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Right arrow Myocardial infarction

Eur J Cardiothorac Surg 2006;29:50-55
© 2006 Elsevier Science NL

Allopurinol/uricase and ibuprofen enhance engraftment of cardiomyocyte-enriched human embryonic stem cells and improve cardiac function following myocardial injury

Theo Kofidis a , d , * , Darren R. Lebl a , Rutger-Jan Swijnenburg a , Joan M. Greeve b , Uwe Klima d , Joseph Gold c , Chunhui Xu c , Robert C. Robbins a

a Cardiothoracic Surgery/Falk Research Center, Stanford University Medical School, Stanford, CA, USA
b Department of Radiology, Stanford University Medical School, Stanford, CA, USA
c Geron Corporation, Menlo Park, CA, USA
d Division of Thoracic and Cardiovascular Surgery, Hannover Medical School, Carl Neuberg Street 1, 30625 Hannover, Germany

Received 27 June 2005; received in revised form 21 September 2005; accepted 7 October 2005.

* Corresponding author. Present address: Division of Thoracic and Cardiovascular Surgery, Hannover Medical School, Carl Neuberg Street 1, 30625 Hannover, Germany. Tel.: +49 511 532 6580; fax: +49 511 532 5404. (Email: kofidis{at}thg.mh-hannover.de).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Statistics
 4. Results
 5. Discussion
 References
 
Objective: A major limitation of stem cell transfer is early donor-cell death. Here, we seek to enhance myocardial repair following injury through transplantation of cardiomyocyte-enriched human embryonic stem cells (hESC) and recipient treatment with cytoprotective (allopurinol + uricase) and anti-inflammatory (ibuprofen) agents. Methods: We injected 106 (15% hESC-derived cardiomyocytes) green fluorescent protein (GFP+) hESC in the infarcted area following left anterior descending artery (LAD)-ligation in SCID-beige mice. In Group I, 1.6 mg allopurinol and 0.2 mg of uricase were injected i.p. for 3 days prior to cell transplantation. In Group II, 0.35 mg/ml of ibuprofen were added to the drinking water before and after cell implantation. In Group III, the LAD was ligated and allopurinol/uricase was administered without cell treatment. In Group IV, ibuprofen was added to the drinking water and the LAD was ligated without additional cell treatment. In Group V, only cells were transplanted. Group VI involved infarcted controls and Group VII involved sham-operated mice (all groups: n = 5). We evaluated heart function (ejection fraction (EF)) by MRI (4.7 T) 3 weeks later. The hearts were harvested for histology. Results: Differentiated hESC formed clusters and expressed {alpha}-sarcomeric actin and Connexin 43. Cell treatment improved heart function, which was best in the ibuprofen- and allopurinol-treated groups (+cell transfer), compared to the infarcted controls [EF: Group I: 76.6 ± 8.6%, Group II: 78.6 ± 7.3%, Group III: 58.1 ± 5.7%, Group IV: 53.9 ± 5.2%, Group V: 57.7 ± 7.5%, Group VI: 43.5 ± 4.3%, and Group VII: 66.3 ± 7.8%]. We did not observe tumors in any group. Conclusions: Allopurinol/uricase and ibuprofen enhance differentiated hESC-engraftment and myocardial restoration following transplantation into the injured heart.

Key Words: Embryonic stem cells • Myocardial restoration • Ischemic heart disease


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Statistics
 4. Results
 5. Discussion
 References
 
Cell and tissue transfer provide novel methods for the treatment of heart disease. Large-scale myocardial regeneration offers the possibility of replacing myocardial revascularization and transplantation in selected patients, once established clinically. A plethora of stem cells and their derivatives have been proposed for myocardial restoration, with controversial results [1–3]. They all share one major limitation: extensive cell death or apoptosis, early after implantation in the diseased myocardium. Although the spectrum of competitively efficient stem cells is growing, little attention is dedicated to the prevention of danger signals and death cascades, which jeopardize the injected stem cells.

