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a Cardiothoracic Surgery, Falk Research Center, and
b Department of Pathology, Stanford University Medical School, Stanford, California, USA
Key Words. Growth substances • Myocardial infarction • Cell transfer • Embryonic stem cells
Correspondence: Theo Kofidis, M.D., Cardiothoracic Surgery, Falk Research Center, 2nd Floor, Stanford University Medical School, 300 Pasteur Dr., Stanford, CA 94305. Telephone: 650-723-5408; Fax: 650-725-846; e-mail: tkofidis{at}stanford.edu
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ABSTRACT |
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-sarcomeric actin ( p = .015) and major histocompatibility complex class I ( p = .01). IGF did not affect the cellular response to the donor cells or tumorigenicity. IGF-1 promotes expression of cardiomyocyte phenotype in ESCs in vivo. It should be considered as an adjuvant to cell transfer for myocardial restoration.
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INTRODUCTION |
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A major determinant of engraftment and host organ–specific differentiation of donor primordial cells is the array of growth factors that are known to promote cardiogenesis and proliferation and to prevent apoptosis and cell death [8–9]. A distinct role is displayed by insulin-like growth factor-1 (IGF-1); it promotes cell mitogenesis and proliferation in early developmental stages and causes eccentric myocardial hypertrophy in the adult. After binding to its receptor on embryonic stem cells (ESCs), IGF-1 induces expression of a number of cardiac-specific transcription factors such as the zinc finger GATA proteins and Nkx-2.5, a coactivator of GATA-4. GATA-4 and Nkx-2.5 are essential for heart development [10–12]. In parallel to adult cardiomyocytes, ESC-derived cardiomyocytes developmentally express cardiac-specific proteins and ion channels. ESC-derived cardiomyocytes have been successfully purified up to 70% from native ESC populations through multiple duplication steps [13]. The stimulation of asymmetric progeny generation toward the cardiac phenotype has been demonstrated recently [14].
Experience with ESC injection into myocardium is very limited, and past efforts have faced the issue of tumorigenicity of these cells in vivo. Their survival and differentiation potential to cardiac-specific cells after transfer in an area of infarction is largely unknown. In the study reported here, we hypothesized that injection of undifferentiated ESCs along with IGF-1 will enhance their potential to harness microenvironmental input in order to survive, differentiate, and improve function of the ischemic heart.
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MATERIALS AND METHODS |
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Enhanced Green Fluorescent Protein (EGFP) ESCs
pEF-1 a-EGFP, containing a GFP gene under the control of human EF1, a promoter, and a neomycin-resistant cassette, was constructed as follows. The promoter region of pEGFP-N3 (Clontech, Palo Alto, CA) was removed by cutting out the Ase I-Nhe I DNA fragment and joining the blunt-ended termini. Human EF1, a promoter from pEF-BOS (a fragment of HindIII and EcoRI DNA) was inserted into the HindII-EcoRI site of the plasmid. D3 ESCs were transfected with pEF-1 a-EGFP by electroporation and selected in the presence of G418. One clone that brightly expresses EGFP was chosen and used for the experiments. The clone was adapted to feeder-free conditions and maintained on gelatin-coated dishes in Dulbecco’s Modified Eagle’s Media supplemented with 15% fetal calf serum, 2 mM sodium pyruvate, 2 mM L-glutamine, 1x nonessential amino acids, 1,000 units of 0.1 mM 2-mercaptoethanol per ml (ESGRO, CHEMICON International, Inc., Temecula, CA), along with 100 units of streptomycin and 100 µg of penicillin per ml. Cells were collected after trypsinization with EDTA and placed in aliquots of the same medium as above for intramyocardial injection 1 hour later.
Animal Groups and Myocardial Injury Model
We injected ESCs originating from 129sv mice into a group of BalbC-recipient hearts (n = 5), a control group that had undergone left anterior descending (LAD) artery ligation only without cell transfer (n = 5), and an additional control group that had undergone LAD ligation and ESC injection without addition of IGF-1 (n = 5).
The mice were pre-anesthetized in an isoflurane inhalation chamber and received an i.p. injection of sodium pentobarbital (25 mg/kg). The animals were then intubated and ventilated for the entire length of the procedure. The surgical approach involved a left lateral thoracotomy, a pericardectomy, and identification of the LAD artery for ligation.
