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Cardiovascular Research 2006 71(1):158-169; doi:10.1016/j.cardiores.2006.03.020
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Copyright © 2006, European Society of Cardiology

Intramyocardial delivery of human CD133+ cells in a SCID mouse cryoinjury model: Bone marrow vs. cord blood-derived cells

Nan Maa,1, Yury Ladilova,1, Jeannette M. Moebiusb, Leelee Onga, Christoph Piechaczekb, Árpád Dávida, Alexander Kaminskia, Yeong-Hoon Choia, Wenzhong Lia, Dietmar Eggerc, Christof Stamma,*,1,2 and Gustav Steinhoffa,1

aDepartment of Cardiac Surgery, University of Rostock, Rostock, Germany
bMiltenyi Biotec GmbH, Research and Development, Germany
cVita34, Leipzig, Germany

* Corresponding author. Deutsches Herzzentrum Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Tel.: +49 30 4591 2109; fax: +49 30 862 3071. Email address: stamm{at}dhzb.de

Received 19 October 2005; revised 22 March 2006; accepted 23 March 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective The regenerative potential of endothelial and hematopoietic progenitor cells in the heart may vary according to their origin. This study was designed to compare the functional effects of CD133+ cells from human cord blood and bone marrow in a mouse model of myocardial injury.

Methods 5 x 105 CD133+ cells from bone marrow (BMCD133) or cord blood (UCBCD133) were injected in the necrosis border zone of NOD/SCID (non-obese diabetic/severe combined immunodeficiency) mice with left ventricular cryoinjury (CI+). Transplanted cells were tracked by immunostaining for hNuclear antigen and by PCR for hDNA. Echocardiography was used to measure contractility. Scar size, capillary density, and cardiomyocyte apoptosis were evaluated by histology. In addition, the myogenic and endothelial differentiation capacity of BMCD133 and UCBCD133 was compared in vitro.

Results DNA was detected 4 weeks after cell injection by PCR, but hNuc+ cells were found by immunostaining only after 48 h. Capillary density in both BMCD133 and UCBCD133 cell-treated CI+ mice was higher than in control CI+ mice, but not different between BMCD133 and UCBCD133 cell-treated hearts. There were no differences in scar size and myocardial mass among BMCD133, UCBCD133 and control CI+ mice, but cardiomyocyte apoptosis was reduced by both BMCD133 and UCBCD133 cells. The post-injury deterioration of shortening fraction (46.2±1% in sham-operated mice and 41.3±0.8% in control CI+ mice) was prevented by BMCD133 cells (45.4±0.9%), but not by UCBCD133 cells (40.8±0.7%). On the other hand, both BMCD133 and UCBCD133 cells abolished post-injury mortality. In vitro, neither cultivated BMCD133 or UCBCD133 cells developed into myocytes, but both readily differentiated towards an endothelial cell phenotype.

Conclusions While both cord blood and marrow CD133+ cells have some beneficial effects on post-injury angiogenesis and survival, only marrow cells appear to improve myocardial contractility.

KEYWORDS Cell therapy; Angiogenesis; Cell differentiation; Stem cells


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The concept of cardiac cell transplantation to improve myocardial function is tremendously attractive, and it has repeatedly been suggested that cells derived from skeletal muscle, bone marrow, blood, or other tissues might have a regenerative capacity in the mammalian heart. Among those, CD34+ or CD133+ progenitor cells are of particular interest, because the corresponding antibodies can be used in clinical enrichment protocols, applying methods for primary cell isolation that comply with standard regulations for clinical use. The CD133 antigen is a 120-kDa 5-transmembrane domain glycoprotein that is expressed in immature hematopoietic stem and progenitor cells [1,2]. In addition, several studies indicate that CD133 also serves as a marker for stem and progenitor cells with non-hematopoietic, pluripotent differentiation capacity, in particular the subset of CD133+ cells that is CD34– [3,4]. CD133+ cell products may also contain mesenchymal stem cell-type cells, which have been designated as multipotent adult progenitor cells (MAPC) and might possess a particularly broad differentiation capacity [5]. The most robust line of evidence, however, indicates that CD133+ cells possess the characteristics of endothelial progenitor cells, which can readily be differentiated into endothelial cells in vitro and induce or at least participate in neoangiogenêsis processes in vivo [6–17]. For those reasons, CD133+ cells were among the first cell types to be used in clinical trials of cell therapy for myocardial regeneration, and they find increasingly wide-spread application [18–23]. Nevertheless, few experimental studies have systematically assessed the myocardial regeneration potential of CD133+ cells in vivo. We therefore sought to determine the functional and histologic consequences following direct intramyocardial delivery of human CD133+ cells derived from bone marrow and from cord blood in a SCID-mouse model of left ventricular cryoinjury.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Cell donors
The study conforms to the Declaration of Helsinki and all cell donors gave their informed written consent to use part of their bone marrow for experimental purposes. Aliquots of bone marrow (BM) were collected from patients undergoing coronary artery bypass (CABG) surgery at Rostock University Hospital (n=36, including 19 male and 17 female donors). The median age of the study group was 72 years (range 48–80 years). Human cord blood (CB) was donated for research and written consent was obtained prior to collection during normal full-term deliveries by Vita34 (Leipzig, Germany). Fifty-one cord blood samples (28 from male and 23 from female donors) were used in this study.