In the context of severe tissue ischemia, a series of events takes place to the disadvantage of the injected stem cells [4–6]. Massive liberation of inflammatory cytokines (such as interleukins and leucotrienes) attracts leucocytes and macrophages to the site of the graft. These cells attack and diminish the inoculated donor cell population. Free radicals lyse cell membranes and promote subsequent cell death. Fibroblasts are triggered to produce scar tissue. Uric acid accumulates and serves as a "danger signal" [5,7]. The resulting vicious circle aggravates cell loss, reverse remodeling and non-ischemic infarct extension. A series of antioxidant agents, free-radical scavengers and cyclooxygenase inhibitors have displayed well-reported beneficial effects on resident cell survival within ischemic tissue and hence could promote survival of transferred stem cells [8].

Allopurinol is an inhibitor of the enzyme xanthine oxidase, the enzyme that converts the purines produced by cells undergoing apoptosis to uric acid; uric acid crystals serve as powerful "danger signals" that induce potent immune reactions [7]. Allopurinol is broadly used in chemotherapy patients to control lysis syndrome. The use of allopurinol and the enzyme uricase, which directly destroys uric acid, has the potential to decrease the immune stimulus provided by dying ischemic cells and thereby limit the extent of myocardial infarction [8,9]. Ibuprofen is a cyclooxygenase inhibitor with anti-inflammatory properties, and directly inhibits leukocyte activation and accumulation [10,11]. Furthermore, it decreases production of leukotriene B4, a leukocyte attractant and activator. A concomitant use of allopurinol/uricase and ibuprofen in the recipient animals might enhance stem cell engraftment and consequently improve the function of injured myocardium.

Embryonic stem cells hold great promise in the field of regenerative medicine and restorative surgery, due to their proliferative capacity and pluripotency, allowing them to give rise to differentiated cells such as cardiomyocytes [12–14]. Even though issues related to their utilization such as tumorigenicity and immunogenicity still have to be addressed, their ability to survive and differentiate makes them attractive for restorative applications into the injured myocardium. We tested this hypothesis using a cardiomyocyte-enriched population of human embryonic stem cells (hESC). In the present study we examine the potential of allopurinol/uricase and ibuprofen to enhance engraftment of this robust and promising cell type, and thereby improve heart function in a mouse model of acute myocardial ischemia.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Statistics
 4. Results
 5. Discussion
 References
 
2.1 Animals
All surgical interventions and animal care were provided in accordance with the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, publication volume 25, number 28, revised 1996) and the Guidelines and Policies for the Use of Laboratory Animals for Research and Teaching of the Department of Comparative Medicine, Stanford University School of Medicine.

Female SCID-beige mice were used for hESC transfer experiments. We injected 106 (15% hESC-derived cardiomyocytes) green fluorescent protein-positive (GFP+) hESC in the infarcted area following left anterior descending artery (LAD) ligation in SCID-beige mice. In Group I, 1.6 mg allopurinol and 0.2 mg of uricase were injected i.p. for 3 days prior to cell transplantation. In Group II, 0.35 mg/ml of ibuprofen were supplemented to the drinking water for 7 days prior, until 7 days post-cell transplantation. In Group III, the LAD was ligated and allopurinol/uricase was administered without cell treatment. In Group IV, ibuprofen was supplemented to the drinking water, the LAD ligated without additional cell treatment (drug dosages were based upon Ref. [7]). In Group V only hESC without additional treatment were transplanted. Group VI involved infarcted controls and Group VII involved sham operated mice (all groups: n = 5). We evaluated heart function (ejection fraction (EF)) by MRI (4.7 T) 3 weeks later. The hearts were harvested for histology.

2.2 Lentiviral vector production and transduction
The lentiviral transfer vector pMIN-Ubi-eGFP-INW contains a deletion of the 3' LTR and includes the human Ubiquitin C promoter driving expression of a multicistronic eGFP-ECMV-IRES-neor transcript that includes the WPRE element from Woodchuck Hepatitis C virus. Expression of the viral genome is driven by the CMV promoter/enhancer.

Human kidney epithelial 293T cells were maintained in DMEM plus 20% FCS and split 24 h prior to transfection to achieve 50–75% confluency on the following day.

VSV-G-pseudotyped lentiviral vectors were made by co-transfecting 293T cells with expression plasmids for HIV-1 Gag-pol, HIV-1 Rev, and VSV-G protein along with the lentiviral genome plasmid pMIN-Ubi-eGFP-INW. After 12 h, media was changed to Ultraculture medium (Bio-Whittaker). Infectious virus was collected 28–42 h post-transfection and used immediately.