Once ligation with a 9-0 ethilon (Optima Inc., Houston, TX) stitch was performed on the proximal 2-mm portion of the LAD, a pale area was demarcated on the surface of the left ventricle. Placement of the ligature resulted in significant left ventricular ischemia, which was immediate and encompassed the middle and apical portion of the ventricle. This area constituted the target for the cells. Twenty-five ng of recombinant mouse IGF-1 (R&D Systems, Minneapolis) were added to the cell suspension shortly before injection. Using a 29G needle, 250,000 ESCs suspended in 25 µ1 were injected into the demarcated area, which consequentially developed a wheal, a reliable sign that cells have been administered intramyocardially and did not accidentally enter the left ventricular cavity. Immediately thereafter, a chest tube (20G Angio cath, Becton, Dickinson, Franklin Lakes, NJ) was inserted, and the chest was closed in layers. Ventilation was maintained until sufficient spontaneous breathing occurred, and extubation and removal of the chest tube followed. The mice were left to recover in a temperature-controlled chamber until they resumed full alertness and mobility. We sacrificed the animals (in deep anesthesia) at various time points after cell transfer and echocardiography.
Echocardiography
Mice were transferred in a portable chamber and kept under inhalational isoflurane anesthesia for the duration of the procedure, which took place immediately before sacrifice. The chests were shaved, and the animals were placed in recumbent position. We used the Acuson Sequoia C256 echocardiography system (Acuson, Mountain View, CA) with a 15.8-MHz probe. The following measurements were recorded: end systolic (ESD) and end diastolic (EDD) diameter in a cross section, ESD and EDD at two different sites of a longitudinal section of the heart (basal and apical), posterior wall thickness (PWT) and septal wall thickness and calculated fraction shortening (FS) as FS = (EDD – ESD)/EDD.
Organ Harvest, Tissue Storage, Immunofluorescence, and Confocal Microscopy
Hearts were excised and fixed in 2% paraformaldehyde in phosphate-buffered solution (PBS) for 2 hours and cryoprotected in 30% sucrose overnight. Tissue was embedded in optimum cutting temperature medium and sectioned at 5 µm on a cryostat. Serial sections were stained with hematoxylin and eosin (H&E) or with Masson’s trichrome, or they were used for immunohistochemistry. Immunostaining was performed as previously described. Briefly, sections were blocked and incubated with primary antibody for 30 minutes to 1 hour at room temperature. Primary antibodies against cardiac, immunological, and GFP proteins were used. These included rabbit anticonnexin-43, mouse monoclonal anti--sarcomeric actin (Sigma, St. Louis), hamster anti-CD3
, hamster anti-CD11c, mouse anti–major histocompatibility complex class I (MHC I) (BD Pharmingen, San Diego), goat anti-GFP antibody (Rockland, Gilbertsville, PA), and rabbit anti-GFP Alexa-488–conjugated antibody (Molecular Probes, Eugene, OR). After brief washing with PBS, sections were incubated with secondary antibodies for 30 minutes to 1 hour at room temperature. Either Texas red–conjugated or streptavidin-Texas red secondary antibodies were used against the cardiac and immunological marker primary antibodies. Goat anti-GFP antibody was recognized by a fluorescein isothiocyanate–conjugated secondary antibody. After brief washing with PBS, sections were mounted with Slowfade antifade reagent with 4', 6-diamino-2-phenylin-dole dihydrochloride (Molecular Probes). Stained tissue was examined with a Leica DMRB fluorescent microscope and a Zeiss LSM 510 two-photon confocal laser scanning microscope.
Connexin-43 and -actinin were used to identify differentiation when expressed on donor GFP+ cells. We used smooth muscle actin (SMA) to identify differentiation of donor cells to smooth muscle cells and participation of those in angiogenic processes. At x 400 magnification, the ratio of relative marker signal (Texas red) to GFP graft was calculated. Using SMA, we calculated vessels per GFP graft area and vessel cross-sectional area. To further assess the restorative and tumorigenic potential of ESC cell grafts, we performed H&E and trichrome stains. These also allowed for identification of vessels and measurement of their cross-sectional area. Trichrome and H&E stains were used to estimate extent, distribution, structure, and kinetics of ensuing scar after the infarction and injection of cells, as well as the mode of their organization, vessel count, and cellular type. H&E staining was helpful for evaluating cellular atypia and nuclear polymorphism as indicators of tumor formation. The trichrome and H&E observations were expressed in an ordinal scale, ranging from 0 (vessels absent, no scar tissue, no cellular atypia, no nuclear polymorphism) to 4 (dense vascularization of the optical field corresponding to the GFP+ graft, extensive scarring, severe cellular atypia and chaotic pattern, and severe nuclear polymorphism). Therefore, latter findings are to be regarded as semiquantitative and are represented in the grafts as "scale" of the individual event.