2.2 CD133+ cell isolation
Bone marrow was obtained during standard CABG surgery by sternal aspiration. The heparinized marrow was diluted with PBS/EDTA and carefully layered over 20 ml of Ficoll (1077 g/ml, Biochrom). After 35 min centrifugation in a swinging bucket rotor, the upper layer was aspirated and the mononuclear cell layer was collected. Following labelling with ferrite-conjugated anti-CD133 monoclonal antibody (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany), two cell separation cycles were run using the Mini-MACS LS column (Miltenyi Biotec) according to the manufacturers protocol. After the separation, the purity of the cell product was determined by flow cytometry.

Cord blood was stored in the vapour phase of liquid nitrogen with citrate phosphate dextrose solution (CPD) and dimethyl sulphoxide (DMSO) added. After thawing, it was diluted with PBS containing 2 mM EDTA and 200 U/ml DNase I. The cells were washed and centrifuged three times. Ficoll density centrifugation and enrichment of CD133+ cells was then performed as described above.

2.3 Flow cytometry
For flow cytometric analysis cell surface markers were blocked with FCR Blocking Reagent (1:5; Miltenyi Biotec) and incubated for 10 min at 4–8 °C with the following antibodies: anti-CD34.PE (clone AC136; 1:11), anti-CD45.FITC (clone 5B1; 1:11) and anti-CD133/2.APC (clone 293C3; 1:11) (all from Miltenyi Biotec). CD117 labeling was performed using anti-CD117 antibody (clone YB5.B8; 1:100; BD Parmingen, BD Bioscience, San Diego, CA, USA). Isotype controls were purchased from BD Pharmingen. After incubation cells were washed with PBS/2 mM EDTA and analysed using a FACS Calibur flow cytometer (Becton Dickinson, Heidelberg, Germany). Dead cells were excluded via propidium iodide staining. Data analysis was performed with BD CELLQuest software. Histograms of cell number vs. logarithmic fluorescence intensity were recorded for 10,000–20,000 cells per sample.

2.4 In vitro plasticity assays
To assess the angiogenesis-relevant differentiation potential of BMCD133 and UCBCD133 cells in vitro, separate series of CD133+ cell products (n=5, each) were cultivated and induced to adopt an endothelial phenotype. Matrigel (Becton Dickinson Labware) basement membrane matrix was mixed 1:1 with EGM medium (Cambrex) and added to a chamber slide (Labtech). After 1 h incubation at room temperature, 2 x 104 cells were added to the chamber slide with 500 µl EGM. Cultures were stimulated with a combination of stem cell growth factor SCGF (100 ng/ml) and vascular endothelial growth factor VEGF (50 ng/ml). All cultures were performed in quadruplicate, incubated at 37 °C in 5% CO2 and 95% humidity, and scored after 14 days of culture by light microscopy. The Matrigel was then dissolved with dispase (Becton Dickinson), and immunostaining for von Willebrand factor (vWF) was performed (monoclonal anti-vWF antibody followed by FITC-conjugated secondary goat anti-mouse IgG). Direct fluorescent staining was used to detect cellular uptake of FITC-acetylated low density lipoprotein (acLDL; Molecular probes). Cells were incubated with acLDL at 37 °C and later fixed with 1% paraformaldehyde for 10 min. After several washing steps, samples were viewed by confocal microscopy (Leica). Cultured human umbilical vein endothelial cells (HUVEC) and NIH3T3 cells served as postive and negative controls, respectively.

Attempts were also made to drive both BMCD133 and UCBCD133 cells towards a myogenic phenotype, using a protocol that has been shown successful in myogenic differentiation of bone marrow cells. Here, CD133+ cells were cultured on matrigel-coated plates in the culture medium containing DMEM/F-12 supplemented with 10% fetal calf serum in the presence of 25 nM bFGF, 10 ng/ml TGF beta-3, 50 ng/ml b-FGF and 50 ng/ml platelet-derived growth factor (PDGF-BB). Separate sets of experiments were carried out with and without epigenetic manipulation by 5-Azacytidine. In regular intervals for up to 14 days, samples were screened for spontaneously beating cells, and were stained with anti-β-myosin heavy chain antibody.

