Objective: We tested the hypothesis that intravenously administered human umbilical cord blood (hUCB) cells contribute to repair processes following myocardial infarction.
Methods: hUCB mononuclear cells containing 0.11% to 1.1% CD34+ cells were injected in the tail vein of NOD/scid mice that had (MI+) or had not (MI−) previously undergone ligation of the left anterior coronary artery (LAD). Homing to bone marrow and solid organs was determined by polymerase chain reaction (PCR) for human DNA (hDNA) using human-specific primers of Locus D7Z1. Immunostaining was used for phenotypic analysis, and capillary density as well as myocardial scar formation was assessed. Moreover, expression of stromal cell-derived factor-1 (SDF-1) was studied in infarcted and in normal hearts.
Results: hDNA was detected in marrow, spleen, and liver of both MI+ and MI− mice 24 h, 1 week, and 3 weeks after cell injection. In the heart, however, hDNA was detected in 10 of 19 MI+ mice but in none of the MI− mice (p=0.002). Infarct size was smaller in cell-treated MI+ mice than in untreated MI+ hearts (38.7 versus 47.8%, P<0.05), and there was also less collagen deposition. In cell-treated MI+ mice, capillary density in the infarct border zone was approximately 20% higher (p=0.03), and clusters of hUCB-derived cells were detected in the perivascular interstitium. Occasionally, chimeric capillaries composed of human and mouse endothelial cells were found, but the vast majority of neo-vessels appeared to consist of mouse cells only. Up to 70% of the cord blood-derived cells in the heart were CD45+. There was no evidence of cardiomyocyte differentiation as determined by co-localization of HNA or HLA-I with GATA-4 or Connexin 43. In infarcted myocardium, expression of SDF-1 mRNA was approximately 7-fold higher than in normal hearts.
Conclusions: hUCB cells migrate to infarcted, not to normal myocardium, where they engraft, participate in neoangiogenesis, and beneficially influence remodelling processes. Cord blood cells may hence be useful for cell therapy of ischemic heart disease.
This article is referred to in the Editorial by Friedrich and Böhm (pages 4–6) in this issue.
Umbilical cord blood contains circulating stem/progenitor cells with cellular characteristics that are quite distinct from those of bone marrow and adult peripheral blood [1,2]. The frequency of hematopoietic stem cells and progenitor cells equals or exceeds that of marrow and greatly surpasses that of adult peripheral blood . Stem cells from cord blood expand longer in culture, produce larger hematopoietic clones in vitro, and have longer telomeres [4,5]. Cord blood also contains mesenchymal progenitor cells capable of differentiating into marrow stroma, bone, and muscle, and the immaturity of neonatal cells compared with adult cells may translate into greater cell plasticity . In addition, cord blood cells are usually not infected with cytomegalovirus or Epstein–Barr virus, as is often the case with marrow cells. For all those reasons, cord blood is considered as a valuable source of cells with a potential for tissue repair in response to injury . However, the homing and migration characteristics of cord blood cells as well as their capacity for transdifferentiation into various target organ cells remain largely unexamined . We therefore studied the organ-specific homing pattern of human mononuclear cord blood (hUCB) cells in a NOD/scid-mouse model and tested the hypothesis that hUCB cells specifically migrate to the heart after myocardial infarction, participate in tissue remodelling, and possibly facilitate regeneration processes. Moreover, we investigated whether stromal cell-derived factor-1 (SDF-1), which has been implicated in stem cell homing processes [9,10], is involved in selective cell migration to ischemic myocardium.
2.1. Preparation of cord blood mononuclear cells
Human cord blood was donated for research and written consent was obtained prior to collection by Vita34 (Leipzig, Germany). The protocol conforms to the principles outlined in the Declaration of Helsinki. Cord blood was stored in the vapour phase of liquid nitrogen with citrate phosphate dextrose solution (CPD) and dimethyl sulphoxide (DMSO) added. After thawing on ice, it was diluted with PBS containing 2 mM EDTA and 200 U/ml DNase I. The cells were washed and centrifuged three times (1000 rpm for 7 min). 15 ml of this cell suspension was carefully layered over 20 ml of Ficoll (1,077 g/ml, Biochrom). After 35 min of centrifugation in a swinging bucket rotor, the upper layer was aspirated and the mononuclear cell layer was collected. Cell viability was determined using the Trypan blue dye exclusion method. Another sample was taken for determination of the hematopoietic stem cell content by standard FACS (see below). Only freshly isolated cells were used, no in vitro cell expansion was performed.