To identify quality and quantity of cellular (inflammatory or immune) response to the injected cells, we stained the same sections for CD3 (an indicator of T lymphocytes) and CD11c (an indicator of dendritic cells). To assess the presence of MHC molecules on the GFP+ cells, we stained for MHC I, which is expressed by all adult cells. We also report on the distribution of the MHC I signal and its behavior over time.
Morphometry
Sections were obtained at five different levels of the harvested heart. The infarct was identified as a dark area that contained the injected GFP conglomerate. For all morphometric evaluations, the focused microscopic field was photographed by an adapted camera (Diagnostic Instruments Inc., Burlingame, CA). The total GFP+ area was measured and related to the infarction area at low magnification. To quantify the extent of expression of specific markers, five random sections of the GFP+ graft were photographed and evaluated using the Spot advanced software, version 3.4.2 (Diagnostic Instruments). The marker-positive area and the area occupied by the GFP graft on every section were measured, and their quotient provided an area ratio, which was expressed as percentage. To avoid missing marker-expressing cells, high magnification was chosen for this portion of the morphometry (x 400).
Statistics
Descriptive statistics included mean and standard deviation of all measured values. Comparison between groups was performed using Student’s t-test for independent variables using Microsoft Excel 2000, and significance was assumed with p < .05.
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RESULTS |
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). The ratio of restored area to infarcted area ranged between 27% and 68%, with an average of 50% ± 18.10% versus 39.3% ± 7.2% in the ESC-only group (Fig. 1A). The cells formed dense conglomerates, which in many cases did not allow for distinguishing individual cells under the GFP filter. This was often possible via comparison to the corresponding H&E, trichrome, or otherwise stained section or by colocalization studies using GFP and the Texas red signal of the individual cell marker such as connexin-43.
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). The area of connexin-43 expression–contained cells aligned in multiple directions, which in only a few cases displayed the typical sarcolemma and myotubular orientation that are morphologically attributable to cardiac cells. Fewer than 10% of the connexin-43 or -sarcomeric actin cells or cell islets in the cardiomyocyte morphology displayed a myotubular form. There was a trend toward stronger expression of connexin-43 (area ratio) in the IGF-1 group without statistical significance. The -sarcomeric actin pattern displayed striations on GFP+ elongated cells (Fig. 1C). The overall area of expression of -sarcomeric actin ranged from 20%–40% of the GFP graft area (mean, 31.6% ± 10.4%) versus 4%–8% (mean, 6.5% ± 2.2%) in the ESC-only group. In most, though not in all, cases, connexin-43 and -actinin–positive cells (signals) colocalized on sequential sections. The -sarcomeric actin expression was more pronounced in the IGF-1 group (p = .015) (Fig. 2A). SMA was expressed in the GFP+ cells that participated in vessel formation. The overall expression of SMA in the GFP+ graft was only very minor and mostly present in the border zones of the GFP+ cellular conglomerate.
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). CD11+ cells were detected primarily within the GFP+ grafts. Their distribution did not follow a specific pattern. None of the inflammatory cells stained for GFP, indicating they were of host origin. In many occasions, the inflammatory cells have infiltrated and surrouded the donor cells. Their number was similar in the two treated groups. Cellular atypia, nuclear polymorphism, and a shift of the ratio of cytoplasmic to nuclear cells was evident in all animals 2 weeks after cell injection. The animals treated with IGF-1 did not show more cellular atypia than those treated with ESC only. Nuclear polymorphism was similar in both treated groups (Fig. 1D).
Functional Effects of ESC and IGF-1 Treatment in Postischemic Hearts
Echocardiography revealed significantly larger EDD diameters in the control group than in the group treated with ESC only (p = .038) and in the IGF + ESC–treated group. PWT was higher in the IFG-1–treated group (p = .032) (Fig. 2B). FS was higher in the IGF-1 and ESC-only groups than in the controls. There was no significant difference between IGF-1–treated animals and ESC controls, but a clear trend for IGF + ESC–treated mice to display better FS was noted (Fig. 2A).