2.5 Surgical procedure
All animal experiments were performed in accordance with the guidelines published in the "Guide for the Care and Use of Laboratory Animals" (NIH publication no. 86 to 23, revised 1985), and under the protocols approved by the Institutional Animal Care and Use Committee at Rostock University. Female NOD/SCID mice weighing 27±3 g were anesthetized with avertin (0.05 mg/kg, i.p.). A left thoracotomy through the fifth intercostal space was performed and the heart was exposed. A cryoprobe (4 mm diameter) cooled to – 190 °C by immersion in liquid nitrogen was applied to the epicardial surface of the anterolateral left ventricular (LV) wall 3 times for 15 s to create a transmural necrosis. Immediately after cryoinjury 5 x 105 cells suspended in PBS were injected into the border zone. Three injections of 7 µl each were performed. Surgical mortality (death occurring within 4 h postoperatively) was similar in all cryoinjury groups. Four weeks after surgery animals were euthanized and hearts were excised for histological analysis. There were four experimental groups: In sham operated mice (sham, n=9) only a thoracotomy was performed. In control animals (control, n=26) an equivalent volume of cell-free phosphate-buffered saline was injected intramyocardially after cryoinjury. In the treatment groups BMCD133 or UCBCD133 cells were injected (BMCD133 n=23, UCBCD133 n=14). To determine whether injected BMCD133 and UCBCD133 cells can be detected by histology early after intramyocardial delivery, a separate set of experiments (n=5 per group) was performed and hearts were excised and prepared for cell-tracking histology 48 h after the operation.

2.6 PCR analysis
To confirm the presence of human DNA in murine myocardium, PCR was performed using a human-specific primer. DNA was extracted from homogenized tissue with phenol/chloroform, precipitated with ethanol/sodium acetate, and analyzed for presence of a human {alpha}-satellite chromosome sequence by PCR using the primers D7A and D7B of {alpha}-satellite of chromosome 7 Locus D7Z1 (a gift from Dr. Michael Cross, Leipzig University). Primer sequences were as follows: 5'AGC GAT TTG AGG ACA ATT GC-3' forward primer 7-A; and 5'-CCA CCT GAA AAT GCC ACA GC-3' reverse primer 7-B [11]. Cycling conditions were: 35 cycles of 94 °C for 30 s, 48 °C for 30 s, 72 °C for 30 s; and 72 °C for 5 min on the DNA thermal cycler. The predicted size of the major amplification product for D7Z1 was 1000 bp.

2.7 Myocyte apoptosis
Apoptotic cardiomyocytes were detected by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) using the Chemicon Apoptag assay (Chemicon) according to the manufacturers instructions. Analysis of apoptosis was performed in LV sections of a given heart that showed the maximum infarct size. The cardiomyocyte origin of an apoptotic body was assumed based on the presence of myofilaments surrounding the nucleus. The amount of apoptotic cardiomyocytes was expressed as the ratio of TUNEL-positive cardiomyocyte nuclei to the total number of DAPI-stained cardiomyocyte nuclei. The frequency of TUNEL-positive nuclei was quantified in three separate sections per heart, and the average value was calculated. Apoptotic cardiomyocytes were counted i) in the center of the necrosis area, ii) in the border zone of the recent necrosis (48 h postoperatively), and iii) in remote non-infarcted myocardium. Myocardium extending 0.5–1.0 mm from the cryolesion was considered to represent the border zone. To avoid contamination of the remote normal myocardium with border zones, a myocardial area extending 2 mm from the border zone area was not included in the statistical analysis.

2.8 Echocardiography
Cardiac ultrasound studies were performed prior to and 4 weeks after surgery using a Philips SONOS 5500 echocardiography system equipped with a 15-6L transducer (6–15 MHz). We used the resolution advantage of the 15 MHz capability at a tissue penetration depth of 2 cm. Mice were sedated and immobilized in supine position on a custom-made procedure surface. The anterior chest wall was shaved, acoustic coupling gel was applied, and the transducer was placed avoiding excessive pressure. Left ventricular shortening fraction (SF) was used as the principal measure of myocardial contractility. Correct position of the transducer was determined in the parasternal long axis view before the parasternal short axis view was visualized. Then, M-mode and 2D-mode recordings were obtained in both the antero-posterior axis and the medio-lateral axis. The the akinetic or hypokinetic area of cryoinjury was usually found anterolateral. Endocardial echoes were identified, and the internal left ventricular diameter was measured in end-diastole and in end-systole. Shortening fraction was calculated based on 3 measurements in different cardiac cycles, and the average value was computed. By using the biaxial method distortion of SF measurements due to compensatory hypercontractility of the nonaffected myocardium was minimized. Heart rate was determined recording the continuous wave Doppler of the aortic flow. All measurements were first performed at rest and then repeated minutes after 200 µg/kg dobutamine had been injected subcutaneously (stress echocardiography). The cardiac response to dobutamine was clearly evident by increasing heart rate. In all experiments, the echocardiographer was blinded with respect to the cell treatment.

