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Keywords:

  • Aging;
  • Heart;
  • Myocardial infarction;
  • Progenitor cells;
  • Stem cells;
  • Tissue therapy;
  • Ventricular remodeling

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. Acknowledgements
  10. References

The use of adult stem cells for myocardial tissue repair might be limited in elderly and sick people because their cells are depleted and exhausted. The present study was conducted to explore the potential of human umbilical cord blood (UCB) CD133+ progenitor cells for myocardial tissue repair in a model of extensive myocardial infarction (MI). CD133+ progenitor cells were isolated from newborn UCB. Cells (1.2–2 × 106) or saline (control) was infused intravenously 7 days after permanent coronary artery ligation in athymic nude rats. Left ventricular (LV) function was assessed before and 1 month after infusion by echocardiography. Tracking of human cells was performed by fluorescent in situ hybridization for human X and Y chromosomes or by immunostaining for HLA-DR or HLA-ABC. One month after delivery, LV fractional shortening improved by 42 ± 17% in cell-treated hearts and decreased by 39 ± 10% in controls (p = .001). Anterior wall thickness decreased significantly in controls but not in treated hearts. Microscopic examination revealed that the UCB cells were able to migrate, colonize, and survive in the infarcted myocardium. Human cells were identified near vessel walls and LV cavity and were occasionally incorporated into endothelial cells in six of nine cell-treated animals but not in controls. Scar tissue from cell-treated animals was significantly populated with autologous myofibroblasts as indicated by colocalization of HLA-DR and α-smooth muscle actin staining. In conclusion, the present work suggests that, after MI, intravenous delivery of human UCB-derived CD133+ cells can produce functional recovery by preventing scar thinning and LV systolic dilatation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. Acknowledgements
  10. References

Ventricular remodeling and ultimately heart failure are the devastating consequences of extensive myocardial infarction (MI). Current options for treatment of MI and subsequent heart failure suffer from specific limitations. In light of the limited efficacy of these current treatment options, alternative, additional long-term therapeutic strategies are needed. Cardiac repair is a new therapeutic option. Through autologous stem cell–based therapies, the concept of “growing” new heart muscle and vascular tissue has promised to revolutionize the treatment of heart disease.

However, the use of adult stem cells for myocardial repair might be limited by low recovery of cells from any adult tissue, thereby leading to difficulties in obtaining the appropriate number of stem cells in a reasonable period [13]. Furthermore, stem cells that are generated by aging bone marrow with or without atherosclerosis risk factors manifest impairment in self-renewal and proliferation, adhesion, and incorporation into vascular structures [16]. These limitations underline the need for an alternative stem cell source for myocardial repair.

The use of umbilical cord blood (UCB)–derived stem cells may provide a solution to impaired stem cell function in the sick and aged population. Their therapeutic effect has been demonstrated in animal models of hindlimb ischemia [7] and stroke [811]. The UCB contains relatively a high number of CD133+ and CD34 progenitor cells [7, 12, 13]. These cells have homing, myogenic, and angiogenic potential that is relevant to myocardial repair [14, 15]. UCB cells can be easily obtained, can be expanded in vitro [16], have the potential for enhanced self-renewal and differentiation, and can be stored for future use [17]. The aim of the present study was to test the hypothesis that human UCB-derived stem/progenitor cells can be used as an alternative cell source to rejuvenate the infarcted myocardium, enhance healing, and improve left ventricular (LV) function.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. Acknowledgements
  10. References

UCB collection was performed with written approval by mothers according to a protocol approved by Sheba Medical Center's committee on ethics of human investigation. Animal studies adhered to the American Physiological Society's Guiding Principles in the Care and Use of Animals.

Isolation of CD133+ Cells from UCB

Fresh placental blood was recovered in EDTA-containing bags immediately after delivery. On the first day, the mononuclear cell (MNC) population from UCB was obtained from Ficoll (IsoPrep; Robbins Scientific Corporation, Sunnyvale, CA, http://www.robbinssci.com) gradient. CD133+ cells were purified from MNCs by magnetic activated cell sorting columns (MACS; Miltenyi Biotec, Auburn, CA, http://www.miltenyibiotec.com) using microbead-conjugated antibodies. Purity of sorted cells was assessed by fluorescence-activated cell sorting (FACS) analysis using anti-CD133 antibodies labeled with phycoerythrin (PE) or fluorescein isothiocyanate (FITC), and cells were stored for future use.

Rat Model of MI and Cell Transfer

Female athymic nude rats (Hsd:RH-Foxn1rnu; Harlan Laboratories, Indianapolis, http://www.harlan.com) were anesthetized with a combination of ketamine (90 g/kg) and xylazine (10 g/kg). We induced MI by permanent left coronary artery occlusion as previously described [18]. Seven days after MI, cells were prepared for infusion by centrifugation (300g for 10 minutes) and re-suspension in phosphate-buffered saline (PBS). The rats were anesthetized as described above, and 0.1 ml of UCB-derived CD133+ progenitors (1.2–2 × 106) or PBS (control) was infused via the femoral vein for 1 minute, as previously described [19]. The dose was based on cell availability and previous reports [15, 19, 20].

