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First published online October 20, 2005
Stem Cells Vol. 24 No. 2 February 2006, pp. 349 -356
doi:10.1634/stemcells.2005-0168; www.StemCells.com
© 2006 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Cartilage-Derived Stromal Cells: Is It a Novel Cell Resource for Cell Therapy to Regenerate Infarcted Myocardium?

Wenjun Sua, Hao Zhanga, Zhuqing Jiab, Chunyan Zhoub, Yingjie Weia, Shengshou Hua

a Research Center for Cardiovascular Regenerative Medicine, the Ministry of Health, and Department of Cardiovascular Surgery, Cardiovascular Institute and Fu-Wai Hospital, CAMS and PUMC;
b Stem Cell Center, Beijing University, Beijing, People’s Republic of China

Key Words. Myocardial infarction • Cartilage • Stromal cells • Cell transplantation

Correspondence: Shengshou Hu, M.D., Research Center for Cardiovascular Regenerative Medicine, the Ministry of Health, and Department of Cardiovascular Surgery, Cardiovascular Institute and Fu-Wai Hospital, CAMS and PUMC, A 167 Beilishilu, West District, Beijing 100037, People’s Republic of China. Telephone: 8610-68334788; Fax: 8610-68313012; e-mail: huss{at}163bj.com


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human cartilage is reported to contain multipotent stromal cells. We evaluated the effect of human cartilage-derived stromal cells (CDSCs) on heart function when transplanted into the infarcted myocardium of rats. CDSCs were isolated and cultured from human articular cartilage and subjected to fluorescence-activated cell sorting (FACS) analysis. The CDSCs were consistently negative for CD14, CD34, CD38, CD45, CD49f, CD104, CD105, CD106, CD117, HLA-DR, and ABCG-2, and positive for CD10, CD44, CD71, CD73, CD90, CD147, and HLA-A, -B, and -C by FACS analysis. Myocardial infarction (MI) was created in rats by ligation of the left anterior descending artery. Three weeks after MI, the CDSCs labeled with Hoechst stain were injected into the infarct and border zone. Echocardiography, histological examination, and reverse transcription-polymerase chain reaction (RT-PCR) were performed 4 weeks after cell transplantation. Echocardiography indicated that CDSC transplantation could improve heart function. The number of capillaries increased in the injection regions in the transplantation group. Histological examination showed that Hoechst-labeled CDSCs in islands within the infarcted region were stained positively for desmin and smooth muscle actin but negatively for alpha-sarcomeric actin and troponin-I. RT-PCR results indicated the expression level of collagen I, collagen III, tissue inhibitor of metalloproteinase-1, transforming growth factor-ß1, and vascular endothelia growth factor were much higher in the scar tissue in the transplantation group than in the medium and control groups. Our findings suggested that CDSCs might promote angiogenesis, prevent left ventricular remodeling, and improve the heart function when transplanted into injured heart in the rat model of myocardial infarction.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The adult heart lacks the potential of effective regeneration. Permanent loss of cardiomyocytes and nonfunctional fibrous scar formation after myocardial infarction (MI) result in irreversible damage of cardiac function, and the subsequent remodeling process leads to expansion of the initial infarct area and dilation of the left ventricular lumen [1–3]. Recent studies have demonstrated that transplantation of multipotent stem cells into damaged myocardium offers a new approach to prevent or even reverse the process of postinfarction remodeling and restore the impaired cardiac function in either cryoinjured or infarcted hearts [4–8]. The human cartilage is reported to contain multi-potent stromal cells that possess the ability of differentiating in vitro into cells of mesenchymal lineage, including adipocytes, osteoblasts, chondrocytes, and neurons [9–11]. It has not yet been proved that human cartilage-derived stromal cells (CD-SCs) could improve the heart function when transplanted into the injured heart. We transplanted CDSCs into the infarct and border zone of injured heart in rat animal model to evaluate the possibility of cell differentiation and the influence on angiogenesis, left ventricle (LV) remodeling, and heart function.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and Culture of Human CDSCs
Cells were isolated from articular cartilage of 12- to 20-week-old spontaneously aborted fetuses as previously described [10]. The Ethical Committee of Peking University Health Science Center and CAMS and PUMC granted permissible use of human tissue. Articular cartilage from glenohumeral, humeroulnar, and knee joints was excised and thoroughly minced. CDSCs were separated by incubation with 0.2% collagenase II for 2 hours with stirring and cultured in Iscove’s modified Dulbecco’s medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 1.25 µg/ml amphorectin B, 2 mM L-glutamine, and 10% fetal calf serum. Cultured cells were seeded at a density of 1 x 105 cells/cm2 and incubated at 37°C with 5% humidified carbon dioxide for 24 hours, nonadherent debris was removed, and adherent cells were cultured at a density of 5 x 103/cm2, with replacement of the medium every 3 to 4 days. When the cells had grown to 80% confluence, they were harvested by trypsinization (0.25% trypsin-EDTA) for 5 minutes at 37°C. Passaging of the cultures (1:3 split) was carried out.