2.9 Histological analysis
At the end of the observation period, animals were sacrificed with an overdose of avertin. Hearts were quickly removed, washed with PBS, weighed and snap frozen in liquid nitrogen. Frozen sections embedded in O.C.T medium (5 µm in thickness) were prepared and stained with sirius red/green fast or hematoxylin/eosin. From every heart, three slices on mid-level of the infarcted region were used for measurement of scar size. Quantification of scar area and length were performed using imaging software ESI-Vision (SIS, Muenster, Germany). The scar area was calculated in percent of the whole area of the left ventricular section.

2.10 Immunostaining
For immunohistologic detection of human cells, frozen tissue sections were incubated with monoclonal mouse anti-human nuclear antigen antibody (MAB 1281; 1:50; Chemicon) or monoclonal mouse anti-HLA-class I antibody W6/32 (Dako M0736). For measurement of capillary density (see below), sections were stained with anti-mouse CD31 antibody (Chemicon). Attempts were also made to identify myocyte-type progeny of transplanted BMCD133 and UCBCD133 cells by counterstaining 10 sections of different levels per heart for myosin-{alpha} heavy chain (Chemicon) After blocking in Envision blocking buffer (DAKO), sections were placed in primary antibody overnight at 4 °C. On the following day, the sections were rinsed and then incubated with secondary antibody (alkaline phosphatase- or FITC-conjugated secondary goat anti-mouse IgG.).

2.11 Capillary density
To determine the capillary density in the border zone of the cryolesion, tissue was prepared as described by Vartanian and Weidner [24]. Sections were stained using monoclonal anti-mouse CD31 antibody (Chemicon) and incubated with biotinylated secondary antibody. For quantification of positively stained vessels, 5 sections from within the necrosis border zone of each animal were analysed by an investigator who was blinded with respect to the cell treatment. Capillaries were counted in 10 randomly chosen high-power fields (HPFs) in 2 sections per heart and 6 hearts per time point. The results were expressed as capillaries per high power field.

2.12 Statistical analysis
All values are presented as mean±standard deviation of the mean. One-way ANOVA with Scheffe's post hoc test for unequal sample sizes was used to compare numeric data between the four experimental groups. Datasets consisting of two groups only (BMCD133 vs. UCBCD133 cell isolation data and in vitro plasticity data) were compared by unpaired Student's t-test. A level of p<0.05 was considered as significant difference. The SPSS software package was used to compute Kaplan–Meier survival curves.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Characteristics of CD133+ cells from cord blood and bone marrow
The average purity of the CD133-enriched cell products was 91.2±4% (CD133+ cells in percent of total mononuclear cells), with no difference between UCB and BM-derived cells. To test whether the CD133-enriched cell products from UCB and BM had a different composition of subpopulations, cells were counted by flow cytometry following labelling of CD34, CD133, CD45, and CD117. Representative results are shown in Fig. 1. Cell viability was quantified by propidium iodide staining, and was consistently higher than 90%. There were no differences in the expression of CD45 and CD34. BMCD133 expressed the CD133 antigen not as strong as UCBCD133 cells. On average, 70% of the BMCD133 cells were also positive for CD117, whereas only up to 35% of the UCBCD133 cells were CD117+. The frequency of BMCD133+/CD117+ cells in every examined BMCD133 cell product was markedly higher than that of UCBCD133+/CD117+ cells among the UCBCD133 cell products (Fig. 1A and B).


Figure 1
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  		Fig. 1 (A) Multicolor flow cytometric analysis of CD133+ cells derived from bone marrow. First row, left: CD133-selected cells were found to have a dim expression of CD133 and are all CD34+; right: isotype control. Second row, left: BMCD133 cells are also CD45 dim; right: isotype control. Third row, left: 68.9% of all BMCD133 cells were found to be positive for CD117; right: isotype control. (B) Multicolor flow cytometric analysis of CD133+ cells derived from cord blood. First row, left: All UCBCD133 positive cells were found to be positive for CD34, right: isotype control; Second row, left: All UCBCD133 cells also express CD45; right: isotype control. Third row, left: 34,1% of all UCBCD133 cells were found to be positive for CD117; right: isotype control.