Histology, Fluorescent In Situ Hybridization

CD133+ cells were deposited onto positively charged glass slides by centrifugation (Cytospin 3; Shandon, Manchester, U.K., http://www.shandon.com). Cytospin slides were processed for two-color fluorescent in situ hybridization (FISH) and subsequent nuclear staining with 4,6-diamidino-2-phenylindole (DAPI).

For FISH, sections on slides were fixed in methanol/acetic acid (3:1), washed in PBS, and dehydrated in 70%, 90%, and 100% ethanol. Cytospin preparations were denatured in 70% deionized formamide (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and dehydrated, and a mixture of denatured human X and Y chromosome probes labeled with green and orange fluorescent dyes, respectively, was added (Vysis, Downers Grove, IL, http://www.vysis.com). After hybridization at 42°C, sections were mounted in Vectashield anti-fade with DAPI (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com).

By 4 weeks after transfusion, the hearts were sectioned into three or four transverse slices parallel to the atrioventricular ring. Each slice was either fixed with 10% buffered formalin, embedded in paraffin, and sectioned with a microtome (5-μm-thick) or embedded for FISH in optimal cutting temperature (OCT) embedding compound fixative and snap-frozen, and 5-μm serial sections were collected on the slides. The presence of human donor cells in the recipient heart was confirmed by FISH using DNA probes specific for human X and Y chromosomes or antibodies against HLA-ABC (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com).

To identify transdifferentiation of the engrafted cells, representative slides with positive X or Y signals were stained with antibodies against troponin I (Chemicon, Temecula, CA, http://www.chemicon.com), sarcomeric actin (Sigma-Aldrich), and von Willebrand factor (vWF) (Chemicon). The entire cell-containing area of each section was screened under an Axioscope fluorescent microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com) equipped with appropriate filters to identify nuclear (DAPI) staining and FISH signals.

After endogenous peroxidase was blocked, immunohistochemical stainings were performed with antibodies against HLA-DR (DakoCytomation): α-smooth muscle actin (Sigma-Aldrich); human CD31—endothelial and hematopoietic cell antigen (DakoCytomation); CD45—leukocyte common antigen (DakoCytomation); CD 68—monocyte/macrophage lineage antigen (DakoCytomation); and CD34—human hematopoietic and endothelial progenitor cell (EPC) antigen (DakoCytomation). Representative cytospin slides were air-dried and immunostained with antibody against HLA-DR (DakoCytomation). The lungs, liver, spleen, intestine, and femurs were removed, and representative sections were fixed in formalin and immunostained with HLA-DR antibodies.

The effect of engrafted cells upon neovascularization in the infarcted and peri-infarcted myocardium was assessed on representative slides, obtained from midheart transverse section, and immunostained with α-smooth muscle actin antibodies (Sigma-Aldrich) to localize periycytes and arterioles [21]. After low-power examination, five consecutive adjacent fields were photographed from each section at a magnification of ×200. The number of vessels was counted, and vessel density (mean number of capillaries and arterioles per mm2) was calculated for hearts of transplanted and control groups.

Echocardiography

Transthoracic echocardiography was performed on all animals 1 day after MI, before infusion (baseline echocardiogram), and 4 weeks after implantation. Echocardiograms were performed with a commercially available echocardiography system equipped with 12-MHz phased-array transducer, Hewlett-Packard Sonos 5500 (Andover, MA, http://www.hp.com) as previously reported [18]. By short-axis view, we measured maximal LV end-diastolic dimension and minimal LV end-systolic dimension; we calculated LV fractional shortening (FS) as a measure of systolic function, which was calculated as [(LVIDd − LVIDs)/LVIDd] × 100, in which LVID indicates LV internal dimension, d is end diastole, and s is end systole. LV fractional area change (%) was calculated as [(EDA − ESA)/EDA] × 100, in which EDA indicates LV end-diastolic area, and ESA end-systolic area [18]. All measurements were averaged for three consecutive cardiac cycles and were performed by an experienced technician who was blinded to the treatment group. Change (%) in baseline parameter was calculated as [(follow-up parameter-baseline parameter)/baseline parameter] × 100.

Statistical Analysis

All values are presented as mean ± SEM. Because each rat in both groups was used as her own control, changes in echocardiography measurements and LV function between baseline and 4 weeks in treated and control groups were assessed with paired t-test using GraphPad Prism version 4.00 for Windows (Graph-Pad Software, San Diego, http://www.graphpad.com). Change (%) in baseline parameters was calculated as [(follow-up parameter-baseline parameter)/baseline parameter] × 100. The difference between cell-treated and control group was compared by unpaired t-test. All tests were two-tailed, and p < .05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. Acknowledgements
  10. References

Overall, 36 athymic nude rats were included in the study. Eleven rats died immediately after the surgical procedure to induce MI, and three rats (two cell-treated and one control animal) died after infusion. Echocardiography studies and analyses were performed on 17 rats. Nine rats were treated with cord blood–derived CD133+ cells (1.2–2 × 106), and the control group (n = 8) received infusion of PBS (control). The remaining five rats were part of a cell tracking study to optimize the FISH method and HLA immunostaining. Hearts were harvested 4 weeks after infusion.

