Embryonic Stem Cell Immunogenicity Increases Upon Differentiation After Transplantation Into Ischemic Myocardium

Abstract

Background— We investigated whether differentiation of embryonic stem cells (ESCs) in ischemic myocardium enhances their immunogenicity, thereby increasing their chance for rejection.

Methods and Results— In one series, 129/SvJ-derived mouse ESCs (ES-D3 line) were transplanted by direct myocardial injection (1×106 cells) into murine hearts of both allogeneic (BALB/c, n=20) and syngeneic (129/SvJ, n=12) recipients after left anterior artery ligation. Hearts were procured at 1, 2, 4, and 8 weeks after ESC transplantation and analyzed by immunohistochemistry to assess immune cell infiltration (CD3, CD4, CD8, B220, CD11c, Mac-1, and Gr-1) and ESC differentiation (hematoxylin and eosin). In a second series (allogeneic n=5, sham n=3), ESC transplantation was performed similarly; however after 2 weeks, left anterior descending artery-ligated and ESC-injected hearts were heterotopically transplanted into naive BALB/c recipients. After an additional 2 weeks, donor hearts were procured and analyzed by immunohistochemistry. In the first series, the size of all ESC grafts remained stable and there was no evidence of ESC differentiation 2 weeks after transplantation; however, after 4 weeks, both allogeneic and syngeneic ESC grafts showed the presence of teratoma. By 8 weeks, surviving ESCs could be detected in the syngeneic but not in the allogeneic group. Mild inflammatory cellular infiltrates were found in allogeneic recipients at 1 and 2 weeks after transplantation, progressing into vigorous infiltration at 4 and 8 weeks. The second series demonstrated similar vigorous infiltration of immune cells as early as 2 weeks after heterotopic transplantation.

Conclusion— In vivo differentiated ESCs elicit an accelerated immune response as compared with undifferentiated ESCs. These data imply that clinical transplantation of allogeneic ESCs or ESC derivatives for treatment of cardiac failure might require immunosuppressive therapy.
Stem cell transplantation has recently emerged as a novel approach for replacement of injured myocardium. The potential of both hematopoietic stem cells, harvested from the adult bone marrow, and embryonic stem cells (ESCs), harvested from the inner cell mass of embryos at the blastocyst stage, to develop into viable heart muscle cells in the damaged heart is now being explored. Despite early encouraging results with hematopoietic stem cell transplantation,1 the ability of these cells to transdifferentiate into cardiomyocytes in vivo and to functionally repair ischemic myocardium has recently been seriously questioned.2,3 Instead, previous studies have reported successful differentiation of ESCs into cardiomyocytes, both in vitro4,5 and in vivo.6,7 This prompted us to investigate the potential of ESCs as a source of cell transplantation for myocardial restoration.
Pluripotent ESCs are capable of spontaneous differentiation into cells of all 3 different germ layers.8 Because of their early stage of development, ESCs are considered “immune privileged,” ie, unrecognizable by the recipient immune defenses.9 This assumption is bolstered by observations in neonatal tolerance. An embryo, consisting of 50% foreign material derived from the father, is usually not rejected by the maternal host. Recent evidence suggests, however, that even in their undifferentiated state, human ESCs express low levels of human leukocyte antigen class I antigen that moderately increase as the cells differentiate.10,11 In addition, rat embryonic stem cell-like cells also have been shown to express minimal, but detectable, levels of major histocompatibility complex (MHC) class I molecules.12 The presence of distinct MHC antigens suggests that developing ESCs can be at risk for immune rejection when introduced in vivo across histocompatibility barriers.13 However, no progressive studies analyzing the immune fate of ESCs during their development in vivo have been reported so far.
In this study, we tested the hypothesis that intramyocardially transplanted ESCs elicit an immune response that results in rejection of the transplanted cells. In addition, we investigated whether the state of cell differentiation affects their immunogenicity and the intensity of the recipient immune response.
Methods
Animals
Six- to 10-week-old female BALB/c (H-2d) and 129/SvJ (H-2b) mice (20 to 25 g) were purchased from The Jackson Laboratory (Bar Harbor, Me) and housed at no more than 5 per cage in our American Association for Accreditation of Laboratory Animal Care-approved facility with 12:12-hour light-dark cycles and free access to standard rodent chow and water. All animal procedures were approved by the Animal Care and Use Committee of Stanford University.