 
3.2 In vitro plasticicty studies
When cultivated on matrigel coated culture dishes and exposed to VEGF, both BMCD133 and UCBCD133 cells formed capillary network-like structures after 14 days of culture (Fig. 2A and B). Approximately 75% of the thus cultivated cells expressed endothelial cell-specific von Willebrandt factor (Fig. 2C and D). Moreover, most of those cells were also positive for uptake of ac-LDL (Fig. 2E–H), further evidence of an endothelial cell-like differentiation commitment. In this respect, no apparent difference in behavior was found between BMCD133 and UCBCD133 cells.


Figure 2
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  		Fig. 2 In vitro differentiation of CD133 cells towards an endothelial cell phenotype. (A) After cultivation on Matrigel for 2 weeks, UCBCD133 cells form capillary-like tubules (scale bars=100 µm). (B) BMCD133 cells display the same behaviour. (C, D) Immunostaining of UCBCD133 (C) and BMCD133 (D) derived endothelial progenitor cells for von Willebrandt factor VWF (red fluorescence, scale bars=50 µm). (E, F) Cellular uptake of acLDL (green fluorescence) by endocytosis in BMCD133 derived endothelial cells (F, differential interference contrast image). (G, H) The same observation in UCBCD133 derived cells (scale bars=20 µm).

 
On the other hand, after up to 21 days exposure to myogenic differentiation conditions, neither with or without 5-Azacytidine, no expression of β-myosin heavy chain and no spontaneously beating cell was detected in any of the samples, indicating that differentiation in myocyte-like cells, at least under the given in vitro conditions, did not occur.

3.3 Survival
Overall, left ventricular cryoinjury produced an early mortality of approximately 30% within the first 4 h after completion of the surgical procedure, and there was no difference in peri-procedural mortality between control and cell-treated groups (Fig. 3). In sham operated animals no mortality was observed. During the subsequent phase of recovery from the operation, another third of the mice in the cryoinjury control group died (late mortality=32%). This delayed mortality was abolished by both BMCD133 and UCBCD133 cell transplantation (p<0.05 vs. Control). In the time-related Kaplan–Meier analysis, survival of mice in the cryolesion control group was 43% (70% confidence interval 33–53%) at 28 days, 75% (64–86%) in UCBCD133 cell treated mice, and 68% (60–74%) in BMCD133 cell treated mice. Mean survival time was computed as 13 days (70% confidence interval 11–16 days) for control mice, 19 (16–22) days for BMCD133 cell treated mice, and 21 (18–24) days for UCBCD133 cell treated mice. Post-mortem histological analysis of mice that died during the observation period revealed the typical transmural cryolesion of the left ventricle.


Figure 3
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  		Fig. 3 Kaplan–Meier estimated survival of mice with 28 days after cryoinjury of the left ventricle. Surgical mortality was similar in all cryoinury groups. Other than that, no mice died late after simultaneous injection of UCBCD133 cells, and only one mouse died after delivery of BMCD133 cells. In contrast, mice without cell treatment (control) had a marked mortality during the first postoperative week. In the sham operated group (thoracotomy without cryolesion) no animals died peri- or postoperatively (data not shown).

 
3.4 Heart function
Left ventricular contractility was measured by transthoracic echocardiography before and 4 weeks after cryoinjury or sham operation. No differences in shortening fraction were found between groups before surgery. At the end of the observation period, LV contractility in the control group was significantly reduced in comparison with sham operated animals (41.3±0.8% vs. 46.2±1%, p=0.01) (Fig. 4). Injection of human UCBCD133 cells in the cryolesion had no beneficial effect on shortening fraction (SF=40.8±0.7%, p=0.7 vs. control). In contrast, treatment with human BMCD133 cells almost completely prevented the decrease of contractility (SF=45.4±0.9%, p=0.02 vs. control). In order to evaluate heart function under stress conditions, anaesthetized mice were treated with dobutamine (200 µg/kg, s.c.). Dobutamine produced a similar increase in heart rate in all groups (from 405±8 to 543±8 beats per minute). Contractility also markedly increased in all groups, however, the relative increase was more pronounced in sham operated mice (18.0±1.5%) than in the cryoinjury groups (10.5±1.6%, p=0.02 vs. sham). Under dobutamine stress, shortening fraction in BMCD133 cell treated mice remained significantly higher than in control mice or UCBCD133 treated hearts.


Figure 4
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  		Fig. 4 Left ventricular shortening fraction as a measure of myocardial contractility measured 28 days postoperatively by transthoracic echocardiography, both at rest ("baseline") and under catecholamine stress provoked by injection of dobutamine ("dobutamine"). Compared with sham-operated animals, cryoinjury led to a significant reduction in shortening fraction ("control"). Injection of UCBCD133 cells had no measurable effect on contractility, but BMCD133 cells completely prevented or reversed to loss of contractile function at rest, and partially under dobutamine stress. *p<0.05 vs. sham group, #p<0.05 vs. control group.

