Stem cell treatment may positively influence recovery and inflammation after shock by multiple mechanisms, including the paracrine release of protective growth factors. Embryonic stem cells (ESCs) are understudied and may have greater protective power than adult bone marrow stem cells (BMSCs). We hypothesized that ESC paracrine protective mechanisms in the heart (decreased injury by enhanced growth factor-mediated reduction of proinflammatory cytokines) would be superior to the paracrine protective mechanisms of the adult stem cell population in a model of surgically induced global ischemia. Adult Sprague-Dawley rat hearts were isolated and perfused via Langendorff model. Hearts were subjected to 25 min of warm global ischemia and 40 min of reperfusion and were randomly assigned into one of four groups: 1) vehicle treated; 2) BMSC or ESC preischemic treatment; 3) BMSC or ESC postischemic treatment; and 4) BMSC- or ESC-conditioned media treatment. Myocardial function was recorded, and hearts were analyzed for expression of tissue cytokines and growth factors (ELISA). Additionally, ESCs and BMSCs in culture were assessed for growth factor production (ELISA). ESC-treated hearts demonstrated significantly greater postischemic recovery of function (left ventricular developed pressure, end-diastolic pressure, and maximal positive and negative values of the first derivative of pressure) than BMSC-treated hearts or controls at end reperfusion. ESC-conditioned media (without cells) also conferred cardioprotection at end reperfusion. ESC-infused hearts demonstrated increased VEGF and IL-10 production compared with BMSC hearts. ESC hearts also exhibited decreased proinflammatory cytokine expression compared with MSC hearts. Moreover, ESCs in cell culture demonstrated greater pluripotency than MSCs. ESC paracrine protective mechanisms in surgical ischemia are superior to those of adult stem cells.
- bone marrow stem cells
- vascular endothelial growth factor
- tumor necrosis factor
- mesenchymal stem cells
cardiogenic shock and subsequent heart failure is a prevalent cause of morbidity in intensive care units. However, it is well established that the heart has insufficient intrinsic repair mechanisms, which heal by formation of noncontractile scar tissue (21, 24). Modern revascularization and pharmacological strategies have also largely been ineffective in preventing the progression of ventricular remodeling. Thus, recent interest has focused on cell-based cardiac repair, with particular emphasis on stem cells to provide acute and/or chronic regenerative solutions to attenuate this disease (6, 14).
Stem cells are not created equally (18). Although bone marrow (31) hematopoietic stem cells (17) and mesenchymal stem cells (MSCs) (35) have shown the ability for conferring protection and repair of damaged tissue, they are limited to differentiating into cell types of their tissue of origin. Adult stem cells also have a lower capacity for self-renewal and can be more difficult to expand in culture. In contrast, embryonic stem cells (ESCs), derived from the inner cell mass of the blastocyst, are pluripotent and can proliferate indefinitely in the laboratory without differentiating (4, 8).
It was recently reported that transplantation of undifferentiated ESCs improves cardiac function after myocardial infarction (22, 25). ESCs could be transplanted into the heart across major histocompatibility and even species barriers. Furthermore, infarcted rat hearts treated with mouse ESCs resulted in a sustained functional benefit for several months (16). However, the mechanisms of this protection and whether ESCs may confer acute cardioprotection remain unknown.
We now appreciate that bone marrow stem cell-derived cardioprotection probably did not result from differentiation into cardiac myocytes. Investigators noted that donor stem cell survival as low as 1% was too few to influence contractile function. Indeed, we have recently demonstrated that acute application of MSCs into injured cardiac tissue attenuated ischemia-reperfusion (I/R) injury without evidence of differentiation (5, 35). When infused into infarcted hearts, MSC-conditioned medium alone also limited infarct size and improved functional myocardial recovery (13). Taken together, it appears that adult bone marrow stem cells may improve cardiac contractile performance not via differentiation but rather via complex paracrine actions (7, 33). Although the particular growth factors that contribute to this beneficial paracrine effect remain to be defined, the list includes the following: vascular endothelial growth factor A (VEGF), a central growth and survival factor for both the endothelium and the myocardium; interleukin 10 (IL-10), an antiinflammatory cytokine inversely associated with myocardial ischemia; and insulin-like growth factor I (IGF-I), an important antiapoptotic growth factor. Similarly, embryonic stem cell transplantation has been characterized by low survival numbers after transplantation into injured tissue. It remains unknown whether ESC-mediated protection is also due to complex paracrine actions.
