(Circulation. 2002;105:93.)
© 2002 American Heart Association, Inc.
Basic Science Reports |
From the Department of Medicine, Division of Cardiology, Johns Hopkins School of Medicine (C.T., P.D.K.), and Osiris Therapeutics, Inc (M.F.P.), Baltimore, Md; and the Powell Gene Therapy Center, Departments of Pediatrics, Molecular Genetics, and Microbiology, University of Florida School of Medicine, Gainesville, Fla (K.S.C., B.J.B.).
Correspondence to Mark Pittenger, PhD, Osiris Therapeutics Inc, 2001 Aliceanna St, Baltimore, MD 21231. E-mail mpittenger{at}osiristx.com
| Abstract |
|---|
|
|
|---|
Methods and Results A small bone marrow aspirate was taken from the iliac crest of healthy human volunteers, and hMSCs were isolated as previously described. The stem cells, labeled with lacZ, were injected into the left ventricle of CB17 SCID/beige adult mice. At 4 days after injection, none of the engrafted hMSCs expressed myogenic markers. A limited number of cells survived past 1 week and over time morphologically resembled the surrounding host cardiomyocytes. Immunohistochemistry revealed de novo expression of desmin, ß-myosin heavy chain,
-actinin, cardiac troponin T, and phospholamban at levels comparable to those of the host cardiomyocytes; sarcomeric organization of the contractile proteins was observed. In comparison, neither cardiac troponin T nor phospholamban was detected in the myotubes formed in vitro by MyoD-transduced hMSCs.
Conclusions The purified hMSCs from adult bone marrow engrafted in the myocardium appeared to differentiate into cardiomyocytes. The persistence of the engrafted hMSCs and their in situ differentiation in the heart may represent the basis for using these adult stem cells for cellular cardiomyoplasty.
Key Words: stem cells genes myocytes heart failure
| Introduction |
|---|
|
|
|---|
The ideal candidate for cellular cardiomyoplasty is likely to be a less committed cell that can undergo full cardiogenic differentiation. Such a cell population might be found in the adult bone marrow. It is now accepted that an adherent population of cells isolated from bone marrow and expanded in vitro represents a potential source of undifferentiated mesenchymal stem cells (MSCs) that can give rise to connective tissue cell types. Such human MSCs (hMSCs) were first isolated and shown to have multiple differentiation potential by Haynesworth et al.7 Recently, we clonally isolated hMSCs, culture-expanded them, and demonstrated their multilineage potential by in vitro methods.8
In vivo evidence supports the idea that MSCs undergo myogenic differentiation. For example, a murine MSC population isolated from mouse bone marrow and injected into the quadriceps of the mdx mouse demonstrated that the cells could locally contribute dystrophin to the muscle fiber sarcolemma.9 When bone marrow cells from a normal male mouse were injected intravenously into the tail vein of affected mdx female mice, skeletal myotubes in the recipients were found to contain Y-positive nuclei.10 Mouse MSC-like cells converted to a myogenic phenotype after infusion and homing to cardiotoxin injured skeletal muscle.11 Also, after treatment with the DNA demethylation agent 5-azacytidine, a murine MSC-like cell line was shown to express cardiac differentiation markers and exhibit spontaneous membrane depolarization in vitro.12 Recently, Bittner et al13 showed that bone marrowderived cells could be recruited to both skeletal and cardiac muscle in the mdx mouse, in which these striated muscles undergo continual remodeling.
In the results reported here, hMSCs were injected into the hearts of mice to test whether hMSCs may undergo "milieu-dependent differentiation." Early passage, multipotential hMSCs were tagged with ß-galactosidase and injected into the left ventricle of immunodeficient mice. The animals were killed at several time points, and the hearts were analyzed for the differentiation of the engrafted hMSCs.
| Methods |
|---|
|
|
|---|
Adenovirus Vectors
Replication-deficient recombinant adenoviruses carrying the ß-galactosidase reporter gene lacZ under the control of either cytomegalovirus immediate-early promoter (CMV) or Rous sarcoma virus long-terminal repeat (RSV-LTR) promoters were purchased from the University of Iowa Gene Vector Core (Dr Richard Anderson). To test the vectors, hMSCs were plated at a density of 10 000 cells per well (subconfluent). Incremental concentrations of virus over the range of 100 to 2000 multiplicity of infection (MOI) were applied overnight in serum-free DMEM to determine an optimal transduction efficiency. The next day, the virus supernatant was removed, and the cells were washed with several changes of DMEM containing 10% FBS. Two days later, the cells were fixed with 0.2% glutaraldehyde for 10 minutes, and X-gal staining was performed according to procedure (Kirkegaard and Perry Laboratories). The ß-galactosidasepositive (ß-gal+) blue cells found in 10 microscopic fields (x20) were counted and expressed as a percentage of the total number of cells in those fields.