 
3.5 Heart morphology
Histological analysis demonstrated that cryoinjury consistently produced transmural myocardial necrosis (Fig. 5). In the injured region profound scar formation and wall thinning was observed after 28 days. The size of scar area did not significantly differ between BMCD133 cell-treated, UCBCD133 cell-treated, and control animals (Fig. 6A). There was also a significant increase in heart weight-to-body weight ratio in all cryoinjured mice, again without relevant differences between the treatment groups (Fig. 6B). The development of LV dilatation and partial compensatory hypertrophy was also mirrored by increased left ventricular end-diastolic dimensions on echocardiography (data not shown). Again, there was no measurable effect of BMCD133 or UCBCD133 cell transplantation.


Figure 5
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  		Fig. 5 (A) Gross morphology following Sirius red staining of a normal left ventricle (transverse section) in the sham operation group, and (B) a heart 4 weeks after cryoinjury (control group) without cell injection. (C) 4 weeks after cryoinjury and injection of UCBCD133 cells, and (D) after injection of BMCD133 cells.

 

Figure 6
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  		Fig. 6 (A) Scar size after 4 weeks in hearts of sham operated animals ("sham") and mice that underwent cryoinjury alone ("control") or with injection of BMCD133 or UCBCD133 cells. Scar size was measured by computerized planimetry on Sirius Red stained sections from mid-level of the lesion. There was no difference between control and cell-treated hearts. (B) Heart weight-to-body weight (HW/BW) ratio in %. Mice with cryoinjury had a higher HW/BW ratio, indicating some degree of compensatory hypertrophy of the remaining viable myocardium. Cell injection had no effect on this parameter.

 
3.6 Identification of transplanted cells
Forty-eight hours after transplantation, human cells could readily be detected by immunostaining using human specific antibodies (hNuc or HLA-1) in both BMCD133 and UCBCD133 transplanted hearts (Fig. 7A). Attempts to identify neo-cardiomyocyte-like cells consistently failed: there was no evidence of transplanted human BMCD133 or UCBCD133 cells that co-expressed myosin-{alpha} heavy chain. One month after cell transplantation, neither BMCD133 nor UCBCD133 derived hNuc+ or HLA-1+ human cells could be visualized in any of the sections studied by immunofluorescence. However, human DNA was clearly present in all cell-treated hearts and was identified by PCR using a human specific primer (Fig. 7B+C).


Figure 7
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  		Fig. 7 (A) Detection of human cells 48 h after cryoinjury and BMCD133 cell injection by immunostaining for human nuclear antigen (hNuc) and counterstaining of all nuclei with DAPI (original magnification 200 x). Both BMCD133 and UCBCD133 cells could readily be detected 48 h after injection. After 4 weeks, however, hNuc staining was negative in all examined sections. (B, C) Detection of human DNA 4 weeks after cryoinury and cell injection by PCR using the human-specific primers D7A and D7B of {alpha}-satellite of chromosome 7 Locus D7Z1. All examined hearts that had been treated with BMCD133 (B) or UCBCD133 (C) cell injection were PCR-positive. MW=molecular weight marker.

 
3.7 Capillary density
Capillary density in the border zone of the cryolesion was determined based on CD31 immunostaining. Representative images are shown in Fig. 8A. Overall, capillary density in cell-treated hearts was approximately 25% higher than in control hearts with a cryolesion. (Fig. 8B), whereas there was no significant difference in capillary density between BMCD133 and UCBCD133 cell-treated hearts (control, 10.7±0.7 vessels/HPF; BMCD133, 13.3±2.3 vessels/HPF, p=0.04 vs. control; UCBCD133, 14.3±2.6 vessels/HPF, p=0.005 vs. control, p=0.2 vs. BMCD133).


Figure 8
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  		Fig. 8 Blood vessel density in the border zone of the cryolesion 4 weeks postoperatively. (A–C) Representative photomicrographs following immunostaining for CD31. (A) Control animal (cryolesion without cell injection); (B) myocardium adjacent to the cryolesion after injection of BMCD133 cells; (C) corresponding area following injection of UCBCD133 cells. Original magnification 100 x . (D) Average blood vessel count per high power field counted in 10 randomly chosen high-power fields (HPFs) in 2 sections per heart and 6 hearts per group. In cell-treated hearts, blood vessel density was approximately 25% higher than in control hearts. *p<0.05 vs. control.