2.2. Flow cytometric analysis
Cells were washed once in PBS with 0.1% BSA and 0.01% sodium azide (FACS buffer) before flow cytometric analyses were performed. Briefly, cells were labelled during 15-min incubation on ice with monoclonal antibody recognizing the various surface molecules. Antibodies used were FITC-conjugated anti-CD45 (clone 2D1), PE-conjugated anti-CD34 (clone 8G12), and PE-conjugated anti-CD14 (clone MφP9) from BD Biosciences and biotin-anti-CD133/1 (clone AC133) from Miltenyi Biotec used in combination with strepavidin-Cy5 (BD Biosciences). Anti-CD45, anti-CD34, and anti-CD133 are of the IgG1 isotype; anti-CD14 is an IgG2b antibody. Cells were washed once again, resuspended in FACS buffer, and analyzed using FACSCalibur and Cellquest software from BD Biosciences. The absolute number of CD34+ cells was calculated based on the percentage of CD34+ cells and the absolute number of hUCB mononuclear cells.
2.3. Myocardial infarction
All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” (NIH Publication No. 85-23, revised 1996). Female NOD/scid mice were obtained from Jackson Laboratories (Durham, NH). Myocardial infarction was induced at 8–12 weeks of age (approximately 20 g body weight) in 33 animals. Mice were anesthetized by intraperitoneal injection of pentobarbital, endotracheally intubated, and mechanically ventilated. The heart was exposed via a left thoracotomy and the left anterior descending coronary artery was ligated with a 7-0 silk suture (MI+). In the control group (n=6), mice were sham operated including a left thoracotomy but without coronary artery ligation (MI−). Twenty-four hours following the operation, 6 × 106 cord blood MNCs containing between 0.11% and 1.1% CD34+ cells were suspended in 20μl PBS and injected into the tail vein of 19 MI+ mice and 6 MI− mice. Six further MI+ mice did not receive cells and served as controls. Animals were sacrificed 24 h, 1 week, and 3 weeks after cell transplantation, and engraftment of human cells was studied in bone marrow, spleen, liver, and heart. Marrow was collected from the femurs and washed with PBS. Liver and spleen tissue was homogenized and prepared for DNA extraction. Hearts were excised, rinsed, weighed, embedded in OCT medium, and stored at −80 °C. Subsequently, 6 μm frozen sections were prepared of the entire heart. Every other section was collected for DNA extraction and PCR analysis; the remaining sections were mounted on slides and used for immunostaining.
2.4. PCR analysis
As a screening test for human cord blood cell migration to various organs, we chose to study the presence of human DNA in murine tissue. DNA was extracted from homogenized tissue with phenol/chloroform, precipitated with ethanol/sodium acetate, and analyzed for presence of a human α-satellite chromosome sequence by PCR using human specific primers D7A and D7B of α-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 . 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 amplification products for D7Z1 was 600 bp and 1000 bp .
2.5. Determination of infarct size
Sections were stained with haematoxylin–eosin and Sirius red F3BA (0.1% picric acid, Aldrich). An increase in collagen content, measured as the Sirius red-positive area on each section, determines the infarct area, which was measured by computerized planimetry. The ratio of scar length and entire circumference defined the infarct extent for the endo- and epicardial surface, respectively. Final infarct size was determined as the average of endo- and epicardial surfaces and was given in percent. Quantitative assessment of collagen depositions was performed with a multipurpose colour image processor (Ensivision). A single investigator blinded to the experimental groups performed the analysis.