2.3 Cell culture
H7 hES cells were maintained on Matrigel in culture medium (CM); preparation of CM and other cell culture methods are described in the web site http://www.geron.com/. Confluent H7 hES cell cultures were incubated with 0.5 mM EDTA in PBS at 37 °C for 8 min, dissociated, resuspended in CM and replated onto Matrigel-coated plates at ~1 x 106 cells/cm2. The cells were exposed to the supernatant containing lentivirus 24 h after plating. DEAE-dextran at a final concentration of 10 µg/ml and hbFGF at 4 ng/ml were added to the viral supernatant immediately before the transduction. The medium was replaced with CM after 12–18 h incubation. The CM was supplemented with 100 µg/ml G418, 5 days after the transduction. The cells were split with collagenase IV when confluent, seeded onto Matrigel-coated plates and maintained in CM supplemented with 100 µg/ml geneticin.

Cardiac differentiation of hES cells was induced through embryonic body (EB) formation as described previously [15]. The cells were then dissociated, resuspended in differentiation medium and loaded onto discontinuous Percoll gradients for isolation of cardiomyocytes. In vitro assessment (prior to injection) had been performed by the cell providers (GERON Corp.) using immunohistochemistry (Troponin I, MF 20, a-sarcomeric actin) and identification of the cardiotypical phenotype.

2.4 Thoracotomy/cell injection
Mice were pre-anesthetized with isoflurane and received an intraperitoneal injection of Ketanest/Xylazine (50 mg/kg, ca. 1.2 mg per animal and depending on the effect). The animals were then intubated and ventilated for the entire length of the procedure. The surgical approach involved a left lateral thoracotomy, pericardiectomy and identification of the left anterior descending artery (LAD) for ligation. Following ligation of the LAD, 106 donor cells in 25 µl culture medium were injected into the resulting area of infarction within the lateral wall of the left ventricle; cell injections were performed as previously described [16]. Following cell injection, the chest was closed in layers and the animal extubated after respiratory recovery.

2.5 Mouse MRI
Images were acquired using a Unity Inova console (Varian Inc., Palo Alto, CA, USA) controlling a 4.7 T, 15 cm horizontal bore magnet (Oxford Instruments Ltd., Oxford, UK) with GE Techron Gradients (12 G/cm) and a volume coil with an inner diameter of 3.5 cm (Varian Inc., Palo Alto, CA, USA). The mice were anesthetized using isofluorane. The ECG gating was optimized using two subcutaneous precordial leads. Respiration was monitored and body temperature controlled between 36 and 37 °C (SA Instruments Inc., Stony Brook, NY, USA). LV function was evaluated using an ECG-triggered spin echo sequence (TR 120 ms, TE 7.5 ms, FOV 3.0 cm2, matrix 128 x 128, slice thickness 1 mm, NEX 16). The acquisition order of seven short-axis slices was iterated in seven sequential acquisitions to obtain seven time points through the cardiac cycle for each slice. Short-axis views were planned from scout images in a coronal plane that were gated to the cardiac and respiratory cycle. Data were analyzed using MRVision software (Winchester, MA, USA). LV ejection fraction (LVEF) was calculated by tracing the endocardial border in end-systolic and -diastolic phases on mid-ventricular short-axis slice.

2.6 Immunofluorescence and confocal microscopy
Six micrometers cryosections were processed for histology and immunohistochemistry following standard procedures. Antibodies used were rabbit anti-Connexin 43, mouse monoclonal anti-{alpha}-sarcomeric actin (Sigma, St. Louis, MO, USA), goat anti-GFP antibody (Rockland, Gilbertsville, PA, USA), and rabbit anti-GFP Alexa-488 conjugated antibody (Molecular Probes, Eugene, OR, USA). As secondary antibodies, Alexa Fluor 555 and 594 were used (Molecular Probes, Eugene, OR, USA). Images were acquired on a Leica DMRB fluorescent microscope and a Zeiss LSM 510 two-photon confocal laser-scanning microscope. Trichrome and H&E stains were used to estimate the extent, distribution, structure and kinetics of ensuing scar after the infarction and injection of cells, the mode of their organization and cellular type. H&E was used to evaluate cellular atypia and nuclear polymorphism, as indicators of tumor formation.

