PRECLINICAL STUDY
Recipient Age Determines the Cardiac Functional Improvement Achieved by Skeletal Myoblast Transplantation
Chung-Dann Kan, MD*,
,
Shu-Hong Li, MSc*,
Richard D. Weisel, MD*,
Shun Zhang, BSc* and
Ren-Ke Li, MD, PhD*,*
* Division of Cardiovascular Surgery, Toronto General Research Institute, University of Toronto, Toronto, Ontario, Canada
Department of Surgery, National Cheng Kung University Hospital Institute of Clinical Medicine, Cardiovascular Research Center, Medical College, National Cheng Kung University, Taiwan, Republic of China
Manuscript received March 20, 2007;
revised manuscript received June 4, 2007,
accepted June 5, 2007.
* Reprint requests and correspondence: Dr. Ren-Ke Li, MaRS Centre, Toronto Medical Discovery Tower, 3rd Floor, Room 702, 101 College Street, Toronto, Ontario, Canada M5G 1L7. (Email: RenKeLi{at}uhnres.utoronto.ca).
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Abstract
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Objectives: The aim of the current study was to evaluate the effect of recipient age on the regenerative response to implantation with young skeletal myoblasts (SKMCs) after a coronary artery ligation.
Background: In contrast with previous findings in animals, the initial clinical trials of cell transplantation after a myocardial infarction have reported only limited improvements in ventricular function. The restricted regenerative capacity of cells isolated from older patients is certainly a factor; however, the present study investigated the impact of another potentially significant factor: recipient age.
Methods: We compared the myogeneic capacities of SKMCs isolated from young rats (3 months old) and older rats (24 months old). Highly myogenic SKMCs derived from young rats (or culture media, in control rats) were then transplanted into the infarcted myocardium of young and older recipients at 1 week after coronary ligation.
Results: In vitro, proliferation and myotube formation were significantly greater in SKMCs derived from young rats than from older rats. In vivo, young and older recipients of SKMCs exhibited increases in cell density, vascular density, and collagen preservation relative to age-matched control animals. However, cell therapy produced significantly greater functional improvements in young recipients than in older, along with relative increases in stem cell factor, cell density, cell survival, and angiogenesis.
Conclusions: Functional improvement after the post–myocardial infarction implantation of young SKMCs was limited in older recipients, likely due to reductions in their cardiac and systemic responses to cell transplantation.
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Abbreviations and Acronyms
| | EF = ejection fraction | | FAC = fractional area change | | FS = fractional shortening | | MHC = myosin heavy chain | | MI = myocardial infarction | | SCF = stem cell factor | | SKMC = skeletal myoblast |
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Accumulated preclinical data demonstrated effective preservation of ventricular morphometry and function in the infarcted heart after implantation of skeletal myoblasts (SKMCs), endothelial progenitor cells, or bone marrow cells (1–3). However, preliminary clinical trials using autologous SKMCs, bone marrow-derived stem cells, or progenitor cells to treat patients suggest that the benefits of implanting cells from middle-aged or older patients are significantly limited compared with those obtained in preclinical studies with cells from young animals (4).
Several reports have proposed that the age of the donor cells may have restricted the effectiveness of cell implantation in some of the clinical trials (5–8). We attempted to determine the contribution of another potentially significant factor, recipient age, on the functional response to the implantation of young, healthy cells after a myocardial infarction (MI).
In the current study, we characterized SKMCs isolated from young and older donor rats, then transplanted highly myogenic SKMCs from young rats into the infarcted myocardium of young and older recipient rats. We hypothesized that an age-related deficiency in the intrinsic regenerative response to injury might limit the effects of cell transplantation in older individuals.
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Methods
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Animals.
Inbred male Fisher rats (3 months old = young; 24 months old = older; U.S. National Institute of Aging) were used as donor and recipient animals. All procedures were performed with the approval of the Animal Care Committee of the Toronto General Research Institute.
SKMC growth and myotube formation studies.
Skeletal myoblast cells were isolated from young and older rats as previously described (8). Cell culture, myotube formation, and cell growth study were carried out as described in Appendix E1. Myotubes formed by the SKMCs were identified by immunohistochemical staining using antibodies against myosin heavy chain (MHC) (1:50) (Santa Cruz, Santa Cruz, California) and desmin (1:2,000) (Sigma, Toronto, Ontario, Canada) performed according to manufacturer’s protocols.
MI.
Myocardial infarction was generated in young and older rats by ligation of the left anterior descending artery as described previously (8) and in Appendix E1.
Cell preparation and transplantation.
Since the focus of the current study was on the effect of recipient age, rather than cell donor age, on the efficacy of cell therapy, recipients from both age groups received highly myogenic SKMCs isolated from young donor rats. The SKMCs were labelled before transplantation for cell identification as described in Appendix E1.