For immunohistologic detection of human cells, frozen tissue sections were incubated with monoclonal mouse anti-human nuclear antigen antibody (HNA, clone 235-1, Chemicon MAB 1281) or monoclonal mouse anti-HLA-class 2 antibody W6/32 (Dako M0736). After blocking in Envision blocking buffer (Dako), sections were placed in primary antibody overnight at 4 °C. On the following day, the sections were incubated with alkaline phosphatase- or FITC-conjugated secondary goat anti-mouse IgG. To minimize the risk of false-positive results, the neighbouring section of every positive anti-HLA-1 slide was stained using anti-HNA antibody, and the slides were directly compared. For assessment of target cell differentiation, double staining was performed using monoclonal anti-CD31 (Dako), monoclonal anti human CD45 (clone 2B11+PD7/26/16 MRC OX-1, Serotec), monoclonal anti-Connexin 43 (Chemicon), and polyclonal anti-GATA 4 (Santa Cruz) antibody. Nuclei were also counterstained with 6-diamidino-2-phenylindole (DAPI; Sigma). For technical reasons, analyses including DAPI staining were done using conventional fluorescence microscopy (i.e., Figs. 5 and 7). Fluorescence microscopy without DAPI visualization was performed using a Leica TCS SP2 confocal microscope.
2.7. Capillary density
To determine the capillary density in infarcted myocardium and in myocardium remote from the infarct, tissue was prepared as described by Weidner et al. . Sections were stained using monoclonal anti-mouse Factor VIII antibody (Novocastra). After incubation with a biotinylated secondary antibody, bromodeoxyuridine staining was performed. For quantification of positively stained vessels, 5 sections from within the infarct 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 animal and 6 animals per time point. The results were expressed as capillaries per high power field.
2.8. SDF-1 expression
A separate set of experiments involving real-time RT-PCR was performed. Six mice underwent myocardial infarction and hearts were collected 24 h later. Six sham-operated mice (thoracotomy only) supplied untreated control hearts. Total mRNA was isolated using the RNeasy Mini Kit (Qiagen). mRNA was reversely transcripted with TaqMan Reverse Transcription Reagents (Applied Biosystem, CA). Detection of SDF-1 and β-actin mRNA levels was performed by using the ABI PRISM 7000 Sequence Detection System (ABI, CA). 25 μl reaction mixture contained 12.5 μl SYBR Green PCR master mix, 10 ng cDNA template, 5′-GGA TGC AGA AGG AGA TCA CTG-3′ forward and 5′-CGA TCC ACA CGG AGT ACT TG-3′ reverse primers for murine β-actin, 5′CGT GAG GCC AGG GAA GAG T-3′ forward and 5′-TGATGA GCA TGG TGG GTT GA-3′ reverse primers for murine SDF-1. mRNA expression was calculated using the comparative threshold cycle (Ct) method.
2.9. Statistical analysis
Numeric data are expressed as mean ± S.D. Differences in quantitative PCR results between two groups were tested for significance using Student's t-test. Comparison of the data regarding capillary density, infarct size, collagen deposition, and HW/BW ratio was performed using one-way ANOVA. The frequency of hDNA-positive hearts in MI+ and MI− mice was compared using the π2-test. A p-value<0.05 was considered to indicate a significant difference.
3.1. Cord blood cell characteristics
Thirty cord blood samples were used for mononuclear cell separation. The mean collection volume was 44.6 ± 4.1 ml, containing between 99 × 106 and 294 × 106 cells by manual counting after Ficoll centrifugation and washing (mean 208 × 106 ± 58 × 106 cells). Cell viability ranged between 42% and 98% (mean value=78 ± 18%). A representative FACS analysis is depicted in Fig. 1, showing the forward versus sideward scatter plot (A) and the proportion of CD14+/CD45+ cells. CD45+ cells are gated (C) and the percentage of CD34+/CD133+ cells among the CD45+ leukocytes is shown in (D). Overall, the percentage of CD45+ cells ranged between 43.8% and 91.1%. Percentages of CD34+ cells (including CD34+/CD133+ and CD34+/CD133neg cells) ranged between 0.11% and 1.1% (0.63 ± 0.5%). The average percentage of CD14+ monocytes was 14.9 ± 0.3%.