Cell Transplantation
Cells obtained from 4 different human donors were used for cell transplantation experiments. First passage hMSCs were plated at low density in DMEM with 10% FBS. Two to 5 days later, the cells were washed with serum-free DMEM and infected overnight with AdRSVlacZ at 1000 MOI. The supernatant was then removed, and cells were washed with DMEM with 10% FBS. The medium was repeatedly changed over 2 days (
5 changes) to ensure complete removal of viral particles and to allow for internalization of any virus particles remaining on the surface. On the day of the surgery, the cells were harvested with 0.02% trypsin-EDTA (Sigma), washed with PBS, and resuspended. The cells were kept on ice until they were implanted, usually within 20 minutes.
Immunodeficient CB17 SCID/beige male adult mice (4 to 10 weeks old) were used as recipients. These mice lack the ability to mount an adaptive immune response, either B- or T-cellmediated, and also lack functional natural killer cell activity. All animal handling and surgical procedures were performed in accordance with the Johns Hopkins University Animal Care and Utilization Committee review and institutional guidelines. Inhalatory methoxyflurane was used for general anesthesia. A volume of 100 µL of cell suspension containing 500 000 or 1 million cells was injected into the left ventricle with a 32-gauge needle through a transdiaphragmatic approach. Once blood backflow was observed in the syringe, the needle was advanced
1 mm farther, and the cell suspension was slowly injected. The abdominal wall incision was then closed with 5.0 silk suture. An aliquot of the cells for injection was replated and histochemically stained to confirm continued expression of ß-galactosidase at 1 day and 1 and 2 months.
Immunohistochemistry
The 16 animals analyzed for cell engraftment in the study were killed with an overdose of methoxyflurane at 30 minutes (n=1) and 4 (n=3), 14 (n=3), 21 (n=2), 30 (n=4), and 60 (n=3) days after surgery. Tissues were harvested, cryoprotected in 30% sucrose for 4 hours, and snap-frozen in melting isopentane/dry ice. The tissues were embedded in Tissue-Tek OCT (Sakura), and 10- to 16-µm cryostat sections were obtained. During cryosectioning of the hearts, 3 adjacent sections were cut and placed on separate Superfrost Plus slides (VWR). The middle section of 3 was used for X-gal staining, and the adjacent sections on each side were processed for immunohistochemistry. Tissue sections to be stained for ß-galactosidase were fixed with 0.2% glutaraldehyde and 2% formaldehyde for 15 minutes at room temperature and then incubated in X-gal solution overnight at room temperature. For immunostaining, the tissue sections were fixed for 2 minutes at room temperature with either acetone or acetone/methanol 50%/50% vol/vol and then incubated with PBS containing 5% goat serum. The sections were incubated for 60 minutes with a polyclonal rabbit antiß-galactosidase antibody (Corning Biochem) at a concentration of 10 µg/mL in 2% goat serum in PBS, followed by FITC-conjugated goat-anti rabbit IgG for 45 minutes. After thorough washing, the second primary antibody was applied for an additional 60 minutes. These included either a mouse monoclonal antibody directed against desmin (clone D33, used at 1:100 dilution, Dako Corp), striated muscle
-actinin (EA-53, 1:100, Sigma), ß-myosin heavy chain (MHC) (A4.1025, 1:10, DSHB, University of Iowa, Iowa City), MyoD (5.8A, 1:50, Novacastra Biochemicals), or phospholamban (MA3-922, 10 µg/mL, Sigma/RBI). This was followed by TRITC-conjugated goat anti-mouse IgG (1:200, Jackson ImmunoResearch Laboratory) for 45 minutes. Cardiac troponin T staining was performed with a goat polyclonal antibody (C19, diluted 1:20); in this case, a TRITC-conjugated donkey anti-goat antibody was applied first, followed by the FITC-conjugated goat anti-rabbit antibody for ß-galactosidase identification. Fluorescence imaging was performed with a Zeiss Axiovert equipped for epifluorescence or a Nikon PCM 200 confocal microscope.