Fluorescence-Activated Cell Sorting
Fluorescence-activated cell sorting (FACS) detection of CDSCs (at passages 3 through 10) was performed according to the procedure of FACS staining described previously [12]. Briefly, cells were detached from the cell culture dish with 0.25% trypsin-EDTA and stained with the following mouse anti-human monoclonal antibodies: fluorescein isothiocyanate (FITC)-labeled antibodies against CD14, CD34, CD45, CD49f, CD90, CD105, CD106, CD 147, HLA-DR, HLA-A, -B, and -C, and phycoerythrin (PE)-labeled antibodies against CD10, CD38, CD44, CD71, CD73, CD104, CD117, and ABCG-2. After staining, cells were washed using 1x phosphate-buffered saline (PBS) and then resuspended in 1 ml of PBS for FACS flow cytometry (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) analysis.

Animals and Myocardial Infarction
Experiments were performed in 8-week-old male Sprague-Dawley rats with an initial body weight of approximately 280 g (n = 90). All procedures were approved by the Animal Care Committee of the Cardiovascular Institute and Fu-Wai Hospital and performed according to the Guide for the Care and Use of Laboratory Animals prepared by the Institutes of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, revised 1996 (NIH publication No. 85-23). Under general anesthesia induced with a ventilation mask with 4%–5% isoflurane and oxygen at 2 to 3 L/min, adult rats were oral-tracheal intubated with an 18-gauge intravenous catheter, and positive pressure ventilation (200 ml/min) was maintained with 2%–3% isoflurane in oxygen using a Harvard ventilator (model 683, Harvard Apparatus, Holliston, MA, http://www.harvardapparatus.com) [13]. Heart rate and electrocardiographic data were monitored during the procedure. After the chest was cleaned and shaved, the rat heart was exposed through a 2-cm left lateral thoracotomy. MI was created by ligation of the left anterior descending (LAD) artery 2 to 3 mm from the tip of the left auricle with 6–0 polypropylene (Ethicon, Somerville, NJ, http://www.ethicon.com) [14, 15]. Successful performance of coronary occlusion is verified by observation of the development of pale color in the distal myocardium and the ventricular arrhythmia in the electrocardiographic monitor after ligation. The muscle layer and skin incision were closed with 3–0 silk sutures, and penicillin G procaine (150,000 U/ml) was given intramuscularly (0.4 ml per rat). Three weeks after MI, the survived rats with left ventricular ejection fraction (LVEF) less than 60% in ultrasonic assessment were randomized into three groups—CDSC group (n = 23), medium group (n = 22), and control group (n = 23).

CDSC Preparation and Transplantation
Before cell transplantation, CDSCs at the fifth passage were harvested and stained with 5 µg/ml Hoechst 33342 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) for 90 minutes at 37°C as described previously [16]. Three weeks after MI, the survived rats with LVEF less than 60% in ultrasonic assessment were randomized into three groups. The infarcted rat heart was exposed through a midline sternotomy under general anesthesia as described previously [13], and 50 µl of suspension containing 5 x 106 cells labeled with Hoechst 33342 was injected into the infarct and border zone with tuberculin syringe (n = 23, CDSC group). Medium-group rats with MI received exactly the same volume (50 µl) of the cell-free medium (n = 22, medium group) [5]. Control rats only underwent the procedure of thoracotomy with the injection of 50 µl of saline (n = 23, control group). After cell transplantation, cyclosporine was administered subcutaneously (10 mg/kg) every day in both CDSC and medium groups for immunosuppression.