2.7 Morphometry
For all morphometric evaluations, the focused microscopic field was photographed by an adapted camera (Diagnostic Instruments Inc., Sterling Heights, MI, USA). Since the transplanted donor cells form dense conglomerates within host tissue, a cell-by-cell count and there exact evaluation of the number of surviving cells is not possible. However, the bright GFP-signal is present only on living donor cells, and the size of the donor cell colony in three dimensions (3-D) is a reliable estimate for the magnitude of myocardial restoration. Our model has been standardized to produce consistent infarcts of 45–55% of mouse heart muscle. The 3-D measurement of the extent of the infarct is beyond our capacities in the lab, and we doubt that it is possible at all accurately enough. We have made the experience that graft/infarct ratio, when calculated for five sections on five different levels of the LV, gives an accurate estimate for the extent of cellular colonies related to the extent of damage, and even tiny colonies, or single green fluorescent cells cannot escape. The total GFP positive area was measured and related to the infarction area at low magnification (ratio in %), and then at five subsequent cross-sectional levels of the left ventricle. The grafts were photographed and evaluated using the Spot advanced software, version 3.4.2 (Diagnostic Instruments Inc., Sterling Heights, MI, USA).


   3. Statistics
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Statistics
4. Results
 5. Discussion
 References
 
Descriptive statistics included mean and standard deviation of all measured values. Comparison between two study groups was performed using Student's t-test for independent variables using Microsoft Excel 2000, and significance was assumed when p < 0.05. Multivariate between-group differences were analyzed by ANOVA with post hoc Bonferroni test. Statistical analyses were performed with StatView 5.0 (SAS Institute, Cary, NC, USA), and significance was accepted at p < 0.05.


   4. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Statistics
 4. Results
5. Discussion
 References
 
The engrafted cells were identified as human by staining for GFP (Fig. 1 ). They expressed {alpha}-sarcomeric actin (Fig. 1A and B) and Connexin 43 (Fig. 1B). Connexin 43 and {alpha}-sarcomeric actin expression was not contiguous throughout the graft (Fig. 1B). Examination of corresponding slides stained with H&E by a blinded pathologist did not reveal tumor formation, malignant cells, cellular atypia, or nuclear polymorphism.


Figure 1
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  		Fig. 1. Human embryonic stem cells form conglomerates within the infarcted tissue (x1000). (A) The engrafted cells partially express {alpha}-sarcomeric actin (arrows) and (A and B) Connexin 43 (arrow heads) as shown by confocal microscopy (GFP/Alexa 488, Conexin 43/Alexa 555 and {alpha}-sarcomeric actin/Alexa 594).

 
The cardiomyocyte-enriched hESC engrafted and formed "islands" of various size. These conglomerates were distributed throughout the injection area. The "island" size was significantly larger in the hearts if animals treated with allopurinol/uricase and cells versus animals treated with cells alone (64,550 ± 12,230 µm2 vs 26,565 ± 8844 µm2, multivariate comparison between all involved groups, excluding the ibuprofen-group: F = 9.49, p < 0.01). The conglomerates of donor cells were also more voluminous in the ibuprofen-treated animal group versus cells-only treated animals (53,430 ± 11,211 µm2 vs 26,565 ± 8844 µm2, p < 0.01, by t-test, multivariate analysis between Group II and other groups, excluding Group I: F = 10.87, p < 0.01 by ANOVA). There was no significant difference between the allopurinol/uricase and ibuprofen treatment groups pertaining to the size of the donor cell conglomerates (by ANOVA, Fig. 2 A). A precise cell count was not possible due to the density of the ensuing grafts and the intense GFP-signal of the donor cells (autofluorescence excluded by examination at various wave lengths and colocalization). The quality of myocardial restoration defined by the graft/infarct area ratio was as follows: Group I: 8.9 ± 2.2%, Group II: 7.7 ± 1.8%, Group V (cells only): 3.4 ± 1.2% (ANOVA did not reveal a difference between Groups I and II). Myocardial restoration was more extensive in both Group I (p < 0.01) and Group II (p < 0.01) compared with Group V (animals treated with hESC only, without drug supplementation) (Fig. 2B).


