Progenitor Cells From the Explanted Heart Generate Immunocompatible Myocardium Within the Transplanted Donor Heart
Rationale: Chronic rejection, accelerated coronary atherosclerosis, myocardial infarction, and ischemic heart failure determine the unfavorable evolution of the transplanted heart in humans.
Objective: Here we tested whether the pathological manifestations of the transplanted heart can be corrected partly by a strategy that implements the use of cardiac progenitor cells from the recipient to repopulate the donor heart with immunocompatible cardiomyocytes and coronary vessels.
Methods and Results: A large number of cardiomyocytes and coronary vessels were created in a rather short period of time from the delivery, engraftment, and differentiation of cardiac progenitor cells from the recipient. A proportion of newly formed cardiomyocytes acquired adult characteristics and was integrated structurally and functionally within the transplant. Similarly, the regenerated arteries, arterioles, and capillaries were operative and contributed to the oxygenation of the chimeric myocardium. Attenuation in the extent of acute damage by repopulating cardiomyocytes and vessels decreased significantly the magnitude of myocardial scarring preserving partly the integrity of the donor heart.
Conclusions: Our data suggest that tissue regeneration by differentiation of recipient cardiac progenitor cells restored a significant portion of the rejected donor myocardium. Ultimately, immunosuppressive therapy may be only partially required improving quality of life and lifespan of patients with cardiac transplantation.
Successful cardiac transplantation has an inherent yearly 5% mortality and most patients die within 10 to 12 years.1 The improved immunosuppressive regimen has resulted in increased short-term survival, but long-term survival has remained largely the same. Postoperative morbidity and mortality is dictated by acute and chronic complications which rapidly affect the performance of the new heart or lead with time to a progressive deterioration of cardiac function. Chronic rejection with accelerated cardiac allograft vasculopathy (CAV) is the major pathological event that determines the fatal evolution of the transplanted heart.2 Although the mechanisms implicated in CAV are unclear, an immune response is involved.3 Graft failure attributable to CAV is characterized by occlusive vessel disease that results in myocardial infarction, focal areas of injury, arrhythmias, sudden death, and congestive heart failure.3 Three interrelated pathological processes appear to be the critical determinants of heart failure in the transplanted heart: rejection, evolving coronary atherosclerosis, and chronic ischemic myocardial injury.4
So far, the explanted heart of the recipient has been used for the characterization of the underlying disease and for research purposes but never for treatment of the donor heart. Evidence in animals and humans indicates that resident c-kit–positive cardiac progenitor cells (CPCs) possess the ability to regenerate cardiomyocytes and coronary vessels in models of acute and chronic ischemia.5–8 CPCs of autologous origin may create healthy immunocompatible myocardium and coronary vessels which may replace extensive portions of the donor heart. The newly formed myocardium will be biologically and functionally similar to the original heart and, with time, may largely replace the transplant creating an organ in which the risks for severe coronary atherosclerosis, vessel occlusion, and ischemic injury may be significantly reduced. The growth potential of CPCs raises the possibility that these cells, if properly implemented, may have a unique therapeutic import for the transplanted heart. Here, we document that this strategy is feasible and may interfere with the dramatic evolution of the cardiac allograft.
Sex mismatched cardiac transplantation was performed in male dogs and the explanted male heart was used to collect CPCs which were then infected with a lentivirus expressing enhanced green fluorescent protein (EGFP). A catheter was inserted into the coronary arteries and male CPCs were delivered to the female donor heart.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Pathology of the Transplanted Heart
The dog model of cardiac transplantation was chosen because of its important preclinical implications. Immunosuppression is well established in dogs,9 and canine c-kit–positive CPCs have been characterized.6 In all cases, a female donor heart was transplanted in a male recipient (Online Figure I). CPCs obtained from the recipient heart were labeled with EGFP and analyzed by FACS and immunocytochemistry (Online Figure II). Male CPCs to be injected did not express hematopoietic markers and were largely uncommitted. The Y chromosome and EGFP were used as markers of the progeny of the injected cells in the donor heart.8,10,11
Transplanted dogs in the absence of CPC administration (n=4) were obtained from 3 to 22 days after surgery. In spite of immunosuppressive therapy, cardiac function deteriorated rapidly and death occurred. Tissue and vascular damage were apparent in the entire allograft, from base to apex, resulting in disruption of the structural integrity of the donor myocardium. Cardiac pathology consisted of foci of myocyte death and scattered myocyte apoptosis and necrosis, together with extensive lymphocytic infiltrates positive for CD19 and CD20 (B lymphocytes; 39/mm2) and CD4 and CD8 (T lymphocytes; 29/mm2). Monocytes and macrophages (CD14-positive; 18/mm2) were present throughout the myocardium. Frequently, the inflammatory reaction surrounded the intramural branches of the coronary arteries and produced local injury of the vessel wall with thrombus formation (Figure 1A through 1E; Online Figure III and IV).