In Vitro Myogenic Differentiation
For comparison, first-passage hMSCs were used for in vitro skeletal myogenic differentiation experiments with MyoD. The cells were plated on 12-well plastic culture dishes at high density (50 000 cells/well) and infected the next day with an adenovirus containing the human MyoD gene under the control of the RSV promoter (kindly donated by Dr Charles Murry, University of Washington, Seattle) at 1000 MOI. The cells were maintained in low-serum medium (2% horse serum) for 1 month and processed for immunohistochemistry with the same antibodies and methods as described.
| Results |
|---|
|
|
|---|
|
In Vivo Engraftment of Stem Cells
The goal of these experiments was to assess the ability of hMSCs to engraft and differentiate in the adult myocardium. For this, 5x105 to 10x105 AdRSVlacZ-labeled hMSCs were injected through the diaphragm into the left ventricular chamber of immunodeficient mice. At 4 days after injection, the majority of the ß-gal+ cells were identified in the spleen, liver, and lungs (Figure 2, B through D). In the heart, we did not identify hMSCs in 4 of 16 animals, probably as a result of the technical difficulties associated with injecting into the beating myocardium. In 12 animals, hMSCs were dispersed throughout the myocardium as individual cells at 4 to 60 days (Figure 2A and Figure 3). Quantification of the ß-gal+ hMSCs from serial sections estimated that
2200 hMSCs survived in the left ventricle 4 days after the injection of 500 000 cells (0.44%). Fewer hMSCs were identified in the myocardium at later time points, but
1 ß-gal+ cell per tissue section was found.
|
|
With time, most of the ß-gal+ cells became morphologically indistinguishable from the surrounding cardiomyocytes. At 14 days and beyond, the persistent ß-gal+ hMSCs were rod-shaped and well aligned with the host cardiomyocytes (compare Figure 3A with 3B through 3D). Moreover, many of the engrafted cells became enlarged from a diameter of 20 to 30 µm at 4 days to a long axis of 50 to 70 µm and a diameter of
20 µm at 2 months. Immunofluorescence staining for ß-galactosidase also confirmed the presence of the engrafted hMSCs, although this method was less sensitive than X-gal staining. Double-label immunofluorescence staining with an antiß-galactosidase antibody was used to identify engrafted cells, and additional antibodies characterized the myogenic protein expression of the engrafted stem cells. Monoclonal antibodies directed against desmin,
-actinin, phospholamban, ß-MHC, cardiac troponin T, and MyoD were used and visualized with a rhodamine-conjugated secondary antibody. None of the hMSCs from 4 different donors used in this study were reactive with these muscle protein antibodies when stained during in vitro culture.
Neither the hMSCs at the injection site nor those dispersed in the myocardium expressed desmin at 4 days (Figure 4, A and B, respectively). Desmin- and cardiac troponin Texpressing ß-gal+ cells were first identified at 14 days, whereas all identified hMSCs were positive for desmin expression at 60 days. Figure 5A clearly shows the double-positive cells in the same optical plane with antiß-galactosidase labeling (fluorescein green) and the cardiac troponin T labeling (rhodamine red) at 14 days. The ß-gal+ cells were also positive for
-actinin, a component of the Z bands shown at 60 days in Figure 5B. Similarly, engrafted cells were positive for MHC (Figure 5C). Engrafted hMSCs were also found to express phospholamban, a phosphoprotein that plays a key role in modulating the cardiac sarcoplasmic reticulum Ca2+-ATPase (Figures 5D, 6E, and 6F). High-magnification imaging of these cells showed sarcomeric organization of the
-actinin (Figure 6, C and D), cardiac troponin T, and desmin (not shown).