We hypothesized that ESCs would be acutely cardioprotective against myocardial surgically induced I/R injury and that embryonic stem cells would confer greater protection than adult MSCs due to increased paracrine effects. The purposes of the present study were to investigate the effect of ESCs or adult MSCs on myocardial I/R as measured by 1) functional indices; 2) myocardial inflammatory cytokine production; 3) myocardial growth factor production; and 4) in vitro growth factor production.
MATERIALS AND METHODS
Normal C57BL/6J male mice (Jackson Laboratory, Bar Harbor, ME) and normal (250–350 g, 9–10 wk old) Sprague-Dawley male rats (Harlan, Indianapolis, IN) were fed a standard diet and acclimated in a quiet quarantine room for 1 wk before the experiments. The animal protocol was reviewed and approved by the Indiana Animal Care and Use Committee of Indiana University. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996).
Preparation of mouse bone marrow stromal cells.
A single-step purification method using adhesion to cell culture plastic was used as previously described (27), with the following modifications: 9- to 10-wk-old male mice were euthanized, and bone marrow stromal cells were collected from bilateral femurs and tibias by removing the epiphyses and flushing the shaft with complete media (Iscove's modified Dulbecco's medium; GIBCO Invitrogen, Carlsbad, CA) and 10% fetal bovine serum (GIBCO Invitrogen) using a syringe with a 23-gauge needle. Cells were disaggregated by vigorous pipetting several times. Cells were passed through 30-μm nylon mesh to remove remaining clumps of tissue. Cells were washed by adding complete media, centrifuging for 5 min at 300 rpm at 24°C and removing supernatant. The cell pellet was then resuspended and cultured in 75-cm2 culture flasks with complete media at 37°C. MSCs preferentially attached to the polystyrene surface; after 48 h, nonadherent cells in suspension were discarded. Fresh complete medium was added and replaced every 3 or 4 days thereafter. MSC cultures were maintained at 37°C in 5% CO2 in air. When the cultures reached 90% of confluence, the MSC culture was passaged; cells were recovered by the addition of a solution of 0.25% trypsin-EDTA (GIBCO Invitrogen) and were replated in 75-cm2 culture flasks.
Preparation of embryonic mouse stem cells.
Male embryonic W4/129S6 stem cells were purchased from Taconic (Hudson, NY). ESCs were harvested and cultured from normal 129S6/SvEvTac mice. Thawing of cells and initiation of culture process were performed according to the manufacturer's instructions. ESCs were plated in 100-mm tissue culture petri dishes (BD Falcon, Fisher, NY) and cultured with Dulbecco's minimal essential medium with high glucose (GIBCO) supplemented with 15% fetal bovine serum (GIBCO), 100 nM nonessential aminal acids, 1 mM sodium pyruvate, 2 mM l-glutamine, 0.5% penicillin-streptomycin (GIBCO), 100 nM mercaptoethanol (Sigma) and 1,000 U/ml leukemia inhibitory factor (Chemicon International) at 37°C, 5% CO2, and 90% humidity. Medium was changed every 3 days.
Light microscopy and yellow-green fluorescent detection of alkaline phophatase (marker of pluripotency) were performed to verify that ESCs remained pluripotent.
Isolated heart experimental groups.
All isolated rat hearts were subjected to the same I/R protocol: 15-min equilibration period, 25-min global index ischemia (37°C), and 40-min total reperfusion. Rats were divided into six experimental groups: 1) control hearts without intervention (n = 9); 2) isolated hearts infused with two million MSCs before I/R (n = 7); 3) isolated hearts infused with three million ESCs before I/R (n = 9); 4) isolated hearts infused with two million MSCs 1.5 min after I/R (n = 3); 5) isolated hearts infused with three million ESCs 1.5 min after I/R (n = 9); and 6) isolated hearts infused with 1 ml ESC-conditioned media prior to I/R (n = 3). MSCs or ESCs were washed with PBS, resuspended in warm (37°C) oxygenated Krebs-Henseleit (KH) solution, and infused (1 ml of 2 or 3 million cells) via the Langendorff apparatus an antegrade intracoronary route 1 min immediately before global index ischemia.
The dose of MSCs or ESCs was determined using a dose-response curve (data not shown). At higher concentrations of MSCs or ESCs, significant coronary cell trapping occurred, causing a dose-dependent decline in myocardial function. Two million MSCs and three million ESCs conferred maximal cardioprotection without a decline in myocardial function.