Evaluation of Left Ventricular Function
Transthoracic echocardiography was performed 3 weeks after MI (as baseline, before cell transplantation) and 4 weeks after cell transplantation according to the protocols described previously [3, 17]. The animals were anesthetized with a ventilation mask with 4%–5% isoflurane during the procedure. Under anesthesia, the chest was shaved and a layer of acoustic coupling gel was applied. A commercially available echocardiographic system equipped with a 12-MHZ probe (Philip 5500; Philip Corp., Beijing, http://www.philips.com.cn) was used in the measurement. Initially, a two-dimensional short-axis view of the LV was obtained at the level of the papillary muscles. After optimizing gain settings and ensuring that the image was on axis, M-mode tracings were recorded through the anterior and posterior LV walls at a paper speed of 100 mm/sec. This orientation was chosen to allow delineation of wall thickness and motion in infarct and noninfarct territories. The results were recorded on optical discs, and the M-mode tracings were analyzed. Relative posterior wall thickness and LV internal dimensions were measured from at least three consecutive cardiac cycles. We also used posterior wall thickening, endocardial fractional shortening (FS), and LVEF as indices to estimate LV systolic function.

Identification of Transplanted Cells and Histological Analysis
Within 3 days after follow-up echocardiography, all animals were killed with an overdose of ketamine and pentobarbital 4 weeks after cell transplantation. The hearts were quickly removed and the ventricles were cross-sectioned into three specimens from apex to base, and each of these was cut into two pieces. The first one was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to yield 8-µm slices. The sections were stained with hematoxylin and eosin as described in the manufacturer’s specifications (Sigma-Aldrich). The second piece of each specimen was embedded in OCT compound (Tissue-Tech; Miles Inc., Elkhart, IN, http://www.bayerus.com) and frozen in isopentane cooled in liquid nitrogen. Eight-micrometer cryosections were fixed in 4% paraformaldehyde. The transplanted CDSCs in frozen sections were identified by fluorescent microscopy. Endogenous peroxidase activity was quenched with 0.3% H2O2 for 15 minutes. After rinsing with PBS three times, the slices were incubated with antibodies against desmin, alpha-sarcomeric actin, smooth muscle actin, troponin-I, collagen types I and II, and von Willebrand factor for 16 hours at 21°C. Negative control samples were incubated in PBS without the primary antibodies. After being rinsed three times for 15 minutes each, the slices were incubated with goat anti-rabbit immunoglobulin G conjugated to FITC and Rhodamine (tetramethylrhodamine isothiocyanate) at 37°C for 45 minutes [7, 14, 18]. The specimens were observed immediately under fluorescent microscope.

Von Kossa Staining
Von Kossa staining was performed to detect abnormal deposits of calcium within the myocardium. The heart tissue slices were incubated in the dark with a 2% (wt/vol) silver nitrate solution. The slices were then washed with distilled water and exposed to bright light (while covered with water) for 15 minutes and then dehydrated with ethanol [11, 19]. The calcium is reduced by a strong light and replaced with silver deposits, visualized as metallic silver. By von Kossa staining, calcium mineral appears black.

Measurement of Capillary Density in the Scar
After staining with antibody of von Willebrand factor, the number of capillary vessels was counted in the scar tissue of all groups using a standard light microscope at a x 400 magnification [5]. Five high-power fields within the scar of each section were randomly selected, and the number of capillaries was averaged and expressed as the number of capillary vessels per high-power field (0.2 mm2).

Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted from the fresh heart scar tissue using TRI Reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) according to the manufacturer’s instructions. After the extraction of total RNA, a reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed as described previously [20]. The cDNA was generated from 2 µg of total RNA using Moloney murine leukemia virus reverse transcription (Promega, Madison, WI, http://www.promega.com) and random primers during 2-hour incubation at 42°C. PCR was performed with 2 µl of cDNA using Taq DNA polymerase (Promega). Preliminary experiments were carried out for each gene to select the optimal number of cycles to enable the amplification reaction to proceed in a linear range. PCR of a constitutively expressed gene, GAPDH, was used as an internal control for the amount of input RNA. RNA samples from each group were assayed using primers for the following genes: collagen I, collagen III, matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1), transforming growth factor-ß1 (TGF-ß1), and vascular endothelia growth factor (VEGF). Ten microliters of each PCR product was run on 1.5% agarose gels, and bands were visualized with a UV transilluminator (Bio-Rad, Hercules, CA, http://www.bio-rad.com).