|
|
|
None of the hMSCs identified on the cardiac sections reacted with the MyoD antibody. To further distinguish between cardiac and skeletal myogenic differentiation of the hMSCs implanted in the heart, we analyzed the expression of these markers to those expressed in myotubes formed by MyoD-transduced hMSCs in vitro. The MyoD-transduced hMSCs expressed high levels of nuclear MyoD shortly after infection with AdRSVMyoD. After MyoD transduction, adjacent hMSCs fused and formed multinucleated myotubes. At 1 month, the differentiated cells expressed desmin,
-actinin, and ß-MHC, but no detectable levels of phospholamban or cardiac troponin T were observed (Figure 7). Again, sarcomeric organization of the contractile proteins was observed with high magnification, indicating differentiation to a mature myotube.
|
| Discussion |
|---|
|
|
|---|
Desmin is known to represent an early marker of myogenic differentiation synthesized by muscle progenitor cells. In these experiments, none of the engrafted hMSCs expressed desmin at 4 days, whereas at 60 days, all of the identifiable ß-gal+ cells expressed desmin. The hMSCs also expressed cardiac-specific troponin T, ß-MHC,
-actinin, and phospholamban (Figure 5), and sarcomeric striations were evident at high magnification. Significantly, the intensity of immunostaining for these proteins in the engrafted hMSCs was indistinguishable from the surrounding cardiomyocytes.
The expression of myogenic markers in the engrafted cells was compared with the expression found in myotubes obtained by hMSCs expressing MyoD in vitro. It has been shown that forced expression of MyoD in cells of mesodermal origin results in myotube formation of both the fast- and slow-twitch types, indistinguishable from those formed by primary myoblasts.16,17 The MyoD-transduced hMSCs expressed desmin,
-actinin, and ß-MHC but no detectable phospholamban or cardiac troponin T, two constitutive proteins restricted mainly to cardiac muscle. Together, the data are suggestive of conversion of engrafted hMSCs to a mature cardiac muscle rather than slow-twitch skeletal muscle. Although it is theoretically possible that the hMSCs have converted to a skeletal muscle phenotype and then adapted to the cardiac environment as suggested for C2C12 cells,3 we find this unlikely, given the data.
Bone marrow MSCs, as well as cardiomyocytes, are derived from early mesoderm, although they form from different embryonic regions. The cardiomyocyte progenitors form from a ventrally situated portion of the mesoderm, becoming committed to their fate under the influence of differentiation factors secreted by the closely adjacent endoderm (see review18). Adult cardiomyocytes actively produce insulin-like growth factor, transforming growth factor (TGF)-ß,19 and heparin-binding epidermal growth factorlike growth factor,20 which act as autocrine growth stimuli for cardiomyocytes. TGF was shown to induce cardiac differentiation in an avian embryonic mesodermal cell line,21 and the TGF-ß signaling molecule activin is involved in early cardiogenesis in amphibians and birds.18 Locally produced neuregulins promote survival and growth of cardiac myocytes via ErbB2 and B4 receptors, which also bind heparin-binding epidermal growth factorlike factor.22 In the present study, the differentiation process observed in vivo most likely involves a combination of paracrine growth signals and the electrical and mechanical stimulation present in the adult heart.
The recent reports of the ability of marrow cells to migrate from bone marrow and integrate and differentiate into the damaged skeletal and cardiac muscle suggest that such a process may normally contribute to tissue maintenance or regeneration,11,13,23 although this process may not be efficient.24 Other recent reports have demonstrated the potential for rat or mouse marrow cells to engraft in the infarcted myocardium14,15,23,25 and offer further support of this possibility. The present study demonstrates the ability of adult hMSCs to integrate and undergo striated muscle differentiation in the adult heart and opens the possibility for using these human adult stem cells for therapeutic cardiomyoplasty.
| Acknowledgments |
|---|
| Footnotes |
|---|
Received June 28, 2001; revision received October 15, 2001; accepted October 25, 2001.