Typically, myocytes were lost and collagen accumulated with time. Tissue fibrosis was frequently found in proximity of coronary vessels (Figure 1E; Online Figure III). Vessel formation is a typical aspect of healing following myocardial damage and the inflammatory response. Collagen increased from 1.4% at 3 days to 2.0%, 11%, and 16% at 5, 21, and 22 days after transplantation, respectively. From 3–5 to 21–22 days following surgery, the size distribution of scarred lesions was shifted to higher values. Similarly, the dimension of acute inflammatory foci increased with time (Online Figure III). Tissue injury involved 9% of the myocardium at 3–5 days and 20% at 21–22 days, defining the characteristics of the animal model and the progression of cardiac damage.
CPC Treatment of the Transplanted Heart
In a separate group of animals (n=4) in which cardiac performance was maintained for ≈3 weeks after transplantation, we tested the possibility to generate immunocompatible myocardium within the donor heart and document whether this intervention attenuated the extent of damage restoring in part the integrity of the organ. Echocardiographic recordings were obtained at the time of CPC administration and thereafter to assess cardiac function over time (Online Figure V; Online Movie I). We elected the intracoronary route of CPC delivery to obviate the need for multiple survival surgeries that would have been required with intramyocardial injections of cells. Additionally, the multifocal nature of myocardial injury would have benefited from a widespread engraftment of CPCs. A premise for this approach was the documentation that CPCs traverse the wall of coronary vessels and reach the myocardium.12
Under fluoroscopy, the catheter was inserted into the left or right coronary artery, and recipient CPCs suspended in Isopaque contrast medium were injected into the left and then into the right coronary artery. The contrast allowed the visualization of the coronary circulation and the proper delivery of cells (Online Figure V). By trypan blue staining, Isopaque did not affect the viability of CPCs (Online Figure VI). During the procedure, CPCs were exposed to Isopaque for less than 5 minutes.
The first injection of cells occurred 15 to 24 days after surgery. This time was necessary for the isolation, expansion, and EGFP infection of recipient CPCs. Although damage occurred during this period, CPCs have the potential to invade and replace scarred myocardium.13 An additional injection of CPCs was performed in 2 dogs ≈1 week later, whereas the other 2 dogs received 3 more injections at approximately weekly intervals. Each CPC delivery consisted of ≈7×106 cells. In all cases, there were no functional alterations associated with cell infusion acutely and chronically (Online Figure V). Animals were euthanized 6, 23, 24, and 30 days after the last cell administration and hearts collected at 30, 45, 70 and 73 days after transplantation (Online Figure VII).
Repair of the Transplanted Heart
To evaluate quantitatively the effects of recipient CPCs on the cardiac allograft, each heart was sliced in 19 to 22 sections, ≈4 mm thick, and several samples were obtained from each section (Online Figure VIII). This analysis included the left and right ventricle and interventricular septum: an average of 117±7 specimens were processed in each heart and examined histologically; 356±130 cm2 of myocardium were sampled. Acute areas of rejection and foci of myocardial scarring were identified throughout the heart.
Clusters of newly formed EGFP-positive myocytes together with coronary vessels were detected in 115±7 specimens of the transplanted heart. The structures of recipient origin were invariably distributed within areas of acute and chronic tissue injury, replacing partly the lost donor myocardium (Figure 2A through 2C; Online Figure IX). These sites ranged from 800 μm2 to 29.3 mm2 of the donor heart. For quantitative purposes, each heart was used as its own control. In this complex model, it is impossible to collect hearts at the same time points. Any type of noticeable discomfort mandates euthanasia. The progression of damage and its repair is the only therapeutically relevant approach that can be used to assess the evolution of the transplanted heart in large mammals.