Data Analysis
All data are expressed as mean ± standard deviation. SPSS (SPSS Inc., Chicago, http://www.spss.com) for Windows 11.0 was used for all analyses. Comparisons of continuous variables among animal groups were studied by a one-way analysis of variance. Longitudinal studies comparing data within each group were achieved by the use of paired t-tests. Statistical significance was assumed at p < .05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell Morphology, FACS Analysis, and Immunohistochemistry
Cells derived from human articular cartilage exhibited spindle-shaped morphology, attached to the culture plate tightly, proliferated in the culture medium, appeared morphologically to be a homogeneous population, and maintained similar morphology with passages (Fig. 1AGo). We were able to passage the cells more than 20 times without detecting any signs of senescence. FACS analysis showed that the cells were consistently negative for CD14, CD34, CD38, CD45, CD49f, CD104, CD105, CD106, CD117, HLA-DR, and ABCG-2 and positive for CD10, CD44, CD71, CD73, CD90, CD147, and HLA-A, -B, -C (Fig. 1BGo). In immunohistochemical analysis, the cultured CDSCs stained negatively for desmin, smooth muscle actin, alpha-sarcomeric actin, and cardiac specific troponin-I (data not shown). Before cell transplantation, CDSCs were detached and stained with Hoechst 33342 (Fig. 1CGo).


Figure 1
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  		Figure 1. (A): Cartilage-derived stromal cells (CDSCs) were spindle-shaped and attached to the culture tightly (original magnification x 400). CDSCs were detached, labeled with fluorescein isothiocyanate-conjugated or phycoerythrin-conjugated monoclonal antibodies, and detected by flow cytometry. Values represent the mean percentage of all assessed cells positively stained by the respective antibodies in the flow cytometry analyses. The results showed that CDSCs were consistently negative for CD14, CD34, CD38, CD45, CD49f, CD104, CD105, CD106, CD117, HLA-DR, and ABCG-2 and positive for CD10, CD44, CD71, CD73, CD90, CD147, and HLA-A, -B, and -C (B). CDSCs were detached with trypsin/EDTA and stained with Hoechst 33342 before transplantation (C).

 
Surgical Outcome of Animal Model
Mortality over the 3-week period after myocardial infarction was 17.8% (16 of 90). Most deaths occurred within 48 hours of MI. Of the survived rats with LVEF less than 60% in ultrasonic assessment, 91.9% (68 of 74) were randomized into three groups—CDSC group (n = 23), medium group (n = 22), and control group (n = 23). The mortality over the 4-week period after the second surgical procedure was 13.0% (3 of 23), 18.2% (4 of 22), and 17.4% (4 of 23) for CDSC, medium, and control groups, respectively. Most deaths also occurred within 48 hours after the second surgical procedure. The survived rats had no apparent signs of abnormalities throughout the experiments.

Evaluation of Ventricular Function
Two-dimensional and M-mode transthoracic echocardiographic images were obtained. Baseline echocardiography was performed 3 weeks after myocardial infarction, the day before cell transplantation, and there was no significant difference in heart function between the three groups. At the 4-week time point after cell transplantation, the heart function stayed unchanged in the CDSC group except the improved left ventricle posterior wall thickening (54.7% ± 17.7% vs. 40.3% ± 9.7%, p = .016) compared with the corresponding values of baseline echocardiography. Four weeks after cell transplantation, the echocardiographic parameter of the CDSC-treated group showed higher LVEF (55.2% ± 6.1% vs. 47.9% ± 8.7% and 50.2% ± 8.1%), FS (25.6% ± 3.6% vs. 21.5% ± 3.7% and 21.6% ± 4.1%), and posterior wall thickening (54.7% ± 17.7% vs. 33.4% ± 15.3% and 31.5% ± 16.2%) compared with medium and control groups (p < .05). The left ventricular diameter in end-systole (6.72 ± 0.71 vs. 7.61 ± 0.81 and 7.32 ± 0.74 mm) and end-diastole (8.73 ± 0.66 vs. 9.72 ± 0.97 and 9.58 ± 0.73 mm) was smaller in the CDSC-treated group than in the medium and control groups (p < .05) (Fig. 2Go). The parameters had no significant difference between medium and control groups. That is, the heart function of medium and control groups became worse 4 weeks after the second procedure compared with baseline.