| References |
|---|
|
|
|---|
This article has been cited by other articles:
![]() |
C. K. Rebelatto, A. M. Aguiar, M. P. Moretao, A. C. Senegaglia, P. Hansen, F. Barchiki, J. Oliveira, J. Martins, C. Kuligovski, F. Mansur, et al. Dissimilar Differentiation of Mesenchymal Stem Cells from Bone Marrow, Umbilical Cord Blood, and Adipose Tissue Experimental Biology and Medicine, July 1, 2008; 233(7): 901 - 913. [Abstract] [Full Text] [PDF] |
||||
![]() |
C. Mias, E. Trouche, M.-H. Seguelas, F. Calcagno, F. Dignat-George, F. Sabatier, M.-D. Piercecchi-Marti, L. Daniel, P. Bianchi, D. Calise, et al. Ex Vivo Pretreatment with Melatonin Improves Survival, Proangiogenic/Mitogenic Activity, and Efficiency of Mesenchymal Stem Cells Injected into Ischemic Kidney Stem Cells, July 1, 2008; 26(7): 1749 - 1757. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. R. Ward and D. J. Stewart Erythropoietin and mesenchymal stromal cells in angiogenesis and myocardial regeneration: one plus one equals three? Cardiovasc Res, June 25, 2008; (2008) cvn153v2. [Full Text] [PDF] |
||||
![]() |
R. Izadpanah, D. Kaushal, C. Kriedt, F. Tsien, B. Patel, J. Dufour, and B. A. Bunnell Long-term In vitro Expansion Alters the Biology of Adult Mesenchymal Stem Cells Cancer Res., June 1, 2008; 68(11): 4229 - 4238. [Abstract] [Full Text] [PDF] |
||||
![]() |
B. Dawn, S. Tiwari, M. J. Kucia, E. K. Zuba-Surma, Y. Guo, S. K. SanganalMath, A. Abdel-Latif, G. Hunt, R. J. Vincent, H. Taher, et al. Transplantation of Bone Marrow-Derived Very Small Embryonic-Like Stem Cells Attenuates Left Ventricular Dysfunction and Remodeling After Myocardial Infarction Stem Cells, June 1, 2008; 26(6): 1646 - 1655. [Abstract] [Full Text] [PDF] |
||||
![]() |
L. A. Mylotte, A. M. Duffy, M. Murphy, T. O'Brien, A. Samali, F. Barry, and E. Szegezdi Metabolic Flexibility Permits Mesenchymal Stem Cell Survival in an Ischemic Environment Stem Cells, May 1, 2008; 26(5): 1325 - 1336. [Abstract] [Full Text] [PDF] |
||||
![]() |
X. Hu, S. P. Yu, J. L. Fraser, Z. Lu, M. E. Ogle, J.-A. Wang, and L. Wei Transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis. J. Thorac. Cardiovasc. Surg., April 1, 2008; 135(4): 799 - 808. [Abstract] [Full Text] [PDF] |
||||
![]() |
S. Dimmeler, J. Burchfield, and A. M. Zeiher Cell-Based Therapy of Myocardial Infarction Arterioscler. Thromb. Vasc. Biol., February 1, 2008; 28(2): 208 - 216. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Kubo, T.-S. Li, R. Suzuki, B. Shirasawa, N. Morikage, M. Ohshima, S.-L. Qin, and K. Hamano Hypoxic preconditioning increases survival and angiogenic potency of peripheral blood mononuclear cells via oxidative stress resistance Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H590 - H595. [Abstract] [Full Text] [PDF] |
||||
![]() |
C.-C. Wang, C.-H. Chen, W.-W. Lin, S.-M. Hwang, P. C.H. Hsieh, P.-H. Lai, Y.-C. Yeh, Y. Chang, and H.-W. Sung Direct intramyocardial injection of mesenchymal stem cell sheet fragments improves cardiac functions after infarction Cardiovasc Res, February 1, 2008; 77(3): 515 - 524. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. Shujia, H. K. Haider, N. M. Idris, G. Lu, and M. Ashraf Stable therapeutic effects of mesenchymal stem cell-based multiple gene delivery for cardiac repair Cardiovasc Res, February 1, 2008; 77(3): 525 - 533. [Abstract] [Full Text] [PDF] |
||||
![]() |