 
3.8 Tunel assay
Numerous Tunel-positive nuclei were identified in the area of the cryolesion 24 h postoperatively (Fig. 9). In general, they also showed condensed nuclei as another typical feature of apoptosis. In control animals, the apoptotic nuclei count was approximately 5-fold higher in the center of the cryolesion than in remote normal myocardium, and 12-fold higher in the necrosis border zone. Compared with BMCD133 or UCBCD133 cell-treated hearts, there was no difference in TUNEL-positive nuclei count in the center of the cryolesion (Fig. 9B). However, injection of BMCD133 cells resulted in a significant reduction of cardiomyocyte apoptosis in the border zone (3.9±1.6% vs. 7.5±1.3% in control, p=0.02). In the border zone of UCBCD133 cell-treated hearts, apoptosis seemed to be reduced to a similar degree, but the difference did not reach statistical significance (4.3±0.7%, p=0.14 vs. control). In the remote normal myocardium as well as in the center of the cryolesion, the percentage of TUNEL-positive cardiomyocytes was not significantly different between the groups (Fig. 10).


Figure 9
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  		Fig. 9 Apoptosis in the myocardium following cryoinjury with or without cell injection. (A) TUNEL staining with DAPI counterstaining of all nuclei, depicting several TUNEL+ nuclei and one TUNEL+ capillary (arrow). (B) Detail magnification of a TUNEL+ nucleus embedded between or within myofibrils. (C) The same image merged with DAPI stain. The TUNEL+ nucleus appears to be part of a multinucleated myofiber, however, it can not be clearly distinguished from an interstitial cell.

 

Figure 10
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  		Fig. 10 Quantitation of TUNEL+ apopototic nuclei, expressed in % of the number of total nuclei (DAPI staining). (A) Percentage of TUNEL+ nuclei in normal myocardium remote from the cryolesion; (B) TUNEL+ nuclei in the center of the cryolesion; (C) TUNEL+ nuclei in the border zone of the cryolesion. *p<0.05 vs. control.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Although the notion of cardiomyocyte-differentiation of unmodified adult stem cells appears to be unrealistic at present [25], a variety of experimental studies in small and large animals have indicated that haematopoietic stem cells and/or endothelial progenitor cells can improve heart function upon delivery to diseased myocardium. While basic researchers have identified very promising adult stem cell types utilizing complex ex vivo cell isolation and manipulation strategies [5,26,27], clinicians are currently limited to using clinically available cell products. In this context, the CD133 antigen has attracted interest because it is expressed on the surface of endothelial progenitor cells and presumably also on cells with a particularly high degree of stemness (i.e. CD133+/CD34 – cells), and because protocols for large-scale isolation by immunomagnetic selection of native cells are readily available. Following the advent of clincial trials using CD133+ bone marrow or blood-derived cells, Agbulut et al. first demonstrated the functional efficacy of human CD133+ marrow cells in an immunodeficient rat model of ischemic myocardial infarction [28]. Similar to their observation, we were not able to detect transplanted BMCD133 cells by immunofluorescence several weeks after implantation, although the persistence of their progeny in the heart was evidenced by PCR. The reasons for the disappearance of histologically detectable cells are not clear. The immunodeficient NOD/SCID mouse model has been extensively used in xenogenic cell transplantation studies [29], but some residual immunologic host activity against the xenogenic cell graft cannot be completely excluded. Alternatively, pharmacologic immunosuppression might be used, but this could also interfere with cellular proliferation and differentiation. It is not clear, either, why hDNA can be detected by PCR in the absence of positive immunostating for human cells. Possible, albeit largely speculative, explanations are fusion of transplanted human cells with host cells [30,31], persistence of hDNA following phagocytosis or lysis of graft cells, or simply the different levels of sensitivity of both detection methods.

Although the echocardiographic assessment of LV contractility in mice may be less accurate than intracardiac recording of pressure–volume loops in rats (as was done by Agbulut et al. [28]), our data confirm the functional benefit derived from BMCD133 cells. The possible reasons for this increase in contractile function are manifold, but, admittedly, the results of our experiments are not sufficiently conclusive to point out one specific mechanism-of-action. Along with many other groups, we did not find convincing evidence of cardiomyocyte-like differentiation of BMCD133 cells, but our in vitro experimental approach was certainly limited. It should not be ruled out that some myocyte differentiation potential of human hematopoietic/endothelial progenitor cells does exist, provided there is direct contact with host cardiomyocytes, as has been indicated in several in vitro co-culture or in vivo studies [32]. Nevertheless, we did detect a significant impact on vascularization of the injured myocardium [13], and found that the presence of BMCD133 cells appears to reduce the number of apoptotic cells in the infarct border zone, although this phenomenon did not result in a measurable difference in infarct size. In fact, we did not expect to see a relevant impact on the size of the necrosis area, since the cryolesion instantly produces an irreversible transmural necrosis that could only be expected to decrease in size when substantial neo-myogenesis would occur. The most solid albeit least specific line of evidence, however, is the beneficial impact on post-injury mortality. Whatever the mechanism may be, some benefit of CD133+ cells in the heart does exist.