3.2. Detection of hDNA
Following intravenous implantation of human cord blood mononuclear cells, human DNA was detected in bone marrow, liver, and spleen of all animals (MI+ and MI−) at all time points. There was, however, a striking difference regarding hUCB cell engraftment in the heart (Fig. 2). In 10 out of a total of 19 MI+ mice PCR confirmed the presence of hDNA, while no hDNA was detected in any of the MI− hearts (P=0.002 by π2 test). (24 h, 4 out of 6 MI+ mice; 1 week, 3 out of 6 MI+ mice; 3 weeks, 3 out of 7 MI+ mice).
3.3. Myocardial infarction
Eight animals (25%) died preoperatively prior to hUCB cell injection and were excluded from further analysis. As determined by light microscopy following H&E and Picrosirus Red staining, LAD ligation consistently resulted in transmural myocardial infarction, exhibiting typical histological changes including thinning of left ventricular free wall and extensive collagen deposition 3 weeks after myocardial infarction. The average infarct size was 47 ± 7% in untreated MI+ mice and 39 ± 3% in the cell-treated MI+ mice, and this difference proved statistically significant (P=0.016; Fig. 4A). Fig. 3 depicts representative left ventricular sections 3 weeks following LAD ligation with (Fig. 3A) or without (Fig. 3B) cord blood cell injection. Within the group of cell-treated MI+ mice, infarct size was compared between hearts that were positive for hDNA by PCR, and those that were PCR-negative. Indeed, we found that infarct size was significantly smaller in MI+/cell-treated/PCR+ hearts than in MI+/cell-treated/PCR− hearts (Fig. 4B). Overall, loss or gain of ventricular tissue was also assessed by determining the ratio of heart weight (HW) to body weight (BW). The body weight did not significantly differ between groups. In MI+ mice with cord blood cell injection, the HW/BW ratio was slightly lower than in MI+ mice without cell injection (Fig. 4C), perhaps indicating less compensatory myocardial hypertrophy in cell-treated mice. As determined by computerized color analysis of the Sirius red stained sections, collagen content was lower MI+ hearts of hUCB-treated mice than in MI+ hearts of mice without cell treatment (P=0.02).
3.4. Morphology of hUCB-derived cells
In hDNA-positive hearts we also traced hUCB-derived cells by immunostaining with anti-human nuclear antigen (HNA) and anti-HLA class I antibodies. Fig. 5 shows human cord blood-derived cells in the heart of a MI+ mouse sacrificed 3 weeks after cell transplantation. HNA+ and HLA+ cells were found widely distributed in the myocardium. Typically, isolated cells and sometimes clusters of more than 10 transplanted cells were found in the subepicardial and subendocardial infarct tissue. In 100 random fields per section, the proportion of HNA+ and HLA+ cells was approximately 0.01–0.1% of the native mouse cells. When examined at higher magnification, HNA+ and HLA-I+ cells were typically localized in the immediate vicinity of myocardial blood vessels (Fig. 6). Double-immunofluorescent staining with anti-HLA class I and anti-CD31 antibodies revealed that at least that some of the hUCB cells appeared to display the phenotype of endothelial cells. Occasionally, small blood vessels of capillary morphology presented a human–mouse micro-chimerism (Fig. 7), consisting of both HLA-I+ and HLA-Ineg endothelial (CD31+) cells. By double staining with HNA and GATA-4 or Connexin 43 antibodies, we did not find any evidence for differentiation of hUCB cells in a cardiomyocyte-like phenotype (data not shown). Between 30% and 70% of the hUCB-derived cells (HLA-I+/HNA+) detected in murine myocardium stained positive for CD45 and had hence retained their hematopoietic lineage commitment (Fig. 8).