P. Au, L. M. Daheron, D. G. Duda, K. S. Cohen, J. A. Tyrrell, R. M. Lanning, D. Fukumura, D. T. Scadden, and R. K. Jain Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels Blood, February 1, 2008; 111(3): 1302 - 1305. [Abstract] [Full Text] [PDF] |
||||
![]() |
R. P. Gallegos and R. M. Bolman III Stem Cell Induced Regeneration of Myocardium Card. Surg. Adult, January 1, 2008; 3(2008): 1657 - 1668. [Full Text] |
||||
![]() |
J. Chen, A. R. Baydoun, R. Xu, L. Deng, X. Liu, W. Zhu, L. Shi, X. Cong, S. Hu, and X. Chen Lysophosphatidic Acid Protects Mesenchymal Stem Cells Against Hypoxia and Serum Deprivation-Induced Apoptosis Stem Cells, January 1, 2008; 26(1): 135 - 145. [Abstract] [Full Text] [PDF] |
||||
![]() |
Z. Pasha, Y. Wang, R. Sheikh, D. Zhang, T. Zhao, and M. Ashraf Preconditioning enhances cell survival and differentiation of stem cells during transplantation in infarcted myocardium Cardiovasc Res, January 1, 2008; 77(1): 134 - 142. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Koyanagi, P. Bushoven, M. Iwasaki, C. Urbich, A. M. Zeiher, and S. Dimmeler Notch Signaling Contributes to the Expression of Cardiac Markers in Human Circulating Progenitor Cells Circ. Res., November 26, 2007; 101(11): 1139 - 1145. [Abstract] [Full Text] [PDF] |
||||
![]() |
R. Tao, C.-P. Lau, H.-F. Tse, and G.-R. Li Functional ion channels in mouse bone marrow mesenchymal stem cells Am J Physiol Cell Physiol, November 1, 2007; 293(5): C1561 - C1567. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. Rajasingh, E. Bord, H. Hamada, E. Lambers, G. Qin, D. W. Losordo, and R. Kishore STAT3-Dependent Mouse Embryonic Stem Cell Differentiation Into Cardiomyocytes: Analysis of Molecular Signaling and Therapeutic Efficacy of Cardiomyocyte Precommitted mES Transplantation in a Mouse Model of Myocardial Infarction Circ. Res., October 26, 2007; 101(9): 910 - 918. [Abstract] [Full Text] [PDF] |
||||
![]() |
M. Zhang, N. Mal, M. Kiedrowski, M. Chacko, A. T. Askari, Z. B. Popovic, O. N. Koc, and M. S. Penn SDF-1 expression by mesenchymal stem cells results in trophic support of cardiac myocytes after myocardial infarction FASEB J, October 1, 2007; 21(12): 3197 - 3207. [Abstract] [Full Text] [PDF] |
||||
![]() |
Y. Wu, L. Chen, P. G. Scott, and E. E. Tredget Mesenchymal Stem Cells Enhance Wound Healing Through Differentiation and Angiogenesis Stem Cells, October 1, 2007; 25(10): 2648 - 2659. [Abstract] [Full Text] [PDF] |
||||
![]() |
A. Y. Sheikh, S.-A. Lin, F. Cao, Y. Cao, K. E.A. van der Bogt, P. Chu, C.-P. Chang, C. H. Contag, R. C. Robbins, and J. C. Wu Molecular Imaging of Bone Marrow Mononuclear Cell Homing and Engraftment in Ischemic Myocardium Stem Cells, October 1, 2007; 25(10): 2677 - 2684. [Abstract] [Full Text] [PDF] |
||||
![]() |
J. Feygin, A. Mansoor, P. Eckman, C. Swingen, and J. Zhang Functional and bioenergetic modulations in the infarct border zone following autologous mesenchymal stem cell transplantation Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1772 - H1780. [Abstract] [Full Text] [PDF] |
||||
![]() |
W. Li, N. Ma, L.-L. Ong, C. Nesselmann, C. Klopsch, Y. Ladilov, D. Furlani, C. Piechaczek, J. M. Moebius, K. Lutzow, et al. Bcl-2 Engineered MSCs Inhibited Apoptosis and Improved Heart Function Stem Cells, August 1, 2007; 25(8): 2118 - 2127. [Abstract] [Full Text] [PDF] |
||||
![]() |
H. Aupperle, J. Garbade, A. Schubert, M. Barten, S. Dhein, H.-A Schoon, and F.-W Mohr Effects of Autologous Stem Cells on Immunohistochemical Patterns and Gene Expression of Metalloproteinases and Their Tissue Inhibitors in Doxorubicin Cardiomyopathy in a Rabbit Model |