One week after ligation, echocardiography was performed and rats were selected to minimize variation (8). Young and older rats with infarct lengths between 0.7 and 1.2 cm and percent fractional area change (%FAC) between 20% and 40% were divided into 4 groups: young and older recipients of SKMCs isolated from young donors; young and older recipients of culture media (control animals). In each rat, cells (3 x 106 cells per rat; n = 9 rats per age group) or culture media (n = 6 rats per age group) were injected into the central infarcted region as previously described (8).
Cardiac function.
Before and 1 week after coronary ligation (before cell transplantation) and 4 weeks after cell transplantation, echocardiography was performed. Left ventricular diastolic and systolic dimensions and areas were measured, and percent fractional shortening (FS), percent ejection fraction (EF), and %FAC were calculated as described in Appendix E1.
Cardiac function and ventricular volumes were also evaluated at 4 weeks after cell transplantation using a pressure-volume catheter, as described in Appendix E1. Pressure–volume loops were used to quantify stroke work.
Histology and immunohistochemistry.
At 4 weeks after transplantation, hearts were fixed in 10% formaldehyde and sectioned. Sections were stained with Masson trichrome or Verheoff’s Van Geison stain, or immunohistochemically stained for von Willebrand factor or bromodeoxyuridine (BrdU), according to manufacturer’s protocols. Four or 5 fields from each of 5 sections per animal were randomly selected for analysis as described in Appendix E1. Counts were expressed as the number of nuclei (cell density), blood vessels (vascular density), or BrdU+ nuclei (implanted cell survival), or the percentage collagen preservation per 0.2 mm2.
Stem cell factor (SCF) protein levels.
Stem cell factor levels were measured in the central and peripheral infarcted regions of animals in all groups (n = 4/group) at 7 days after cell or media implantation using a commercially available enzyme-linked immunoabsorbent assay kit (R&D Systems, Minneapolis, Minnesota) according to manufacturer’s protocols.
Statistical analyses.
All data are expressed as mean ± standard error (SEM). Multiple group comparisons were performed by analysis of variance, using the SPSS software (version 12.0, SPSS Inc., Chicago, Illinois). When F values were significant (p < 0.05), the differences were specified by a post-hoc evaluation with Tukey’s and/or least squares difference multiple range tests. Survival data were depicted with Kaplan-Meier survival curves, and differences between groups were determined by log-rank statistics. Statistical significance was assumed when p < 0.05.
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Results
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In vitro study.
SKMCs Isolated from Young Donors Exhibited the Greater Myogenic Potential
The morphologies of SKMCs cultured from young and older rats were similar (Figs. 1A to 1J). Proliferation rates of cells derived from young animals were significantly greater (p < 0.05) than those of cells from older animals at passages 1 and 2, but were similar between groups at later passages (n = 5/group/time point) (Fig. 1K).
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Figure 1 SKMCs in Culture
Light photomicrographs (100x) illustrating the morphology of cultured skeletal myoblast cells (SKMCs) derived from young (A to E) and older (F to J) rats, at passages 1 through 5 (P1 to P5). (K) Growth rate of SKMCs isolated from young and older rats at P1 to P5. Arrows = myotube structures.
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To evaluate cell differentiation, cells from each passage at 80% confluency were cultured in a 5% fetal bovine solution medium for 7 days. In both young and older animals, cells positive for MHC or desmin decreased from more than 90% to 80% or nearly zero, respectively, by passage 5 (Figs. 2A to 2H). Myotube formation was observed in all cell cultures, though numbers were significantly greater (p = 0.01) in cells derived from young relative to older animals. Numbers of myotubes and spontaneously beating myotubes decreased significantly (p = 0.01) by passage 5 in both age groups (n = 5/group/time point) (Fig. 2I).
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Figure 2 Cell Differentiation and Myotube Formation
Light photomicrographs (100x) showing cultured cells derived from young and older rats, stained for myosin heavy chain (MHC) and desmin at passages 1 (P1) (A to D) and 5 (P5) (E to H). (I) The number of myotubes in cell cultures derived from young and older rats at passages 1 and 5. *p = 0.01 compared with same group at P1.
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In vivo study.
The Functional Effects of Young SKMCs Were Dependent on Recipient Age
Survival: all of the young rats survived for the 35-day period after MI. In comparison, the survival rate was significantly lower (p < 0.05) for older control rats (<70%). Skeletal myoblast cell implantation appeared to increase survival in older recipients relative to control animals, but the improvement did not reach significance (p = 0.07) (Fig. 3).
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Figure 3 Post-Infarction Survival Rates
Survival rates over 5 weeks (35 days) after myocardial infarction (MI) in young rats implanted with young skeletal myoblasts (young SKMC) or culture media (young control), and older rats implanted with young SKMCs (older SKMC) or media (older control), expressed as a percentage of total animals in each group.