3.5. Capillary density
Capillary density in the infarct border zone as well as in myocardium remote from the infarct was determined following immunostaining with anti-mouse Factor VIII antibody. Representative images are shown in Fig. 9. Infarcted myocardium of mice without hUCB cell transplantation contained few capillaries, whereas capillary density in MI+ mice that had undergone hUCB cell transplantation was approximately 20% higher (Fig. 7). In the cell transplanted group, the average vascular density within the infarct border zone was 6.19 ± 0.2 vessels per high power field (HPF; 400 ×), while in control hearts (MI+, no cell treatment) there were 5.33 ± 0.5 vessels/HPF (p=0.033). In myocardium remote from the infarct area, capillary density was 5.41 ± 0.49 per HPF in MI+ mice with hUCB cell transplantation, which was again significantly higher than in MI+ mice without cell injection (4.3 ± 0.57/HPF, p=0.013). Most of the vessels exhibiting Factor VIII-positive endothelium were capillaries with an internal diameter ≥ 20 μm. The size of the capillaries in the infarct area ranged between 5 μm and 20 μm, with a greater variation in diameter than observed in normal myocardium (10 μm to 20 μm). We also compared peri-infarct capillary density in MI+ mice with cord blood cell injection that were PCR positive with those that were negative for hDNA. Capillary density was indeed higher in PCR-pos cell-treated MI+ mice than in PCR-neg cell-treated MI+ mice (6.6 ± 0.2 vs. 5.8 ± 0.5 vessels/HPF, P=0.028).
3.6. SDF-1 expression
To test the hypothesis that upregulation of SDF-1 occurs in infarcted myocardium and hence may contribute to hUCB-derived cell migration in ischemic hearts, we compared the expression level of SDF-1 mRNA in hearts 24 h after myocardial infarction with that in untreated control hearts. Fig. 10 shows that the level of the SDF-1 mRNA in myocardium is approximately 7-fold higher after myocardial infarction (p<0.0001).
Our study demonstrates that intravenously administered human umbilical cord blood cells migrate to and engraft in the heart of (NOD)/scid mice only following myocardial infarction. While there was no evidence of cardiomyocyte formation, hUCB cells appear to be involved in angiogenic processes. Following cell administration, capillary density in hearts with myocardial infarction is moderately increased, but few hUCB-derived cells actually incorporate in neo-vessels, whereas most of the human cells are located in the perivascular interstitium. Cord blood cell injection also appeared to beneficially influence infarct size and collagen deposition, provided there is effective homing of hUCB cells to the ischemic heart. Since myocardial infarction also led to a marked upregulation of SDF-1 expression in the heart, we assume that SDF-1 is at least partly responsible for the selective hUCB cell trafficking to ischemic hearts, although the study design does not allow for proof of a cause–effect relationship.
Cell therapy for cardiac regeneration is currently under intensive investigation. The initial enthusiasm regarding the cardiomyogenic potential of adult stem cells  has faded [14,15], but substantial evidence obtained in large animal studies and even clinical pilot trials indicates that hematopoietic cells nevertheless exert beneficial effects in ischemic myocardium [16–18]. Trafficking of bone marrow-derived cells to the heart is a phenomenon that has been demonstrated in various experimental models  as well as in human heart transplant patients . The vasculogenic potential of cord blood cells has previously been assessed in several studies. In in vitro experiments using CD34+/CD133+ human cord blood cells, Pomyje and colleagues demonstrated that those express angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor as well as their receptor mRNAs, supporting a role of these cells in regulation of angiopoiesis . Muohara et al. have described that transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization in the ischemic hindlimb , and Pesce et al. observed endothelial and also myogenic differentiation of hUCB-derived stem cells in ischemic limbs . Recently, Le Ricousse-Roussanne et al. reported that ex vivo differentiated endothelial and smooth muscle cells from human cord blood progenitors home to the angiogenic tumor vasculature . In our model, the inoculated hUCB cells appear to be a source of neovascularization of ischemic myocardium, since they differentiated into endothelial cells, formed chimeric capillaries, and presumably participated in post-infarct remodelling, angiogenesis, and maturation of the scar. To what extent these processes will translate into functionally relevant myocardial regeneration as has been described using human marrow-derived cells in athymic rats  remains to be determined. At first glance, our observation regarding the formation of a human/mouse vascular micro-chimerism appears to contradict the report by Ziegelhoeffer et al., who demonstrated that bone marrow-derived cells do not incorporate into the adult growing vasculature . However, it should be noted that (i) we used cord blood cells, not bone marrow-derived cells, (ii) we utilized a myocardial infarction model, not a hindlimb ischemia or tumor model as these authors did, and (iii) we found rather few neo-vessels that stained positive for human cell markers, whereas most of the myocardial neovascularization appeared to originate from native mouse endothelial cells, with donor cells mainly located in the perivascular interstitium. Although we found evidence that hUCB-derived cells are able to incorporate in growing vessels in principle, much of the angiogenic host tissue response is most likely do to local secretion of cytokines and growth factors [17,25]. Since 30–70% of the cord blood cells that we detected had maintained their hematopoietic phenotype, they may very well have been able to modulate the immune/inflammatory response to myocardial infarction.