Stem cell therapy most often entails a postinjury treatment protocol. We have previously demonstrated that a pretreatment protocol, which is particularly useful in surgically induced ischemia, may not only allow the ultimate recovery of viable tissue following injury, but may also act as a stabilizing/protective “helper cell” during I/R. Thus, both a preischemic and a postischemic protocol were used in the present study.
Cell culture experimental groups.
For in vitro cell culture experiments, after five passages and 70% confluency, MSCs and ESCs were plated in 12-well plates (Corning, Corning, NY) in a concentration of 0.1 × 106 cells·well−1·ml−1 for each cell type. Cells were divided into groups (n = 4 in each group, each cell type): control; 24-h TNF (50 ng/ml); 24-h LPS (200 ng/ml); or 24-h hypoxia (1.0% O2; IN VIVO2 300 hypoxia chamber without substrate deprivation). After 24-h incubation, supernatants were harvested for VEGF. With the use of a Nikon TE2000U microscope at ×200 magnification, cell morphology was also assessed.
Conditioned media for isolated heart experiments were generated using identical conditions for the hypoxic stimulus in cell culture experiments. After 24 h of hypoxic exposure, supernatants from ESCs or MSCs were harvested for isolated heart infusion.
Isolated heart preparation (Langendorff).
Hearts were isolated as previously described (35). Briefly, rats were anesthetized (60 mg/kg ip pentobarbital sodium) and heparinized (500 units ip), and hearts were rapidly excised via median sternotomy and placed in 4°C KH solution. The aorta was cannulated, and the heart was perfused in the isolated, isovolumetric Langendorff mode (70 mmHg) with KH solution (in mM: 11 dextrose, 110 NaCl, 1.2 CaCl2, 4.7 KCl, 20.8 NaHCO3, 1.18 KHPO4, and 1.17 MgSO4) at 37°C. The KH solution was bubbled with 95% O2-5% CO2 (Medipure) to achieve a Po2 of 450 to 460 mmHg, Pco2 t and −dP/dt
Myocardial protein expression.
Heart tissue was homogenized in cold buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, and 1 mM PMSF and was centrifuged at 12,000 rpm for 5 min (centrifuge 5417R, Eppendorf, Westbury, NY). Production of VEGF, TNF-α, IL-1β, and IL-6 from myocardial tissue infused with MSCs and ESCs was determined by enzyme-linked immunosorbent assay (ELISA) using commercially available ELISA kits (R&D Systems, Minneapolis, MN, and BD Biosciences, San Diego, CA). ELISAs were performed according to the manufacturers' instructions. All samples and standards were measured in duplicate.
Presentation of data and statistical analysis.
All reported values are means ± SE (n = 3–8/group). All measures of myocardial function were compared using two-way analysis of variance (ANOVA) with post hoc Bonferroni test. All measures of protein were compared using Student's t-test. A two-tailed probability value of less than 0.05 was considered statistically significant.
I/R resulted in markedly decreased LVDP in all groups. Postischemic recovery of LVDP (expressed as percentage of preischemic function) was significantly higher (P < 0.05, ANOVA and Bonferroni) in hearts with preischemic MSC or ESC infusion than control hearts (35.7 ± 3.5%) at end reperfusion (Fig. 1A). However, preischemic infusion of ESCs conferred significantly greater protection (P < 0.05) of LVDP (75.2 ± 8.4%) compared with MSC infusion (56.6 ± 5.9%) at 3 × 106 cells/ml (Fig. 2E) but not at 2 × 106 cells/ml (Fig. 2A).
Left ventricular EDP was elevated in response to I/R (Fig. 2, B and F). Hearts infused with MSCs or ESCs before ischemia demonstrated significantly lower EDP at end reperfusion than hearts with no intervention (Fig. 1B). Furthermore, hearts with ESCs exhibited significantly improved EDP recovery compared with hearts with MSC infused at 3 × 106 cells/ml (Fig. 2F) but not at 2 × 106 cells/ml (Fig. 2B).
Maximum positive and negative dP/dt were impaired at the start of reperfusion (Fig. 2, C and G). Control hearts demonstrated more depression of +dP/dt and elevation of −dP/dt compared with hearts infused with MSCs or ESCs at end reperfusion (Fig. 1, C and D). However, hearts with ESCs exhibited improved contractility compared with hearts with MSCs at 3 × 106 cells/ml (Fig. 2, G and H) but not at 2 × 106 cells/ml (Fig. 2, C and D).