Figure 2
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  		Figure 2. Effect of CDSC transplantation on cardiac function. CDSCs (CDSC group, n = 20), medium alone (medium group, n = 18), and saline (control group, n = 19) were injected into ischemic myocardium. LVEF, FS, LVEDd, LVEds, LVPWd, LVPWs, and LVPW thickening were calculated 3 weeks (baseline) after myocardial infarction and 4 weeks after cell transplantation (*p < .05, **p < .01). Abbreviations: CDSCs, cartilage-derived stromal cells; LVEF, left ventricle ejection fraction; FS, fractional shortening; LVEDd/LVEDs, left ventricular diameter in end-diastole and end-systole; LVPWd/LVPWs, left ventricle posterior wall thickness in end-diastole and end-systole; LVPW thickening, left ventricle posterior wall thickening.

 
Histological Examination
The LAD ligation led to a transmural infarction in the anterior wall of all examined rats, and fibrous scar tissue developed in the infarction area (Figs. 3A, 3BGo). Hoechst-labeled CDSCs were detected in islands within the infarct region under fluorescent microscopy (Fig. 3CGo) and formed vessel lumen–like structure in the scar tissue of cell transplantation rats (Figs. 4A, 4BGo), but they could not be found in the hearts of medium and control groups. Von Willebrand factor staining indicated that the transplanted CDSCs were involved in the formation of microvessels, and endothelial cells that stained positively were found at the lumen side of the vessel-like structure formed by CDSCs (Figs. 4C–4EGo). By immunofluorescent examination, the CDSCs that transplanted into the scar and border zone were stained negatively for alpha-sarcomeric actin and cardiac-specific troponin-I (data not shown) and positively for desmin and smooth muscle actin (Figs. 4F, 4GGo). There are neither calcium deposits nor chondroplasia in the heart tissue 4 weeks after CDSC transplantation by von Kossa staining and immunohistochemical staining of collagen II (data not shown).


Figure 3
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  		Figure 3. The morphology of the heart 3 weeks after myocardial infarction. The left anterior descending ligation led to a transmural infarction in anterior wall of all examined rats, and fibrous scar tissue developed in the infarction area (A, B). Hoechst-labeled cartilage-derived stromal cells (CDSCs) were detected in islands within the infarct region under fluorescent microscopy 4 weeks after transplantation (C) (original magnification x 200). Hematoxylineosin staining in the tissue slice adjacent to slice C (D).

 

Figure 4
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  		Figure 4. Grafted Hoechst-labeled CDSCs were detected within the infarct region and formed vessel lumen-like structure 4 weeks after cell transplantation (original magnification x 200 [A] and x 400 [C]). Von Willebrand factor staining in the tissue slice adjacent to slice A indicated that the transplanted CDSCs were involved in the formation of microvessels (B) (original magnification x 200). Immunohistofluorescent staining indicated that grafted cartilage-derived stromal cells (CDSCs) were negative for von Willebrand factor. Endothelial cells stained positively for von Willebrand factor were found at the lumen side of the vessel-like structure formed by CDSCs (C, Hoechst fluorescent of grafted CDSCs; D, immunohistofluorescent staining of von Willebrand factor in the same tissue slice; E, merged picture of C and D; original magnification x 400). The transplanted CDSCs were stained positively for desmin (F) and smooth muscle actin (G). Arrows indicated the positive staining.