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DISCUSSION |
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-sarcomeric actin expression is consistent with previous reports that IGF promotes myocyte mitogenesis and proliferation. The trend for connexin-43 expression to be higher suggests that IGF-1 might not have any specific effect on expression of gap-junction proteins in vivo, a property that has been attributed to fibroblast growth factor-b. Overexpression of MHC I in the group treated with ESC and IGF-1 offers further evidence of the potential of IGF-1 to promote differentiation. It has been proposed that ESCs might encounter the immune system because they express small amounts of MHC I in culture in the process of differentiation [18–20]. Our data contradict this notion, in that the degree of MHC I expression in vivo is very significant, encompassing as much as 70% of the injected cells. In parallel series of immunological experiments in our laboratory, we obtained evidence of progressive humoral response to donor cells using fluorescence-activated cell sorter (FACS) analysis. In parallel series of immunological experiments in our laboratory, we obtained evidence of progressive humoral response to donor cells using FACS analysis. Furthermore, functional tests by means of enzyme-linked immunospot assay revealed increased production of interferon-gamma, interleukin-2 (IL-2), IL-4, and IL-5 by activated splenocytes of the host, in response to ESC injection into the heart (data not shown). Finally, the histologically observed MHC I signal on GFP+ donor cells within the graft becomes more extensive with time in vivo, in concordance with expression of cardiac markers on subsequent sections, suggesting that MHC I expression and differentiation are coinciding and cross-dependent processes. In conclusion, we have solid reason to believe that ESC transplantation will have to face similar issues of allorecognition as solid organ transplantation, that ESCs are not immune-privileged, and that ESC therapy will have to involve immunosuppressive therapy. Still, the superior robustness and differentiation potential of this cell line defines its suitability for the treatment of ischemic organ diseases.
Given the limitations associated with mesenchymal and bone marrow stem cells, ESCs seem to promote more substantial recovery of infarcted myocardial function. In the absence of sufficient reperfusion, even large numbers of mesenchymal stem cells or bone marrow stem cells have been reported to die or differentiate to target nonspecific cells [21, 22]. ESCs, however, retain their initial volume for 2 weeks, indicating that their self-renewal and local ischemia tolerance are superior to other cell types. A sustained and robust myocardial restoration of this scale seems to be feasible only with ESCs and is enhanced by the administration of IGF-1.
The high degree of in vivo differentiation to an organ-specific phenotype compared with in vitro response with or without stimulants such as 5-azacytidine, indicates that highly regulative mechanisms at severe ischemic conditions educate ESCs to selectively give rise to more committed progenitors. The phenomenon of target-specific asymmetry operates in the ischemic myocardium to sustain an increasing amount of committed cells. It has been reported that populational asymmetry facilitates the response to variable physiological needs [23, 24]. The herein-involved multiple feedback controls and reciprocal cellular interactions need to be further investigated. The distinct effect of paracrine pathways (including growth factors) and coinciding liberation of cytokines and the exact sequence of events remain to be resolved as well. Also, a series of evolutionarily conserved transcription factors are believed to control stem cell fate [21, 24]. Furthermore, the microenvironmental niche regulates stem cell destiny by a wide range of secreted factors, cell–cell interactions mediated by integral membrane proteins, and integrins and extracellular matrix modulations. Finally, homeostatic controls, which should be envisioned as of secondary significance in a myocardial scar, are reported to regulate production of progeny by mechanisms that depend on positive or negative feedback loops [24, 25].
However, it remains unknown how these complex interactions become operable in a myocardial scar. A possible option would be that at the time of injection, remodeling is still at an early stage and that the myocardial reorganization cascade provides the microenvironmental determinants for "selective" asymmetric differentiation to premature cardiomyocytes. In spite of the significant degree of myocyte marker expression and its enhancement by IGF-1, we observed few spindle-like cells within the graft, and they were always positive for connexin-43 and -actinin. The vast majority of cardiac marker–expressing cells displayed a disorganized distribution of connexin-43 surface fluorescence during their residency in a dense conglomerate of GFP+ cells. Connexin-43 and GFP colocalization findings suggest that connexin-43 is being expressed in an early stage of cell differentiation, prior to establishment of the myotubular phenotype. A question still remains to be answered: What portion of the improvement of myocardial function should be ascribed to differentiated progeny of the injected ESCs, and which portion of functional improvement should be attributed simply to the better tissue viability after cell transfer, which, in consequence, prevents myocardial thinning and worsening of hemodynamic performance? Longitudinal studies using advanced imaging techniques in comparison, such as bioluminescence and echocardiography or magnetic resource imaging, should have the potential to resolve this issue.
Further studies of ESC differentiation in vitro in presence of various growth factors must be conducted to evaluate the timeline of phenomena that occur in the process of cardiomyocyte development. For instance, it is unknown whether ESCs express growth factor receptors in early differentiation stages and, if they do, in which sequence [24]. The present study is limited by the fact that it was not designed to address this question. It presupposes that the injected ESCs will express the receptor sequences on their surfaces while residing in the area of ischemic lesion. Moreover, the injected cell population is a mixed one, and it is not optimal to isolate the events that are orchestrated by the ischemic microenvironment toward one single cell type. Transplantation of a differentiated, replicating progeny and post-explant genetic microarray experiments will facilitate determination of sequence of developmental steps and help isolate premature cardiomyocytes in the desired differentiation phase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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