Three measurements were obtained morphometrically in each case: (1) number of foci of tissue damage per square millimeter of myocardium; (2) size of damage foci; and (3) percentage of damaged myocardium. The latter is the product of the other 2 variables. Because the regenerated myocardium was represented by EGFP-labeled cardiomyocytes and coronary vessels located within areas of injury, the number and size of regeneration foci and the percentage of reconstituted myocardium was determined. This information was used to establish the therapeutic effects of CPCs on myocardial injury, ie, damage minus regeneration. CPC-mediated cardiac repair resulted in a marked decrease in the number of damage foci, and, when regeneration occurred, almost the entire site of injury was replaced by newly formed tissue. Collectively, myocardial injury in the transplanted heart was decreased by >50% by CPC treatment (Figure 2D).
Cardiac pathology in untreated dogs at 21 to 22 days after surgery involved 20% of the myocardium. In contrast, in dogs injected with CPCs and euthanized 30 to 73 days after transplantation, only 6% of unrepaired tissue was detected. Scarring accounted for 14% and 5% of the donor myocardium in untreated and treated dogs, respectively. Although these values cannot be directly compared, data in cell-treated hearts suggest that damage was attenuated by delivery of recipient CPCs. However, original tissue injury was most likely less severe in treated than in untreated dogs which died shortly after surgery.
Differentiation of Recipient CPCs
The ability of EGFP-CPCs to commit to the myocyte, smooth muscle cell (SMC), and endothelial cell (EC) lineage was evaluated first by the detection of transcription factors specific of myocytes (GATA4 and Nkx2.5), SMCs (GATA6), and ECs (Ets1) (Online Figure X). The engraftment and survival of male EGFP-positive CPCs was confirmed by polymerase chain reaction (PCR) for EGFP DNA and the Sry gene located in the Y chromosome (Figure 3A and 3B; Online Figure XI). The positivity for the Sry gene, however, cannot exclude that some male circulating cells homed to the myocardium, pointing to the presence of EGFP DNA as the most accurate assay for this analysis. Random sampling of tissue from kidney, spleen, lung, and liver did not reveal DNA sequences for EGFP. These observations indicate that in the absence of tissue damage progenitor cells may be initially retained within distant organs but failed to undergo long-term engraftment with host cells dying by apoptosis/anoikis. The importance of myocardial damage and its milieu for the engraftment and survival of CPCs has previously been shown.5,11
Differences were noted when the 2 dogs, which received 2 CPC injections and were euthanized 30 and 45 days after surgery, were compared with the 2 dogs, which received 4 CPC injections and were studied at 70 and 73 days after transplantation. At the earlier time points, the newly formed myocytes were small and resembled fetal-neonatal cells (Figure 3C). Conversely, at the later intervals, some of the EGFP-positive myocytes reached the adult phenotype and were indistinguishable from resident cells (Figure 3D).
Regions of complete cardiac repair were found together with areas of rather immature myocytes, suggesting that tissue replacement was ongoing and these cells represented, respectively, myocytes formed earlier and later in the regeneration process (Figure 4A through 4D). This possibility was confirmed by the detection of the cell cycle protein Ki67 in most of the developing myocytes (Online Figure XII). Mature myocytes were predominantly Ki67 negative and expressed connexin 43 and N-cadherin (Figure 4C and 4D). Additionally, small coronary vessels and large conductive arteries of recipient origin were detected. Several vessels contained red blood cells documenting that they were connected with the primary coronary circulation (Figure 5A through 5D). The new coronary vasculature was composed of EGFP-positive SMCs and ECs. CPCs have the ability to invade scarred tissue by secreting matrix metalloproteinases and forming tunnels defined by fibronectin.13 Subsequently, both coronary vessels and cardiomyocytes are generated within the fibrotic myocardium.