 
Capillary Density in the Scar
To examine whether transplanted CDSCs induced an angiogenetic effect in injured myocardium, the density of blood vessels was calculated in the infarcted area with von Willebrand factor staining. The number of capillaries increased significantly after 4 weeks in infarcted myocardium in rats with CDSC transplantation compared with the medium and control groups (5.58 ± 1.12 vs. 3.14 ± 0.91 and 2.03 ± 0.82 vessels per 0.2 mm2; p < .05, respectively). The difference between medium and control group was also significant (p = .021) (Fig. 5Go). This result indicates that engrafted CDSCs may promote neovascularization in infarcted myocardium, and the injection of cell-free medium could also induce angiogenesis in the heart.


Figure 5
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  		Figure 5. Effects of cartilage-derived stromal cell (CDSC) transplantation on neovascularization. Endothelial cells were stained with antibody of von Willebrand factor, and vessel numbers within infarcted myocardium of rat hearts were counted. Total numbers of vessels in five areas were evaluated in each animal (A, CDSC group, n = 20; B, medium group, n = 18; C, control group, n = 19; original magnification x 200). The number of capillaries increased significantly after 4 weeks in infarcted myocardium in rats with CDSC transplantation compared with the medium and control groups (D) (*p < .05, **p < .01).

 
Reverse Transcription-Polymerase Chain Reaction
RT-PCR results indicated that the expression levels of collagen I, collagen III, TIMP-1, TGF-ß1, and VEGF were much higher (p < .05) in the CDSC-treated group than in the medium and control group 4 weeks after cell transplantation, whereas the expression of MMP-1 was similar in all three groups (Fig. 6Go). No difference was observed between medium and control groups.


Figure 6
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  		Figure 6. The mRNA expression levels semiquantified by reverse transcription–polymerase chain reaction. Amounts of cDNAs from different RNA samples were normalized using the levels of GAPDH as an internal control (A). Relative intensities of polymerase chain reaction bands relative to those GAPDH bands amplified in the experiment were determined and summarized (B). There was a clear increase in the expression levels of collagen I, collagen III, TIMP-1, TGF-ß1, and VEGF 4 weeks after CDSC transplantation compared with medium and control groups (*p < .05, **p <.01 vs. medium group; #p < .05, ##p < .01 vs. control group). Expression levels of MMP-1 were similar in all three groups. Abbreviations: CDSCs, cartilage-derived stromal cells; mRNA, messenger RNA; cDNA, complement DNA; MMP-1, matrix metalloproteinase-1; TIMP-1, tissue inhibitor of metalloproteinase-1; TGF-ß1, transforming growth factor ß-1; VEGF, vascular endothelia growth factor.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Adult cardiac myocytes are thought to be terminally differentiated and to therefore lose their proliferative capacity after birth. Recent studies have shown the presence of cardiac stem cells residing in the heart [21–25]. Resident cardiac primitive cells seem to be the most promising candidate for novel, cell-based treatment of patients with heart disease. However, their number is very limited, and how to activate the resident stem cells to regenerate damaged myocardium and restore the heart function after myocardial infarction remains a question. Therefore, multipotent stem cell transplantation could be an alternative treatment modality for cardiac regeneration at present.

There is a threshold of the number of stem cells needed to generate adequate heart muscle to contribute to cardiac function. Adult stem cells are limited in supply in each patient and therefore are difficult to isolate and purify. On the other hand, the stem cells derived from patients must be isolated and expanded in culture to obtain a sufficient amount of stem cells for transplantation. However, it is not practical to wait a long time for the manipulation of stem cells in vitro before transplantation in clinical use [26]. Largely because of the ease of harvest and apparent lack of requirement for ex vivo manipulation, there has been a rapid increase in the number of clinical trials using bone marrow (BM) cells or the mononuclear fraction of BM cells isolated directly from the patients with ischemic heart diseases, yet late-onset toxicity may occur when using the whole populations of BM cells or BM mononuclear cells [27]. The nonessential cells from BM may incorporate into regenerating myocardium, resulting in the generation of noncardiac tissues. Yoon et al. [28] reported that direct transplantation of unselected BM cells into the acutely infarcted myocardium might induce significant intramyocardial calcification. Therefore, it is necessary to find more pure candidate cell sources from available human tissue to meet the amount of stem cell transplantation or combined cell transplantation and to avoid side effects aroused by impure cell sources [8, 14].