Cell Culture
ES-D3 embryonic stem cells, a line developed from the 129/SvJ mouse strain, were kindly provided by Dr I.L. Weissman (Department of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, Calif). Undifferentiated ES-D3 cells were maintained on gelatinized tissue culture dishes in Dulbecco’s modified Eagle medium (Specialty Media) supplemented with 15% fetal bovine serum (Hyclone), 100 μg/mL penicillin, 100 U/mL streptomycin, 1 mmol/L sodium pyruvate, 2 mmol/L L-glutamine, 1 × nonessential amino acids, 0.1 mmol/L 2-mercaptoethanol (all Gibco) and 103 U/mL ESGRO (Chemicon). Cells were maintained at 37°C in a humidified atmosphere of 5% CO2. Monolayers were passaged by trypsinization at confluence of 70% to 80%. At the day of transplantation, ESC were harvested and aliquoted in culture medium.
Left Coronary Ligation and ESC Transplantation
Mice were premedicated with ketamine (100 mg/kg intraperitoneally) and anesthetized with inhaled isoflurane (2% to 3%). Mice were intubated and ventilated with a mouse respirator (model 687, Harvard Apparatus, Inc) and anesthesia was maintained with inhaled isoflurane (1% to 2.5%). In each animal, a left thoracotomy was performed in the 5th intercostal space, the left lung was retracted, and the pericardium was opened. The left anterior descending (LAD) artery was permanently ligated with a 9-0 Ethilon suture just distal to the level of the left atrium. Infarction was visually confirmed by blanching of the anterolateral region of the left ventricle along with dyskinesia. After 5 minutes, 1.0×106 ESCs were transplanted by injection into the injured myocardium (volume 25 μL) of a series of allogeneic (BALB/c, n=20) and syngeneic (129/SvJ, n=12) recipients. Similar surgical procedure, with injection of cell free culture medium, was performed on sham control animals (BALB/c, n=8). A thoracostomy tube was placed and lungs were re-expanded using positive pressure at end expiration. The chest cavity was closed in layers with 5.0 Vicryl suture, and the animal was gradually weaned from the respirator. Once spontaneous respiration resumed, the endotracheal and thoracostomy tubes were removed, and the animals were placed in a temperature-controlled chamber until they resumed full alertness and motility.
Heterotopic Transplantation of LAD-Ligated and ESC-Injected Hearts
To study the immune response against in vivo matured ESCs, a second series of animals (allogeneic n=5, sham controls n=3) underwent LAD ligation and ESC transplantation; however, after 2 weeks, their hearts were explanted and heterotopically transplanted into the abdomen of naive syngeneic BALB/c recipients. Heterotopic cardiac transplantation was performed according to the method of Corry et al14 with some modifications. Anesthesia was induced and maintained as described above. Cardiac graft viability was assessed daily by abdominal palpitation.
Tissue Collection, Immunofluorescence, and Histological Analysis
Subsets of allogeneic, syngeneic, and sham-operated animals from the first series were euthanized at 1, 2, 4, and 8 weeks after ESC transplantation. Animals from the second series were euthanized at 2 weeks after heterotopic heart transplantation. Hearts were perfused with cold saline and rapidly excised. They were fixed in 2% paraformaldehyde for 2 hours at room temperature and cryoprotected in 30% sucrose overnight at 4°C. Tissue was frozen in optimum cutting temperature compound (OCT compound, Sakura Finetek USA, Inc) and sectioned at 5 μm on a cryostat. To evaluate inflammatory cell infiltration, immunostaining was performed with a panel of hematolymphoid antibodies. Serial sections were blocked and incubated with hamster anti-CD3 (clone G4.18), rat anti-CD4 (H129.19), rat anti-CD8 (53 to 6.7), rat anti-CD45R/B220 (RA3–6B2), hamster anti-CD11c (HL3), rat anti-Mac-1 (M1/70), or rat anti-Gr-1 (RB6–8C5) antibody (BD Pharmingen) for 1 hour at room temperature. Primary antibodies were detected by incubation of the slides with goat anti-hamster Texas Red (Santa Cruz Biotechnology Inc), goat anti-rat Alexa 488, or goat anti-rat Alexa 594 (Molecular Probes) for 45 minutes at room temperature. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Molecular Probes) and analyzed with a Leica DMRB fluorescent microscope (Leica Microsystems). Images were acquired with a SPOT RT Color camera and electronically merged with SPOT RT software (Diagnostic Instruments). To detect differentiated structures within the ESC grafts and to evaluate morphological changes of the left ventricular wall, sections were stained with hematoxylin and eosin, Masson’s trichrome, and Mucin stain (all Sigma-Aldrich Corp) according to established protocols and carefully analyzed by a pathologist blinded to treatment (H.V.).