MSC infusion postischemia did not improve postischemic recovery of function, whereas ESC infusion 1.5 min into reperfusion did confer protection at end reperfusion as measured by EDP, +dP/dt, and −dP/dt (Fig. 1, E–H). Using repeated-measures ANOVA, neither MSC nor ESC postischemic infusion improved myocardial function significantly (Fig. 3).
ESC-conditioned media alone (without cells) also conferred protection of myocardial recovery but not to the same degree as ESC infusion (with cells) as measured by LVDP (59.7 ± 11.9%) at end reperfusion (Fig. 1, I–L). MSC-conditioned media at end reperfusion did not demonstrate significant improvement of myocardial function. Furthermore, ESC- and MSC-conditioned media alone (without cells) demonstrated a trend of increased myocardial function, which did not prove significant (Fig. 4).
Measure of pluripotency.
To objectively confirm whether ESCs remained undifferentiated, ESC and MSC alkaline phosphatase activity was measured. ESCs demonstrate elevated levels of alkaline phosphatase, an enzyme associated with undifferentiated pluripotent stem cells (data not shown). Alkaline phosphatase was not detectable in MSCs, however, confirming their more limited potency.
ESC and adult MSC morphology.
MSCs exhibit greater cell diameter and differing morphology than ESCs viewed under a Nikon TE2000U microscope at ×200 magnification (Fig. 5).
ESC and MSC activation in cell culture.
Stem cell production of paracrine factors may be important components of stem cell-mediated acute repair. To determine whether differences in paracrine factor production exist between ESCs and MSCs, basal and stimulated ESC or MSC growth factor production was measured. Hypoxia, LPS, or TNF resulted in significant activation of ESCs and MSCs (Fig. 6). ESC VEGF and IL-10 as well as MSC VEGF production was increased compared with control groups in response to hypoxia, LPS, or TNF. However, basal levels of VEGF production were greater in MSCs in culture compared with ESCs in culture.
Myocardial response to stem cell infusion and I/R.
Myocardial production of TNF, IL-1β, IL-6, IL-10, VEGF, and IGF-I was measured via ELISA. ESC-infused hearts exhibited decreased TNF, IL-1β, and IL-6 expression (P < 0.05) compared with MSC-infused hearts as shown in Fig. 7, A–C. Conversely, ESC-infused hearts demonstrated increased IL-10, VEGF, and IGF-I production (P < 0.05) compared with MSC-infused hearts as shown in Fig. 7, D–F.
Interestingly, hearts treated with ESC-conditioned media exhibited greater inflammatory cytokine and VEGF production and similar IL-10 production compared with ESC-treated hearts (Fig. 7).
These results constitute the initial demonstration that pretreatment with murine embryonic stem cells protects native myocardium from injury. Furthermore, we have shown that embryonic stem cell infusion provides greater postischemic functional recovery and decreased myocardial tissue proinflammatory cytokine production compared with adult MSCs. These effects may be mediated at least in part by paracrine growth factor production of VEGF and IL-10 and modulation of local inflammation.
Stem cell therapy is a promising treatment modality for injured cardiac tissue (28). However, we now appreciate that this stem cell-mediated protection may not result from differentiation into cardiac myocytes (3, 23). We previously demonstrated that human and murine MSC administration immediately before cardiac ischemia improved functional recovery, decreased proinflammatory cytokine production, and decreased proapoptotic signaling (35). In the present study, we found that acute delivery of ESCs into injured cardiac tissue before ischemia provided greater attenuation of I/R injury and postischemic decrease of LVDP, +dP/dt, and −dP/dt compared with adult MSCs. The preischemic stem cell delivery protocol used previously and in the present study allowed the MSCs and ESCs to increase recovery of myocardial function after ischemia. Interestingly, ESC-treated hearts exhibited a different rate of recovery in comparison with MSC-treated hearts, which may suggest different mechanisms of action. In addition to our established preischemic stem cell delivery protocol in the isolated heart, a postischemic MSC delivery protocol was also used; the acute use of stem cells and measure of function in both protocols precluded immediate stem cell differentiation as a cause of myocardial protection. Hearts treated with postischemic ESCs demonstrated improved recovery of myocardial function compared with MSC hearts at end reperfusion. However, we found that the postischemic protocol conferred less cardioprotection than the preischemic protocol; indeed, both ESC- and MSC-treated hearts demonstrated no difference throughout ischemia using repeated-measures ANOVA compared with vehicle-treated hearts. This difference in cardioprotection between preischemic delivery of stem cells and postischemic delivery may have occurred for two reasons: 1) the exposure to beneficial stem cell effects is shorter (reperfusion only vs. ischemia + reperfusion) and 2) less powerful stimulus for stem cell activation (I/R only vs. ischemia + I/R injury). These data suggest that embryonic stem cell treatment may provide greater preservation of myocardial function compared with adult MSCs. Furthermore, both embryonic as well as adult stem cells may acutely improve cardiac contractile performance and limit myocardial infarct size by mechanisms other than differentiation. Also, preischemic delivery of stem cells, applicable in the clinical scenario of surgically induced ischemia, may be more effective than postinjury stem cell treatment. However, it remains unclear through which acute mechanisms ESCs and MSCs exert their myocardial protection following ischemia and reperfusion.