In our experiment, human CDSCs were spindle-shaped, proliferated in the culture medium, appeared morphologically to be a homogeneous population, and maintained similar morphology with passages. The CDSCs could be passaged more than 20 times without detecting any signs of senescence. Most of the markers expressed by CDSCs coincided with mesenchymal stem cells [29, 30]. When transplanted into infarcted rat hearts, the CDSCs could promote angiogenesis, prevent LV remodeling, and then improve the heart function. Our study results indicated that transplantation of the CDSCs might provide a novel and promising approach to improve the injured heart function following myocardial infarction.

The cellular and molecular mechanisms that mediate the functional benefits observed in the failing heart after cell transplantation are not clear. First, an active process whereby the engrafted cells replace lost contractile elements and contract synchronously with the host myocardium may occur. Second, it is possible that some of the beneficial effects of cell transplantation may be the result of a reorganization of the structural elements surrounding the engrafted cells. In our experiment, there is no evidence indicating that CDSCs transplanted into the infarcted rat hearts differentiated into myocardium and were involved in myocardial contraction, whereas the CDSCs were positive for desmin and smooth muscle actin. The implanted CDSCs participated in the formation of microvessels and significantly elevated VEGF expression levels accompanied by increased vascular density and regional blood flow in the infarct zone.

It has been well documented that BM stromal cell transplantation can improve infarcted heart function of rat, porcine, and human [5, 6, 14–15, 31]. Rangappa et al. [8] also reported that adult mesenchymal stem cells isolated from fatty tissue can be transformed into cardiomyocytes in vitro. In our experiment, there is no evidence indicating that CDSCs transplanted into the infarcted rat hearts differentiated into myocardium and were involved in myocardial regeneration. Compared with mesenchymal stem cells from BM or fatty tissue, the beneficial effects of the transplanted CDSCs were mainly due to enhancing neovascularization and preventing heart remodeling. The property of CDSCs in promoting angiogenesis was similar to that of BM-derived mesenchymal stem cells [31]. It is interesting that single injection of cell-free medium could also induce the angiogenesis in the heart compared with the control group, although the angiogenic effect induced by cell-free medium is not as strong as that induced by CDSC transplantation. Growth factors and/or cytokines supplemented in the medium may be responsible for the angiogenic effect induced by cell-free medium.

The interstitial or extracellular collagen matrix (ECCM) provides the structural framework for coordinated muscle cell contraction and sequesters growth factors and cytokines that interact with local cell receptors to influence cellular behavior and survival. Remodeling of the ECCM after MI plays a major role in LV remodeling, whereby decrease, disruption, and/or defective composition of the ECCM promote LV dilation and rupture [32]. Covell [33] and Cleutjens et al. [34] reported that although twofold to threefold increase in myocardial collagen above the normal level results in increased LV stiffness and mild dysfunction, a very small decrease in collagen below normal can lead to drastic consequences, including LV dilation and rupture. It thus seems that ECCM is a double-edged sword for the heart, as both ECCM deficiency and overproduction may lead to the damage of heart.

Collagen types I and III composed most of the macromolecules of ECCM. The data presented in this study showed that the expression levels of collagen types I and III in infarcted zone were much higher in the transplantation group than those in the medium and control groups. Fedak et al. [35] reported that cell transplantation might alter the matrix components of the injured heart region as well as the normal myocardium to prevent the thinning and dilatation that constitutes remodeling after myocardial infarction. The increase in expression levels of collagen types I and III in infarcted zone of rats may protect the heart from LV remodeling and dilation. In view of the fact that ECCM overproduction may lead to damage of the heart, in our long-term study project, we will pay more attention to the negative effects induced by ECCM accumulation and search for a new strategy to recover the normal level of collagen in the damaged heart.

A fine balance between MMPs that degrade the ECCM and the endogenous TIMPs maintains normal remodeling and function, and an imbalance can result in adverse remodeling [35, 36]. CDSCs grafted to infarcted myocardium can induce changes in the ECCM and adverse remodeling, and these changes may be significantly enhanced by targeting the MMP/TIMP ratio as a key of the remodeling cascade. RT-PCR results revealed the expression levels of TIMP-1 and TGF-ß1 in the hearts were much higher in the CDSC-treated group than those in the control group, whereas expression level of MMP-1 was similar in the two groups, reflecting a protective effect of ECCM induced by decreased MMP-1/TIMP-1 ratio after CDSC transplantation [32]. The increase in TGF-ß1 expression can also influence the MMP-1/TIMP-1 ratio, promote the biosynthesis of collagen, and protect the ECCM [37]. The functional improvement of rat hearts could also be explained by the ability of the engrafted CDSCs to protect ECCM and limit infarct expansion.