Myocardial regeneration was 2.7-fold greater at the later than at the earlier intervals, resulting in 73% and 27% replacement of damaged tissue, respectively. In the 4 transplanted hearts, there were 1.17±0.24×109 newly formed myocytes with an average volume of 1400±500 μm3; 7% of regenerated myocytes had a volume of 5000 μm3 or larger. In the older dogs, 4% of myocytes were 10 000 μm3 or larger. Vascular growth included coronary vessels from large arteries, 0.3 to 1.2 mm in diameter, to capillaries (Online Figure XIII). However, this structural organization points to the rather immature phenotype of the regenerated myocardium.14 The specificity of EGFP labeling of regenerated cardiomyocytes and coronary vessels was confirmed by spectral analysis which discriminated background tissue autofluorescence from the native fluorescence of EGFP (Figure 6A through 6D).
To account for this magnitude of myocyte formation, it is relevant to indicate that ≈20 000 CPCs were isolated from each explanted heart; ≈9 population doublings were required to obtain ≈10×106 CPCs; 7×106 were injected and the remaining ≈3×106 cells were expanded further. For each subsequent delivery, 1 to 2 additional population doublings were necessary. To reach the final number of regenerated cells in the transplanted heart, 1.17×109, 7 population doublings were needed if 2 injections were made, and 6 population doublings were involved if 4 injections were performed. Assuming that 10% of delivered CPCs engrafted in the donor heart, 3 population doublings had to take place to compensate for the 90% loss of cells; ≈20 population doublings may have occurred throughout the process. We have shown that average telomere length in the dog heart is 14 kbp and each population doubling leads to 130-bp telomere attrition.8,15 Twenty population doublings would result in a shortening of telomeres of ≈2.5 kbp. This degree of telomere shortening does not lead to replicative senescence and growth arrest which occurs when telomere reach 1.5 to 2.0 kbp.16
Because the infection efficiency of CPCs with the EGFP lentivirus varied from 50% to 80%, the recognition of EGFP-positive structures represented an underestimation of the actual amount of regenerated recipient myocardium. The detection of the Y chromosome confirmed the male genotype of EGFP-positive myocytes and vessels and identified areas of regeneration derived from male EGFP-negative CPCs (Figure 7A through 7C). However, we cannot exclude that bone marrow cells from the recipient contributed to the formation of male EGFP-negative structures within the female myocardium. Whether activated resident CPCs resulted in the formation of donor myocardium in response to injury is a likely possibility.
The presence of inflammation in areas of cardiac repair was evaluated for the recognition whether the regenerated recipient myocardium was affected by the rejection process of the organ or the new structures were protected from the immune response. Tissue sections containing clusters of EGFP-positive/Y chromosome–positive myocytes and coronary vessels were labeled for CD19, CD20, CD4, CD8, and CD14. B lymphocytes, T lymphocytes, monocytes, and macrophages were absent in the regions of myocyte and vessel formation (Online Figure XIV), suggesting that the regenerated structures were not attacked by the immune system of the organism. The foci of cardiac regeneration induced by CPCs created restricted immunoprivileged areas of tissue repair but did not prevent the accumulation of inflammatory cells in the distant donor myocardium. This is consistent with the ability of transplanted CPCs to reduce locally the level of proinflammatory cytokines in proximity of acute myocardial injury,13 mimicking the immunomodulatory activity of mesenchymal stem cells. The numbers of B lymphocytes (34/mm2), T lymphocytes (28/mm2), and monocytes and macrophages (20/mm2) in the myocardium remote from the regenerated tissue were similar to those found in untreated donor hearts (see above).
A molecular-based histocompatibility typing protocol was developed to document the presence of recipient myocardium within the donor heart. The polymorphism of the dog leukocyte antigen (DLA)-DRB1 was analyzed.17,18 DLA-DRB1 was chosen because it represents the most highly polymorphic class II locus. Importantly, this DLA-class II antigen was detected by quantitative RT-PCR in CPCs and isolated canine cardiomyocytes from explanted hearts (Online Figure XV). DNA was extracted from the liver of a recipient dog and from EGFP-positive and EGFP-negative cardiomyocytes and coronary vessels present in tissue sections of the paired donor transplanted heart. Following amplification of the highly polymorphic exon 2, PCR products were subcloned into plasmids to obtain separately maternal and paternal alleles; the alleles were then subjected to DNA sequencing.