Histological Evaluation
Shortly after immunofluorescent histology, sections were evaluated and graded a score for degree of inflammatory cell infiltration by 3 independent observers (R.J.S., M.T., and F.G.). Scores related to the following descriptions: – indicates absent, no infiltration;+/−, trace, few infiltrating cells; +, mild, scattered infiltrate or focal accumulation; ++, moderate, modest infiltrate progressing into diffuse; and +++, severe, intense, and diffuse cell infiltration.
Results
Transplantation of Undifferentiated Embryonic Stem Cells Elicits Progressive Inflammatory Graft Infiltration
After successful LAD ligation and ESC injection, ESCs were detected in all transplanted animals at 1 week after injection. (hematoxylin and eosin staining, Figure 1) Masson’s trichrome staining confirmed the location of ESCs within the infarcted left ventricular wall (Figure 2A). Immunofluorescent histological analysis demonstrated that allogeneic ES-D3 cell transplantation elicited progressive graft infiltration of various types of immune cells, involved in both adaptive and innate types of immunity. As shown in Figure 2B, immune cells strictly infiltrated the area of infarction and ESC transplantation. The Table summarizes the immunohistology data obtained over the 8-week time course.
Figure 1. Intramyocardial engraftment of ESCs. Hematoxylin and eosin staining of the infarcted left ventricular wall revealed ESC grafts in all injected animals at 1 week after transplantation. Arrows indicate th borders of the ESC graft. Original magnification: 200×.
Figure 2. ESC graft infiltration of immune cells after allogeneic transplantation. A, Masson’s trichrome staining shows the ESC graft surrounded by infarcted (blue) and healthy myocardium (red). B, Immunofluorescent staining of a corresponding section shows infiltration of CD4+ T cells (green) into the area of ESC transplantation. Note that the T cells are only found within the ESC graft. Healthy myocardium stains positive for the cardiomyocyte marker α-actinin (red). Counterstaining was performed with (blue). Original magnification: 200×. C, Representative higher magnification images taken from the area of ESC transplantation (as shown in B) of progressive ESC graft infiltration of CD3+, CD4+, and CD8+ T cells over time. At 4 and 8 weeks after transplantation, severe graft infiltration of T lymphocytes was observed. D, In comparison, limited numbers of infiltrating T lymphocytes were present in syngeneic ESC grafts at all time points. Original magnification: 400×.

Cellular Composition of Graft Infiltrates Over Time After Intramyocardial ESC Injection

At 1 week after injection, allogeneic ESC grafts displayed mild CD3+ T lymphocyte infiltration within the ESC allograft, which was composed predominantly of CD4+ T helper cells. At that time point, sparse CD4+ T cell clusters could be seen at the border of the graft area, whereas at weeks 4 and 8 after transplantation, massive infiltration of both CD4+ and CD8+ T cells was observed throughout the ESC graft, including the inner area (Figure 2C). Limited numbers of infiltrating T lymphocytes were present in syngeneic ESC grafts (Figure 2D) as well as in sham control hearts over the whole tested time period. These results show that over time, progressive T lymphocyte graft infiltration occurs after injection of allogeneic ESCs. The intensity of T cell infiltration depends on MHC incompatibility between donor ESCs and recipient. These data suggest that alloantigens presented by developing ESCs are recognized by allospecific host T cells.
Dendritic cell (DC) infiltration was absent in sham control hearts, showing that, at least at the time points tested, the performed surgical procedures did not cause non–antigen-specific DC activation/migration. Also, no or minimal numbers of infiltrating DCs were found in syngeneic hosts, demonstrating that MHC disparity is required for DC migration into the area of transplanted ESC grafts. Similar to alloreactive T cells, the numbers of graft-infiltrating DCs increased progressively over time in full-mismatched hosts, and peaked at 4 and 8 weeks after ESC transplantation. Overall, small numbers of ESC graft-infiltrating B cells were detected as compared with T cells.
Inflammatory cells mediating innate immunity, including macrophages and granulocytes, were also detected in the ESC graft area. Although more profound in the experimental (ESC injection) hearts, we found that macrophage and granulocyte cell infiltrates were also present in sham control hearts. This indicates that after transplantation, innate response was not exclusively related to ESC antigens but could also be due to surgical trauma after transplantation (LAD ligation and/or medium injection procedures).