There have been concerns about the potential for adverse effects or unregulated differentiation after stem cell transplantation in vivo [28]. CDSCs were isolated and cultured from cartilage, and it has been reported that CDSCs can differentiate into osteoblasts and chondrocytes in the specific microenvironment [9, 11]. It is therefore necessary to verify the safety of CDCS transplantation. In our study, there was no evidence of calcium deposits or chondroplasia in the heart tissue 4 weeks after CDCS transplantation. Further studies are still required to evaluate the long-term safety.

Recent reports have provided substantial new insights into stem cell populations in a variety of adult tissues. In addition to BM, other sources of stem cells with mesenchymal potential include adipose tissue [8, 38], cartilage [9–11], skeletal muscle [39], peripheral blood [40], trabecular bone [41, 42], and synovium [43]. The possible therapeutic mechanisms involved myogenic and/or angiogenic and paracrine effects of the stem cells. In our study, mesenchymal stem cells could be isolated from cartilage tissue. The cells might differentiate into smooth muscle and secrete cytokines to repair the damaged heart. The cells can be used as a new cell source to be applied in cell-based therapy. In view of the fact that the mesenchymal stem cells from cartilage tissue can be easily cultured and amplified in vitro, we can establish a cell bank for allotransplantation and use the mesenchymal stem cells from cartilage tissue for the treatment of patients with heart disease but not suitable for BM harvesting procedure.

To investigate the influence of CDSC transplantation on heart function, angiogenesis, and ventricular remodeling after myocardial infarction, the rats were usually killed and examined 4 weeks after the transplantation of CDSCs in our experiment. Indeed, long-term observation is necessary to track the survival of the rats with repaired heart function to evaluate the long-term efficacy and adverse events. We do have data about the survival of the rats (data not shown). In our experiment, six rats with repaired heart function survived for 12 weeks with CDSC transplantation before euthanasia (four rats received daily inoculation of cyclosporine for 4 weeks, and two rats received daily inoculation for 12 weeks). The general status of the rats was similar to the healthy rats during the 12 weeks when they were fed in the cages.

Although statistical analysis showed cardiac function improved in the stem cell transplantation group, the difference was not impressive. We used a single dose (5 x 106 cells) for cell transplantation. Previous studies have demonstrated that cell dose has a significant influence on the beneficial effects of cell transplantation on ventricular function [44]. We believe that more improved functional effects could be achieved with an optimal dose of cells and optimal time point for transplantation after myocardial infarction. To determine the optimal number of cells and optimal time point for transplantation, a dose-response and a time-response curve would need to be conducted. However, it is likely that attempts to enhance cell survival after implantation may be more effective in increasing the number of viable engrafted cells than increasing the number of cells implanted by means of combination of cell transplantation with gene therapy of growth factor and cytokines that have functions to enhance survival of the implanted cells.

In conclusion, the present study showed that stromal cells from human articular cartilage may promote angiogenesis, prevent LV remodeling, limit infarct expansion, and improve the heart function when transplanted into infarcted rat hearts. The CDSCs might become a new candidate cell source for cell transplantation in the treatment of myocardial infarction.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
W.S. and H.Z. contributed equally to this work. This research was supported by grants from National Outstanding Young Foundation (30125039), Beijing Science Technology Committee (H020220010490), and National 863 Program. We would like to thank Dr. Xi Chen and Dr. Weiquan Zhu for their valuable advice and technical assistance in the experiments of molecular biology. We also thank Dr. Ping Chen and Yinan Liu for cell culture and fluorescence-activated cell sorting analysis.

DISCLOSURES
S.H. has performed contract work within the last 2 years for National Outstanding Young Foundation, Beijing Science Technology Committee, and National 863 Program.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received on April 11, 2005; accepted for publication on August 10, 2005.




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