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Cardiac function: cardiac function was evaluated by echocardiography (before ligation, before transplantation, 4 weeks after transplantation) and pressure-volume catheter measurements (4 weeks after transplantation).
Echocardiography: %FS, %FAC, and %EF decreased significantly in all groups at 1 week after ligation (Table 1), with no differences among groups. After media implantation, cardiac function continued to decrease in both control groups. This deterioration was diminished in the older recipients of SKMCs, and was reversed in the young recipients after SKMC implantation.
Differences between each functional measure before and after implantation were calculated for each rat, and showed that function deteriorated progressively (p < 0.05) after implantation in both young and older control groups, but the heart failure was more severe (p < 0.05) in the older rats. Cardiac function improved in all recipients of SKMCs, but the improvement was greater (p < 0.05) in the young compared with the older recipients (Fig. 4).
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Figure 4 Cardiac Functional Change by Echocardiography
Changes in percent fractional shortening (FS) (A), percent fractional area change (FAC) (B), and percent ejection fraction (EF) (C) measured immediately before cell or media implantation (pre-TX) and 4 weeks after implantation (post-TX), in young rats implanted with young skeletal myoblasts (young SKMC) or culture media (young control), and older rats implanted with young SKMCs (older SKMC) or media (older control). *p < 0.05 compared with older SKMC group; #p < 0.05 compared with corresponding control group; p < 0.05 compared with older control group.
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Pressure-volume measurement: functional evaluation confirmed improved (p < 0.05) stroke work and negative maximum derivative of pressure divided by derivative of time in the young compared with the older recipients of SKMCs. Cell implantation decreased both left ventricular end-systolic volume and left ventricular end-diastolic volume (p < 0.05) in the young recipients of SKMCs, but not in the older recipients (online Fig. E1 [see Appendix]).
Older Recipients of SKMCs Exhibited a Limited Regenerative Response to Cell Therapy After an MI
Cellularity: Masson trichrome staining suggested that the density of cells within the infarcted region was significantly greater (p < 0.05) in young and older rats that received SKMC implantation compared with the control animals. Quantification indicated greater (p < 0.05) cell density in young compared with older SKMC recipients (Fig. 5).
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Figure 5 Histologic Study of Implanted Cells at 4 Weeks After Cell or Media Implantation
(A to D) Light photomicrographs (100x) illustrating cellular density (Masson trichrome staining; arrows = cell clusters) in the central infarcted region of young and older rats implanted with young skeletal myoblasts (SKMCs) or media (controls). (E) Quantification of cellular density in each group: young rats implanted with young SKMCs (young SKMC) or culture media (young control), and older rats implanted with young SKMCs (older SKMC) or media (older control).
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Implanted cell survival rate: BrdU immunostaining identified the implanted SKMCs 4 weeks after transplantation. BrdU signals were identified within the infarcted regions of SKMC recipients only, and positive cell counts suggested greater (p < 0.05) implanted cell survival in the young compared with the older recipients (Fig. 6).
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Figure 6 Cell Survival at 4 Weeks After Cell or Media Implantation
(A to D) Light photomicrographs illustrating the localization of bromodeoxyuridine (BrdU) prelabeled skeletal myoblasts (SKMC) (arrows) isolated from young donors, implanted into the central infarcted myocardium of young and older rats. Areas inside black borders in A and B (100x) are shown at higher power (400x) in C and D. (E) Quantification of surviving BrdU+ cells in each group: young rats implanted with young SKMCs (young SKMC), and older rats implanted with young SKMCs (older SKMC).
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Stem cell factor: stem cell factor protein levels measured in the infarcted myocardium at 1 week after implantation were significantly greater (p < 0.05) in young compared with older control animals (media recipients). The implantation of SKMCs significantly increased (p = 0.01) SCF levels (relative to control animals) in both young and older recipients, but protein levels were greater (p = 0.01) in the young than in the older recipients (online Fig. E2 [see Appendix]).
Vasculogenesis: von Willebrand factor staining identified more vascular structures in the peripheral infarcted region of young than older recipients of SKMCs (p < 0.05) at 4 weeks after implantation, and both groups exhibited greater (p < 0.01) vascular densities than the control groups (Fig. 7).
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Figure 7 Vasculogenesis at 4 Weeks After Cell or Media Implantation
(A to D) Light photomicrographs (100x) illustrating blood vessels (von Willebrand factor staining; arrows) in the peripheral infarcted region of young and older rats implanted with young skeletal myoblasts (SKMCs) or media (control). (E) Quantification of vascular density in each group: young rats implanted with young SKMCs (young SKMC) or culture media (young control), and older rats implanted with young SKMCs (older SKMC) or media (older control).
