allogeneic mesenchymal STEM cells results in improved cardiac performance in a swine model of myocardial infarction. Circulation 2001;104(Suppl):II Chen J, Li Y, Wang L, Zhang Z, et al. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke 2001;32: Bittner RE, Schofer C, Weipoltshammer K, et al. Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice. Anat Embryol 1999;199: Orlic D, Kajstura J, Chimentl S, et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410: Bittira B, Kuang J-Q, Piquer S, Shum-Tim D, Al-Khaldi A, Chiu RCJ. The pathophysiological roles of bone marrow stromal cells (MSCs) in myocardial infarction. Circulation 2001;104(Suppl)II-523. INVITED COMMENTARY In this very interesting paper from Dr Ray Chiu s group, Saito and colleagues report that intravenously-injected mouse bone marrow stromal cells successfully engrafted into the bone marrow of rats in the absence of any immunosuppressive therapy. Furthermore, following ligation of one of the rat s coronary arteries, mouse cells could be detected in the infarcted myocardium. In similarly treated rats that had not undergone coronary ligation, mouse cells could not be identified in the heart. The conclusion drawn is that the marrow stromal cells were adult stem cells with a unique tolerance to immunologic injury. This allowed their engraftment in a xenogeneic environment while preserving their ability to be recruited to an injured myocardium (via the bloodstream) and to undergo differentiation to form a stable cardiac chimera. These results are certainly remarkable but, as with all experimental studies, leave many questions unanswered. How did the mouse cells escape immunologic injury? The authors invoke the danger model hypothesis that is currently topical, and suggest that the cells might not have presented danger signals to the rat recipient, and thus escaped immune destruction. However, one would anticipate that the presence of any foreign cells would be interpreted by the host as being potentially dangerous. Why were xenogeneic cells chosen for infusion, and not allogeneic or autologous cells? The authors discuss the logistic disadvantages of the use of autologous cells, but do not adequately explain why they chose xenogeneic over allogeneic cells. One could hypothesize that xenogeneic cells are so different that they escape an immune response, but this has not been the case in other mouseto-rat models. Their failure to be injured by the host, therefore, must presumably be related to the nature of the stem cells used. Was the number of mouse cells detected in the infarcted rat myocardium sufficient to have a clinically-useful effect on its recovery or on the strength of its coordinated myocardial contractions? No truly quantitative data are provided but, from the paper s excellent histochemical figures, the answer is probably not. Nevertheless, despite the intriguing and, as yet, unanswered questions, the study provides some encouragement that we shall one day not only be able to treat myocardial infarction by a cell therapy approach, but that we may also be able to overcome the barriers to xenotransplantation. David K. C. Cooper, MD Transplantation Biology Research Center Massachusetts General Hospital Harvard Medical School Boston, MA by The Society of Thoracic Surgeons /02/$22.00 Published by Elsevier Science Inc PII S (02)