Stem cells may mediate their acute protective effects via complex paracrine actions. In response to stress like TNF, stem cells release greater levels of protective factors VEGF and IGF-I (9, 19, 34, 36). In congruence with these findings, we found that both ESC and adult MSC stimulation with hypoxia, TNF, or LPS resulted in significantly increased release of VEGF in cell culture. Moreover, ESCs and MSCs with or without stimulation produced antiinflammatory IL-10 (10). This growth factor production in the face of stress may represent the stem cell acting to release substances that limit local inflammation to enhance its own survival (11). Concurrently, these growth factors may play an important paracrine role in the MSC-induced myocardial protection from ischemia (15). Indeed, embryonic stem cells and adult mesenchymal stem cells both significantly elevated myocardial production of VEGF compared with control hearts. Furthermore, ESC-treated hearts demonstrated greater VEGF production than MSC-treated hearts, which may be associated with the greater ESC protection of myocardial function. Determining whether cell release of VEGF confers protection to surrounding tissue via reduced apoptosis (1, 2, 30, 37), decreased proinflammatory cytokines (29, 38, 39), or other mechanistic pathways (20, 32) requires further investigation.
In vitro studies of cardiomyocytes injured in response to monocyte chemoattractant protein-1 (26) or hypoxia (12) demonstrated protection with MSC-derived conditioned medium that did not contain stem cells. In the current study, ESC-conditioned media also elicited increased myocardial functional recovery and myocardial production of VEGF compared with controls. However, in the present study, embryonic stem cells produced lower basal levels of growth factor production than adult mesenchymal stem cells. In addition, hearts treated with ESCs demonstrated less paracrine factor production than hearts treated with ESC-conditioned media. These interesting findings suggest that further study is needed to determine the role of cell-cell interactions and paracrine effects in myocardial stem cell-mediated protection.
While this cardioprotection may be due in part to paracrine growth factor release by stem cells, other stem cell protective mechanisms include salvage of tenuous or malfunctioning cardiomyocytes at the infarct border zone; alteration of the extracellular matrix, resulting in more favorable postinfarct remodeling; and activation of resident cardiac stem cells. Indeed, in the present study, stem cells may have employed cardioprotection via modulation of local inflammation; embryonic and adult mesenchymal stem cells increased myocardial antiinflammatory IL-10 and attenuated myocardial proinflammatory production of TNF, IL-1β, and IL-6. However, hearts treated with ESCs further exhibited decreased myocardial inflammatory cytokines compared with hearts treated with MSCs. Thus, increased myocardial protection in hearts treated with ESCs may be associated with increased attenuation of myocardial inflammation during I/R.
These results demonstrate that murine embryonic stem cells are superior to adult mesenchymal stem cells in attenuation of surgically induced warm global myocardial I/R injury. Planned ischemic events such as those that occur during cardiac surgery, angioplasty, or transplantation may allow an additional opportunity to observe the potential paracrine clinical benefit of adult stem cell pretreatment. Stem cells are a novel potential agent for myocardial protection against I/R, but further investigations of the limitations are necessary to maximize this protection.
This work was supported in part by National Institutes of Health Grants R01-GM-070628, R01-HL-085595, K99/R00-HL-0876077, and F-32-HL-085982, by an American Heart Association grant-in-aid, and by American Heart Association Post-Doctoral Fellowship 0725663Z. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Program Grant C06-RR-015481-01 from the National Center for Research Resources, National Institutes of Health.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2008 by the American Physiological Society