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Collagen: Verhoeff’s Van Geison staining indicated significantly more (p = 0.01) collagen in the central infarcted region of SKMC recipients compared with control animals at 4 weeks after implantation, but the magnitude of preservation was not affected by age (Fig. 8).
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Figure 8 Collagen Preservation at 4 Weeks After Cell or Media Implantation
(A to D) Light photomicrographs (100x) illustrating collagen content (Verheoff’s Van Geison staining) in the central infarcted region of young and older rats implanted with young skeletal myoblasts (SKMCs) or media (control). (E) Quantification of collagen content (percentage of the total scar area per section stained positive for collagen) in each group: young rats implanted with young SKMCs (young SKMC) or culture media (young control), and older rats implanted with young SKMCs (older SKMC) or media (older control).
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Discussion
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Bone marrow stem cells and circulating progenitor cells isolated from older individuals demonstrate weakened regenerative capacities that may contribute to a decline in the cardiac functional response to cell therapy (5–8). In the current study, we demonstrated that recipient age is another important determinant of this response. The implantation of young, healthy SKMCs produced significantly greater functional improvements in young than in older cell recipients.
Of several reports that age decreases the number and proliferative potential of stem cells and contributes to the variable results of clinical cell therapy trials in aging patients, most have focused on bone marrow stem cells, circulating stem cells, or cardiac cells (6–9). Skeletal myoblast cells have been employed effectively to restore cardiac function in preclinical studies (10), and have been evaluated in clinical trials, but few studies have reported the effect of age on their myogenic capacity. The transplantation of cells from older individuals was not the focus of this study. However, we demonstrated the diminished proliferative and myogenic capacity of SKMCs isolated from older rats. These findings are in agreement with those reported by Conboy et al. (11).
The mechanisms responsible for the limited regeneration in older recipients after the transplantation of young SKMCs likely include both cardiac and systemic responses to cell implantation. After an MI, we observed no morbidity or mortality in young rats, but ventricular rupture and heart failure were frequent in older rats even after cell implantation. Limitations in systemic responses such as endothelial progenitor cell mobilization may have restricted ventricular functional recovery in the older rats through changes to the following processes:
- 1 SCF levels: SCF is important for progenitor cell recruitment after an MI (12). Here, lower overall SCF protein levels in the older compared with the young recipients of SKMCs could have limited bone marrow stem cell mobilization, homing and engraftment in the damaged myocardium, thereby reducing neovascularization (12).
- 2 Angiogenesis: blood vessel density was considerably less in the older compared with the young recipients of SKMCs, suggesting that a limited angiogenic response to cell transplantation may have restricted functional recovery in the older recipients.
- 3 Survival of implanted cells: the number of cells that survive to engraft in the infarcted region determines the extent of the paracrine signals that induce progenitor cell recruitment, angiogenesis, and improved cardiac function. We found fewer BrdU-positive cells in the infarcted region of older compared with young recipients of SKMCs. Limited implanted cell survival might have diminished the paracrine signals and limited functional improvement.
Myoblast-induced preservation of the collagen-integrin-myocyte cytoskeletal complex may also account for the functional improvements associated with cell therapy. Collagen matrix preservation after cell implantation was demonstrated in a hamster cardiomyopathy model (13). In the present study, SKMC implantation was associated with enhanced collagen preservation compared with media injection. However, we did not observe a significant age effect.
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Conclusions
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The beneficial effects of post-MI cell therapy using young SKMCs were decreased in older recipients. Future studies will be required to fully describe the nature of the age-related limitation of regenerative capacity, especially because clinical trials involving the transplantation of cells from younger patients (allogenic mesenchymal stromal cells or SKMCs) into older individuals have been proposed. An understanding of the older recipients’ response to the implantation of healthy cells will be important to interpret the results obtained.
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Appendix
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For a list of the supplementary methods, the cardiac function by pressure-volume catheter, and the stem cell factor expression at 7 days after cell or media implantation, please see the online version of this article.
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Acknowledgments
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The authors acknowledge Heather McDonald Kinkaid for her assistance with manuscript editing.
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Footnotes
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Support for this study was received from the Heart and Stroke Foundation of Ontario (NA 5294, T 5206) and the Canadian Institutes for Health Research (MOP 62698, MOP 14795).
Dr. Li is a Career Investigator of the Heart and Stroke Foundation of Canada and holds a Canada Research Chair in cardiac regeneration.
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References
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- Menasche P, Hagege AA, Vilquin JT, et al. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction J Am Coll Cardiol 2003;41:1078-1083.[Abstract/Free Full Text]
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- Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA. Rejuvenation of aged progenitor cells by exposure to a young systemic environment Nature 2005;433:760-764.[CrossRef][Medline]
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Abstract
Methods