Embryonic Stem Cells Differentiate Into Teratomas in Ischemic Myocardium
In both allogeneic and syngeneic ESC grafts, tumor formation was observed (Figure 3). Masson’s trichrome staining of the infarcted left ventricular wall showed intramural tumors over time increasing in size (Figure 4). At 1 and 2 weeks after transplantation, hematoxylin and eosin staining revealed no evidence of ESC differentiation. At 4 weeks, however, both in the allogeneic and syngeneic ESC grafts, differentiated structures originating from all 3 different germ layers could be observed (Figure 5A through 5D). At this time point, tumors could be characterized as teratomas. At 8 weeks, apart from little intramural cartilage, no differentiated or undifferentiated ESCs were found in the allogeneic hearts, whereas in the syngeneic hearts, both cell types were still present. This finding suggested that at 8 weeks after transplantation, the allogeneic ESC grafts had been destroyed as a result of the host inflammatory alloimmune response.
Figure 3. Transplantation of ESCs causes tumor formation. Tumors were found extending from the left ventricular wall after both syngeneic transplantation (A, white arrow) and allogeneic transplantation (B, black arrow). In sham operated hearts (C), as expected, no tumors were detected.
Figure 4. Morphological changes of left ventricular wall over time. Masson’s trichrome staining of heart sections at different time points after allogeneic (allo) and syngeneic (syn) ESC transplantation revealed intramural tumors over time increasing in size. Note that at 8 weeks after allogeneic transplantation, the allogeneic ESC grafts had been destroyed as a result of the host inflammatory alloimmune response. The left ventricular walls of the sham control hearts are shown in the lower panels.
Figure 5. Intramyocardially transplanted undifferentiated ESCs differentiate into teratomas. Four weeks after ESC transplantation, structures originating from all 3 different germ layers were found in both allogenic and syngenic recipient hearts. Hematoxylin and eosin staining shows formation of intramyocardial cartilage (A, mesodermal derivative, arrowheads, 200×), squamous epithelium (B, ectodermal derivative, 400×), and glandular epithelium (C, endodermal derivative, 400×). Secretion of mucus (pink) by the intramyocardial glands was confirmed by mucin staining (D, 400×).
Embryonic Stem Cell Immunogenicity Increases on Differentiation
In the present study, progressive host-anti ESC graft immunity correlated with ESC differentiation. Thus, differentiated structures could be found within the ESC grafts at 4 weeks after transplantation. At the same time, severe ESC graft infiltration of inflammatory cells was observed. Taken together, these findings suggest that once ESCs reach a more differentiated state, they can be more effectively recognized and rejected by the recipient immune system.
To further investigate this hypothesis, we designed a model to study the immune response against in vivo matured ESCs. A group of BALB/c mice underwent a similar procedure of myocardial infarction and allogeneic ESC transplantation. In this case, however, after 2 weeks, their hearts were explanted and heterotopically transplanted into naive syngeneic BALB/c recipients. Daily assessment of heterotopic cardiac grafts by abdominal palpation confirmed viability of the grafts throughout the study period. Interestingly, as early as 2 weeks after heterotopic transplantation of the isografts containing partly developed allogeneic ESC grafts, we found severe graft infiltration of various types of immune cells, including T and B-lymphocytes. (Figure 6 and the Table, right column) Therefore, ESCs that have matured and differentiated in vivo for 2 weeks elicit a more potent and immediate alloimmune response as compared with undifferentiated ESCs. These data indicate that the immunogenicity of ESC transplants increases on in vivo differentiation.
Figure 6. Graft infiltration of immune cells after transplantation of in vivo differentiated ESCs. Representative images of ESC graft infiltration of T (red) and B (green) lymphocytes at 2 weeks after undifferentiated ESC transplantation (A) versus 2 weeks after in vivo differentiated ESC transplantation through heterotopic transplantation of the LAD-ligated and ESC-injected hearts (B). In vivo differentiated ESCs elicited a vigorous and more immediate immune response as compared with undifferentiated ESCs. Counterstaining was performed with DAPI (blue). Original magnification: 400×.
Discussion
This study was designed to investigate whether ESCs elicit an immune response when transplanted into genetically identical or full MHC-mismatched ischemic myocardium. These data demonstrate that there is progressive infiltration by various types of inflammatory cells within the ESC graft after transplantation across histocompatibility barriers. Severe cellular invasion was observed 4 weeks after intramyocardial injection, followed by disappearance of the ESC allografts between 4 and 8 weeks after transplantation, presumably because of a robust alloimmune response. When transplanted into a naive recipient after 2 weeks of in vivo maturation, ESCs triggered an accelerated infiltration of immune cells, indicating that the immune response toward developing ESCs in allotransplant settings increases over time.