In the typical clinical target population, older patients with advanced ischemic heart disease, the regenerative capacity of autologous adult stem cells is probably reduced [33], and cord blood-derived cells have been suggested as an attractive alternative with enhanced angiogenic and hematopoietic potential [34–38]. In a previous study, we have shown that human cord blood mononuclear cells injected intravenously migrate to the ischemic mouse heart and facilitate angiogenesis processes [39]. Similarly, Leor et al. described beneficial functional effects of CD133+ cord blood cells after intravenous infusion in athymic rats with myocardial infarction [40], and Yang et al. reported on the angiogenic capacity of expanded UCBCD133 in ischemic hind limbs [17]. We therefore decided to directly compare the efficacy of BMCD133 and UCBCD133 cells in the present study, and found a similar effect on post-injury survival and blood vessel density. In vitro, both BMCD133 and UCBCD133 cells were able to form capillary-like structures and to differentiate into endothelial cells and/or their immediate progenitors. Besides primary endothelial differentiation of CD133+ cells, the increase in blood vessel density in vivo might also be attributed to the generation of angiogenic growth factors. In this context, Pomyje et al. demonstrated the expression of angiopoietin-1, angiopoietin-2 and vascular growth factor as well as their receptor mRNAs in CD34+/CD133+ human UCB cells in vitro, supporting the notion of a paracrine action of these cells in the regulation of angiogenesis [15]. The increase in blood vessel density most likely increases local blood flow in the necrosis border zone, thus delaying or preventing the onset of apoptosis. Alternatively, direct anti-apoptotic actions due to secreted cytokines or other graft-host cell interactions may also play a role.

On the other hand, there was no measurable impact of UCBCD133 cells on LV contractility, and the reduction of apoptosis did not reach statistical significance. Taken together, UCBCD133 cells appear to less potent than BMCD133 cells in preventing or reversing myocardial injury, at least in the model used here, although in vitro they appear to be similarly potent endothelial progenitor cells. In an attempt to find an explanation for this counterintuitive finding, we compared the expression profile of a number of "stemness"-related surface markers. The only difference we found is an approximately 50% lower percentage of CD117+ cells among UCBCD133 cells. The proto-oncogene c-KIT/stem cell factor receptor/CD117 is thought to modulate cellular differentiation and proliferation processes but also to inhibit apoptosis, and one might assume that less CD117+ cells in a given cell product indicate an overall lower functional capacity [41]. However, our study is only observational and does not provide data to prove this notion. There is a multitude of other confounding factors that might also help explain the differences between BMCD133 and UCBCD133 cells, including the UCB preservation protocol and possibly a higher content of mesenchymal-type stem cells in BM-derived cell products.

In summary, we found that CD133+ cells from human bone marrow clearly have beneficial effects on the hearts subjected to an extensive cryolesion, improving survival, contractility and vascularization, and inhibiting myocardial apoptosis. The experimental support for the ongoing clinical pilot trials using similar cell products therefore seems to grow. The corresponding cell product derived from cord blood appears to be less potent, and further studies are required to elucidate the reasons for this discrepancy.

4.1 Limitations of the study
We used a cryoinjury model because it results in a distinct area of myocardial necrosis with quite uniform impairment of left ventricular contractility, facilitating comparison of changes in function. On the other hand, the nature of the injury is different from that produced by ischemic myocardial infarction. Most importantly, the local cytokine milieu, which is probably relevant for host-to-stem cell interactions, may not be comparable. Furthermore, measurement of LV contractility in mice is clearly challenging, and transthoracic echocardiography is known to have substantial inter-observer variability. In our experiments, one investigator who was blinded with respect to the group assignment of the animals, performed all the studies. However, we plan to use intracardiac catherization for more sensitive measurement of LV function in the future.


   Acknowledgement
 
The authors gratefully acknowledge the technical help of M. Nickel and M. Fritsche, and thank Vita34 (Leipzig, Germany) for the cord blood samples. Parts of this work have been done within the START-MSC network, and the input of the network partners is highly appreciated.


   Notes
 
1 Both authors equally contributed to this article. Back

2 Current address: German Heart Institute Berlin, Cardiac Surgery, Augustenburger Platz 1, 13353 Berlin, Germany. Back

Time for primary review 25 days

* This work was supported in part by the German Minister of Research (BMBF FKZ 01ZZ0108 and FKZ 01GN0536) and the Minister of Economy of Saxony (SAB 7522/1193).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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