The alleles of DLA-DRB1 gene present in the regenerated EGFP-positive myocardium were identical to those of the liver of the recipient dog. Conversely, a different combination of alleles was found in the DNA from the EGFP-negative donor myocardium (Figure 8A; Online Figure XVI, A). Although we cannot exclude that some circulating EGFP-negative cells from the recipient homed the heart and may have participated in cardiac repair,19 the lack of recipient alleles in the EGFP-negative donor myocardium strongly suggests that this potential confounding factor did not alter the validity of the protocol. Thus, our results document that CPCs generated immunocompatible myocardium within the nonimmunocompatible heart.
We have identified a previously unrecognized allele (GenBank accession no. FJ415066) of the DRB1 gene in EGFP-positive myocardial structures and recipient liver. This is not surprising in view of the high degree of polymorphism of DRB1.17,18 The possibility of a PCR error was excluded by using a proof-reading polymerase in the first and second PCR rounds. If a PCR error occurred in the third PCR round, the likelihood of detecting exclusively incorrect amplicons was very low; an identical sequence was found in samples from 2 different organs, making it extremely improbable that the same substitution of nucleotides occurred at the same location of the PCR product in separate reactions. More than 20 PCR clones derived from the 2 samples shared identical sequence. Conversely, the sequences of the 3 registered alleles (DRB006, DRB25, and 03701), which share a high degree of homology with the new allele, were never found. The difference between the new and the 3 other alleles involved 4 nucleotides which were shared in various combinations (Online Figure XVI, B), documenting further that the mismatched nucleotides did not represent the product of amplification error. Modifications in the 4 nucleotides resulted in distinct amino acid sequences (Online Figure XVI, C). Thus, the variance in the newly characterized DNA sequence provides a relevant immunologic antigen of DRB1.
An important question was whether the newly formed myocardium was the product of fusion events. The merge of a recipient CPC with a differentiated myocyte or vascular cell would result in the formation of a binucleated heterokaryon or a mononucleated hyperploid synkaryon.20 However, the destiny of the hybrid cell would be regulated by the nucleus of the mature cell, which would acquire the ability to divide. To evaluate this possibility, DNA content per nucleus was determined in EGFP-positive myocytes, SMCs and ECs. In all cases, 2n DNA content was found in each of these cell populations excluding cell fusion (Online Figure XVII). Most importantly, only 1 Y chromosome and 1 X chromosome were detected in EGFP-positive myocytes (Figure 8B through 8D), and SMCs and ECs organized in coronary vessels showing their diploid male genotype. Conversely, the EGFP-negative donor cells showed at most 2 X chromosomes reflecting a diploid female genotype. Thus, CPCs from the recipient form de novo myocardium independently from cell fusion.
The results of the present study raise the possibility that the major problems associated with the unfavorable evolution of cardiac transplantation in humans21,22 may be corrected partly by a novel strategy that implements the use of CPCs from the recipient as a source of multipotent progenitors capable of repopulating the donor heart with immunocompatible cardiomyocytes and coronary vessels. Rejection and severe atherosclerosis with marked thickening of the vessel wall develop with time in the transplanted heart and together lead to a progressive defect in myocardial perfusion, scarring, and ischemic heart failure.23 Human CPCs can form functionally competent myocardium8 and may offer an alternative or complementary treatment of the cardiac allograft acutely and chronically.
To obtain information with clinical import, a model of cardiac transplantation in dogs was developed and our initial data suggest that nearly 50% of the rejected nonimmunocompatible donor myocardium was restored by immunocompatible tissue. A large number of cardiomyocytes and coronary vessels were created in a rather short period of time from the delivery, engraftment, and differentiation of CPCs from the recipient. Although most cardiomyocytes possessed an immature phenotype, a proportion of these cells acquired adult characteristics and was integrated structurally and functionally within the transplant. Similarly, the regenerated large, intermediate, and small coronary arteries and arterioles and capillary network were operative and contributed to flow distribution and oxygenation of the chimeric myocardium. The attenuation in the extent of acute damage by repopulating cardiomyocytes and vessels decreased significantly the magnitude of myocardial scarring preserving partly the integrity of the donor heart.