In contrast to tissue allografts, ESC transplants are devoid of highly immunogenic mature dendritic cells, or any other type of specialized antigen presenting cells. Thus, the transplanted cells do not express the MHC class II molecules required for effective priming of CD4+ alloreactive T cells through direct recognition.10,15 Therefore, the direct pathway of alloresponse is likely to be dominated by MHC class I-reactive CD8+ T cells, whereas the indirect alloreactive pathway (presentation of processed allogeneic peptides by host antigen presenting cells) would engage both CD4+ and CD8+ T cells.15 In the present study, similar amounts of both CD8+ and CD4+ infiltrating T cells were detected within the ESC grafts. Similarly, the numbers of graft-infiltrating DCs increased progressively over time. Colocalization of antigen-presenting DCs and T lymphocytes within the ESC graft indicate ongoing T cell–antigen presenting cell interaction and suggest that both direct and indirect allorecognition were involved in immune reactions against allogeneic ESC antigens. Furthermore, ESC graft infiltration of B cells was observed after allogeneic ESC transplantation. CD4+ T cells primed by indirect allorecognition could provide contact-dependent help for B cells to produce and alloantibody by a classical cognate T cell-B cell interaction. Alloantibody might mediate further graft damage by various mechanisms, including complement-dependent target-cell injury.13
ESC transplantation is being widely investigated as a potential novel approach to regenerate infarcted myocardium. A review of the current literature on transplantation of ESCs, however, revealed a surprising lack of concern for potential immunological conflict.13 The issue of immune recognition by the host is often circumvented by syngeneic transplantation,16 immunosuppressive therapy,17 or transplantation into an immune privileged site.18 Moreover, previous studies in xenogeneic transplant models provide no evidence for immune rejection of engrafted ESCs.6,7 This may create an impression that the problem of immune rejection does not apply to ESC transplantation. The present study, which was designed to address this question, does not support this notion. Thus, we detected potent progressive inflammatory ESC graft infiltration, indicating efficient recognition of ESC alloantigens by the host immune system.
One could argue that ESC immunogenicity could be due to inflammation of the host heart tissue caused by LAD-associated ischemia injury. Histological analysis after syngeneic ESC transplantation revealed no progressive infiltration, however, demonstrating that observed ESC immunogenicity in vivo could not be attributed to ischemia-induced inflammation of the recipient heart. In addition, it has been reported that transfection of ESCs with a genetic marker such as green fluorescent protein could alter their immunogenicity.19,20 Meanwhile, green fluorescent protein transfection of ESCs is frequently used in quantitative analysis of ESC survival and differentiation. To exclude potential immune reactivity toward exogenous proteins, we used a nonmanipulated ESC line.
It is known that undifferentiated ESCs may form teratomas when transplanted under the skin of immunodeficient severe combined immunodeficient mice.8 In addition, teratoma formation in the host retroperitoneum has been observed after ectopic transplantation of allogeneic ESC-derived cardiomyocytes.21 On the other hand, it has been suggested that the infarcted heart possesses paracrine signaling pathways that are capable of directing in vivo differentiation of transplanted ESCs into functional cardiomyocytes.6 In the present study, intramyocardial teratomas were observed in both allogeneic and syngeneic hearts at 4 weeks after ESC transplantation. Although this study was not designed to evaluate cardiomyocyte differentiation parameters, detection of ESC derivatives originating from various germ layers demonstrates that, at least under the current experimental conditions, the host environment could not guide differentiation of all transplanted cells into the cardiomyocyte lineage. Careful removal of undifferentiated elements from ESC derivatives before transplantation may possibly help to circumvent formation of intracardiac teratomas.
In conclusion, these data provide a clear demonstration of the induction of an alloimmune response against developing ESCs in an experimental animal model. Furthermore, it offers a novel experimental approach, in which immunogenicity of partly differentiated ESCs can be assessed after heterotopic transplantation of previously LAD-ligated and ESC-injected hearts into naive syngeneic hosts. This model could provide further insight into the characterization of anti-ESC immunity in animal models. In the present study, the immunogenicity of ESCs was analyzed by immunohistological evaluation of graft infiltration by host immune cells. Intragraft migration and infiltration represents an important step in the sequence of immune reactions. It indicates that other systemic immune events, such as peripheral lymphocyte activation in the spleen and/or lymph nodes, cytokine release, and production of circulating alloantibodies, likely are present in this model. Experiments evaluating these parameters are in progress in our laboratory.
In summary, we report that ESCs elicit an alloimmune response after transplantation into MHC-mismatched ischemic myocardium. On in vivo differentiation, ESC immunogenicity increases, resulting in efficient recognition of ESC antigens by the host immune system and in alloimmune rejection. These results imply that clinical transplantation of ESCs or ESC derivatives harvested from allogeneic donors for treatment of cardiac failure might require immunosuppressive therapy.