The mechanisms underlying homing and engraftment of stem cells and progenitor cells in various target organs are not fully understood, but in situ chemokine and cytokine expression probably plays a role. A noted chemokine is stromal cell-derived factor-1, also known as pre-B cell growth stimulating factor. Under normal conditions, bone marrow stromal cells such as bone-forming osteoblasts produce and secrete high levels of SDF-1. SDF-1 and its receptor CXCR-4 are believed to facilitate stem cell adhesion to microvessel endothelium and trans-endothelial migration in the infarct border zone of infarcted myocardium . We assume that after myocardial infarction, the local expression level of SDF-1 is upregulated, increasing the myocardial concentration of SDF-1 and attracting circulating CXCR4 positive cells . However, the molecular mechanism of SDF-1 secretion by ischemic heart cells and its chemoattractant action on stem cells is still under investigation. Cord blood cell trafficking to the ischemic heart is not an all-or-nothing phenomenon, since hDNA was detectable only in 10 out of 19 MI+ hearts. In intact biological systems subjected to complex pathophysiologic stimuli, the molecular and cellular response is usually gradual, and chemokine expression may need to reach a certain threshold to attract relevant numbers of progenitor cell. The variables involved are manifold: Initial infarct size, magnitude of cytokine expression per unit ischemic tissue, or the responsiveness of injected hUCB cells may vary considerably. The necessity of relevant hUCB cell trafficking to the ischemic heart for a protective or regenerative effect on the myocardium is underscored by our finding that infarct size is significantly smaller and capillary density is higher in hearts of cell-treated MI+ mice that were PCR+ for hDNA than in those that were PCR−.
Naturally, our study possesses several methodological limitations. We did not determine the functional relevance of myocardial hUCB cell migration in terms of left ventricular contractility or relaxation properties. Furthermore, the co-localization studies that showed the incorporation of hUCB-derived cells into murine neo-vessels were performed using fluorescence microscopy, which has been implicated in producing false-positive results [15,25]. Another possible pitfall is the complete absence of hUCB-derived cells in normal control hearts and the presence of hUCB-derived cells in approximately 50% of the ischemic hearts. Would these data rely on microscopy-based methods such as immunohistology or in situ hybridisation only, one could argue about an inherent lack of sensitivity. Therefore, we chose to perform PCR for detection of human DNA in mouse hearts, and used human chromosome specific α-satellite primers to track cells. In preliminary experiments, we amplified human alpha satellite DNA from in vitro somatic cell hybrids containing only a handful of human cells. PCR analysis with centromere-specific primer sets can be performed to characterize somatic cell hybrids more effectively and perhaps more accurately than by cytogenetic means. To our knowledge, this is the first time that a chromosome specific α-satellite DNA primer has been used to distinguish different genetic backgrounds, and we are confident that PCR-negative murine myocardium does in fact not contain a relevant number of hUCB-derived cells.
We thank Vita34 for donating human cord blood samples, and appreciate the excellent technical help of Nadine Hoffmann, Claudia Fahle, and Anna Schmicker. This work was supported in part by the German Minister of Research (BMBF FKZ 01ZZ0108) and the Minister of Economy of Saxony (SAB 7522/1193).
- myocardial infarction
- polymerase chain reaction
- human leukocyte antigens
- cd34 antigens
- cd45 antigens
- severe combined immunodeficiency
- blood capillaries
- umbilical cord blood
- stromal cell-derived factor 1
- mononuclear cells