The interaction between donor and recipient cells after transplantation has received great attention in an attempt to identify the basis of rejection and graft-versus-host disease.24 Cell migration from the allograft to the recipient results in systemic chimerism, whereas cell migration from the host to the transplant results in chimerism of the organ.24 Several studies on cardiac chimerism following sex-mismatched organ transplantation have provided consistent results concerning the migration of progenitor cells from the host to the graft.25–27 Although there is little disagreement on the occurrence of this phenomenon, the magnitude of cardiac chimerism varies significantly in different reports and the discrepancy involves not only cardiomyocytes but also coronary vessels.25,26 Additionally, the origin of the host progenitor cells continues to be a matter of debate. However, in all cases, cardiomyocytes of recipient origin never replaced areas of damaged myocardium and the newly formed vessels were small and, thereby, incapable of attenuating the devastating consequences of the allograft vasculopathy. The approach used here experimentally may overcome, in part, these limitations leading to the generation of a degree of immunocompatible myocardium which may have important clinical consequences.
A relevant question on cardiac chimerism and the formation of myocardial structures by adult stem cells is whether tissue regeneration is a primary event mediated by progenitor cell commitment and differentiation or a secondary process triggered by the merging of exogenous stem cells with resident parenchymal cells, resulting in the formation of hybrid cells which then divide.20 The formation of heterokaryons that contain both donor and recipient genes would be a required intermediate step for the subsequent activation of cell growth and the generation of a differentiated progeny. The newly formed myocardium would be of donor origin and thereby nonimmunocompatible, raising critical problems as to the therapeutic efficacy of CPCs from the recipient. In this study, the possibility that regenerated myocytes and vessels were the product of fusion events between CPCs from the recipient and myocytes, SMCs, and ECS, from the donor has been excluded. Our strategy appears to be successful; it does not involve cell fusion and the reconstituted myocardium is comprised of immunocompatible coronary vessels and cardiomyocytes, which largely replace the nonimmunocompatible diseased arteries and myocytes. The outcome is the coexistence of recipient-derived cells with donor-derived cells which together participate in the preservation of myocardial function in the transplanted heart.
In the present work, a normal male dog was transplanted with a normal female heart immediately after dissection. The explanted heart from the recipient had an intact CPC reserve, which facilitated the collection and expansion of these cells for therapy. The scenario is much more complex in humans with terminal cardiac failure in which the poor condition of the patient is dictated by the extensively damaged heart. The number of functionally competent CPCs is reduced in the severely decompensated human heart but portion of healthy myocardium are present and contain a restricted pool of CPCs which express telomerase and have long telomeres.28 Based on recent results, it appears that this restricted CPC compartment can be isolated and expanded in vitro from samples of diseased hearts collected from patients undergoing extracorporeal circulation and complex cardiac surgery.8 These human CPCs possess a significant growth reserve and repopulate the infarcted heart with cardiomyocytes and coronary vessels integrated within the recipient myocardium.8 Additionally, large conductive coronary vessels of human origin can be formed in immunosuppressed dogs with critical functional stenosis.29 Thus, CPCs may be obtained from the explanted human heart for subsequent delivery to the donor myocardium.
Caution has to be exercised in the interpretation of the present findings. Long-term studies remain to be performed to establish whether the donor heart actually provides the scaffolding for the generation of a new heart in which recipient-derived cells become the predominant component of the allograft with minimal quantities of donor-derived cells. Theoretically, CPCs and other progenitor cells capable of acquiring the cardiomyocyte and vascular lineage can be delivered locally and repeatedly to the donor heart over the years. These cells may repair areas of damage resulting from rejection and ischemic injury and may promote the progressive replacement of donor myocardium with structures of recipient origin. Ultimately, the recipient-derived myocardium may govern the behavior of the transplanted heart. Immunosuppressive therapy may be only partially required improving quality of life and lifespan of patients after cardiac transplantation.
Sources of Funding
This work was supported by NIH grants. S.B. was supported by a grant from Cardiocentro Ticino, Lugano, Switzerland.
Original received August 14, 2009; revision received September 30, 2009; accepted September 30, 2009.
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