Figure 2
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  		Fig. 2. (A) Treatment with differentiated hESC plus allopurinol/uricase (p < 0.01 by t-test, F = 9.49, p < 0.01 by ANOVA) or cells plus ibuprofen (p < 0.01 by t-test, multivariate analysis: F = 10.87, p < 0.01 by ANOVA) results in increased graft volume (groups without cell treatment are not included) and (B) graft/infarct area ratio, compared to animals treated with hESC only, without additional drug treatment (groups without cell treatment are not included). (C) Cardiac function (ejection fraction (EF), measured by 4.7 T MRI) is better in the groups treated with cells plus ibuprofen (p < 0.01 by t-test, F = 15.44, p < 0.01 by ANOVA, excluding Group I) or cells plus allopurinol/uricase (p < 0.01 by t-test, F = 11.52, p < 0.01 by ANOVA, excluding Group II), compared to the hearts treated with hESC alone. (D) Lateral wall thickness is higher in the groups treated with cells plus allopurinol/uricase (p < 0.01) or cells plus ibuprofen (p < 0.01), compared to the group treated with hESC only (measured by echocardiography).

 
The measurements of ejection fraction obtained with MRI were as follows: Group I: 76.6 ± 8.6%, Group II: 78.6 ± 7.3%, Group III: 58.1 ± 5.7%, Group IV: 53.9 ± 5.2%, Group V: 57.7 ± 7.5%, Group VI: 43.5 ± 4.3%, and Group VII: 66.3 ± 7.8% (ANOVA did not reveal superiority of Group II over Group I). The animals treated with allopurinol/uricase and cells displayed a significantly better heart function compared to control mice (p < 0.001 by t-test) and those treated with allopurinol/uricase alone (p < 0.01 by t-test, F = 11.52, p < 0.01). The animals treated with ibuprofen and cells had a better heart function compared to control mice (p < 0.001 by t-test) and those treated with ibuprofen alone (p < 0.01 by t-test, F = 15.44, p < 0.01 by ANOVA). We did not find a significant difference between allopurinol/uricase and ibuprofen treatment in cell using MRI evaluation (Fig. 2C). Finally, lateral wall thickness was higher in the allopurinol/uricase (0.8 ± 0.05 mm, p < 0.05) and ibuprofen (0.8 ± 0.07 mm, p < 0.05) treated groups compared to the controls which received hESC only (0.72 ± 0.06 mm, Student's t-test) (Fig. 2D). Lateral wall thickness was also higher in Groups I and II compared to the groups that had been treated with the drugs only, i.e., Groups III and IV (p < 0.05 and p < 0.05, respectively, comparisons between two groups using Student's t-test for independent samples) (Fig. 2D). The 4.7 T magnet-based MRI provides accurate figures throughout the cardiac cycle and allows for calculation of the EF and regional discrimination of wall thickness in the mouse model (Fig. 3 ). Findings of wall thickness were comparable between Groups I and II (by t-test).


Figure 3
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  		Fig. 3. Small animal MRI using a 4.7 T magnet allows for accurate measurements of ejection fraction (EF) and evaluation of wall thickness throughout the cardiac cycle. Following infarction the left ventricle appears dilated and features thinner left ventricular wall; these changes are less pronounced in the animals receiving differentiated hESC.

 

   5. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Statistics
 4. Results
 5. Discussion
References
 
The restorative effect of a cardiomyocyte-enriched population of embryonic stem cells within injured myocardium is enhanced by concurrent therapy with allopurinol/uricase or ibuprofen. Even though cell survival within ischemic tissue still requires improvement, this study provides evidence that allopurinol/uricase or ibuprofen treatment along with cell transplantation might have a synergistic effect on the resulting heart function. Obviously, the mouse heart is small enough to be significantly restored with 106 and higher populations might not merit heart function further (as indicated by preliminary comparative studies in our laboratory). The main motivation to inject 1,000,000 hESC-derived cardiomyocytes is that the likelihood of cell survival and the extent of the ensuing intramyocardial conglomerates is higher and facilitates histology and characterization of the injected cells. Compared to other types of cells human ESC still process the potential to form myotubes and revitalize/stabilize the area of injury. There may be a threshold (at least in the present model) for a cell population to be injected into the infracted LV wall. It might be of no benefit to exceed it. Injection of higher populations may further jeopardize viability of the graft. Yet, these observations remain speculative. Cardiac function might have also benefited from higher lateral wall thickness in the groups treated with drug + cells, by prevention of further wall thinning through the cell injection (according to the Laplace law of the heart). A viable graft which maintains the thickness of the otherwise reversely remodeling, scarred lateral wall prevents an increase of circumferential wall stress within the wall, thereby decreasing free radical formation, cell death, non-ischemic extension of the infarct and loss of heart function. Hence, the improvement of heart function is not only the result of the transfer of "viable matter" (the cells) but also the structural support within the area of ischemia that prevented thinning of the LV wall in these animals. According to the law of Laplace, the circumferential wall stress increases greatly within the diseased myocardium that becomes aneurysmatic. Concurrent experiments in our laboratory, which involve transplantation of cell-free collagen matrices within an area of acute myocardial injury underscore this effect (data not shown).

One could argue that, had we measured EF multiple times, we would have obtained an analytical trend of heart function until 3 weeks post-cell injection. The 3 weeks EF, however, remains the same, even if you check upon it just once. By designing a control group of non-infarcted animals we provide a before–after assessment of heart function. Of note, the present study was not designed to evaluate the mechanism of functional improvement of the injured heart, hence we do not claim that an optimized regimen involving multiple drugs or cells injected at later time points would not display a more beneficial effect. Moreover, the major criteria for choosing these agents to enhance myocardial restoration of embryonic stem cells were their well-described cytoprotective action and the significant amount of leukocyte and macrophage infiltration we have observed within similar grafts in preliminary experiments [17] without this supplemental treatment. Inflammation and rejection partially develop over common pathways. Therefore, the anti-inflammatory action of ibuprofen might mask or attenuate a possible immune response to the donor cells.

There are a plethora of substances which might enhance stem cell engraftment in various combinations. We wished to focus on the specific impact of one drug. It will require extensive studies to identify the optimal combination, with unlimited combinatory approaches; and still, the optimal adjuvant treatment would not be found.

Our preliminary experience with primordial cell types such as hematopoietic stem cells, myoblasts and embryonic stem cells strongly favors the cell type used in this study [15,16]. Embryonic stem cells retain in vivo plasticity and are capable of giving rise to target organ-specific progeny. The utilization of hESC population enriched for hESC-derived cardiomyocytes was selected to avoid tumor formation. Malignancy within the graft was not observed at the time of organ harvest for histology, but such a fate of the donor cells cannot be excluded in the long term. We are skeptical toward the clinical use of human embryonic stem cells in the future, unless this pre-eminent issue is resolved, down to the last tumorigenic cell. Further studies will address tumorigenicity and may involve down regulation of oncogenes in tissue culture. Also, the immunogenic potential of embryonic stem cells needs to be further addressed and eventual immunosuppressive strategies need to be developed [18–20].

The reason for the utilization of MRI, despite of the additional labor (compared to echocardiography) is the higher resolution and more accurate measurements that can be obtained. The entire cardiac cycle can be "frozen" and stored, to be evaluated later on by blinded examiners. The image acquisition can be further synchronized with ECG and respiration recordings. A magnet of 4.7 T is a powerful tool for cardiac imaging in mice, and mathematical physical optimization and extrapolations are underway to facilitate complex hemodynamic measurements. MRI-magnets of 7 and 9 T are being built and installed in few institutions in the US and Europe and would allow for superb image quality.

ESC emerge as the panacea for advanced disease of parenchymatous organ. Reports cumulate on their use for the functional restoration of pancreas and central nervous system. For the heart, ESC can be used either via direct and guided injection into the target area following myocardial injury, they could be enriched with growth factors or transfected to overexpress a specific protein and then injected. Furthermore they might serve as a substrate for tissue engineering, through inoculation into 3-D matrices. The optimal mode of cell transfer and the post-infarction phase they should be administered at, still remains to be defined. The more profound study of differentiation processes can only be guaranteed if the microenviromental niche which hosts the injected cells allows them to survive and interact with the host tissue. Anti-inflammatory and cytoprotective drugs are very likely to be crucial parts of cell-based restorative procedures in the future.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Statistics
 4. Results
 5. Discussion
 References
 

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