Characteristics of Isolated Cord Blood-Derived CD133+ Cells

The CD133+ population was derived from a pool of cord blood samples from female and male newborns. The isolated CD133+ progenitor cells demonstrated a purity of >95%, as estimated by FACS analysis (Fig. 1), and a viability of >90%, as assessed by the trypan blue exclusion test. Cytospin slides were processed for two-color FISH and subsequent nuclear staining with DAPI. The cells presented two green signals, XX, or one green and one orange signal, XY, respectively (Fig. 2A). Immunostaining of cell samples, before infusion, showed that more than 90% of the cells were stained positive for HLA-DR (Fig. 2B).

Colonization of the Infused CD133+ Progenitor Cells in the Infarcted Heart

One month after transplantation, the presence of FISH- or HLA-positive human cells was identified in representative slides from six of the nine cell-treated animals but in none of the eight controls.

Using FISH with human sex chromosome probes, we identified accumulations of human cells in close contact with the vascular walls or LV cavity (Fig. 2). The shape and the size of the human cells were similar to those of hematopoietic cells. We were unable to identify positive FISH signals in cardiomyocytes. In several cryosectioned slides, we observed cell clusters that had been detached from the host myocardial tissue into the LV cavity, indicating that the cell graft did not integrate with the host myocardium (Fig. 2E). Counterstaining of representative slides containing human cells with antibodies against troponin, sarcomeric actin, CD31, or vWF was negative, suggesting that the engrafted cells did not transdifferentiate into relevant cardiac cells.

Immunostaining for HLA-DR identified a few positive cells around blood vessels or near the LV cavity (Fig. 2F). Overall, we detected a maximum of 35 FISH- or HLA-DR–positive human cells per heart slide from treated animals.

Immunostaining for human hematopoietic and EPC antigen-CD34 revealed specific signals, confined occasionally to endothelial cells, in cell-treated hearts but not in controls (Fig. 3). Immunostaining with anti-human monocyte/macrophage lineage antigen-CD68, and anti-leukocyte common antigen-CD45 revealed a few positive cells in cell-treated hearts but not in controls (Fig. 3). The shape, localization, and morphology of the positive cells suggest that the donor cells kept their hematopoietic lineage.

In the cell-treated hearts, we identified extensive areas of α-smooth muscle actin-positive cells, namely myofibroblasts, that significantly populated the scar tissue (Fig. 4) and could contribute to scar thickness and strength. In control hearts, positive α-smooth muscle actin staining was limited mainly to the subendocardium (Fig. 4B). Colocalization of the HLA-DR and smooth muscle actin was examined in serial micrograph sections. Four weeks after injection, none of the nine hearts in the cell group showed the presence of myofibroblasts expressing HLA-DR (Fig. 4). This indicates that the myofibroblasts at the scar tissue did not originate from a human source. Total vessel density or functional vessels with a diameter of 10–100 μm (vessel/mm2 ± SEM) in the infarcted myocardium did not differ between CD133+-treated animals and controls (131.0 ± 16.4 vs. 175.4 ± 12.5/mm2, p = .06, or 12.8 ± 2.7 vs. 17.4 ± 3.2/mm2, p = .3, respectively). HLA-DR immunostaining of tissue samples from remote organs identified infused human cells that had migrated to and colonized the spleen (Fig. 5A) and the liver (Fig. 5B) but rarely the lungs (Fig. 5C), intestine (Fig. 5D), and bone marrow (Fig. 5E).

Echocardiography Functional Study

There was no significant difference in baseline values between cell-treated and control animals. Four weeks after injection, the infused CD133+ progenitor cells attenuated the classic course of LV systolic dilatation, dysfunction, and anterior wall thinning, which complicated extensive anterior MI, as was seen in controls (Table 1). The cell infusion did not influence LV diastolic dilatation and mass. One month after transfusion, LV FS improved by 42 ± 17% from baseline in cell-treated hearts and decreased by 39 ± 10% in controls (p = .001; Fig. 6A; Table 1). This effect was primarily the result of a smaller increase in end-systolic diameter from baseline values in the cell-treated group (11 ± 10%) compared with controls (51 ± 12%) (p = .02). Cell delivery prevented scar thinning, and anterior wall thickness decreased by 12 ± 4% in controls (p = .01; Fig. 6B; Table 1). The effect of UCB cells on the changes in systolic anterior wall thickness, from baseline values, was more remarkable: It was increased by 6 ± 9% in the CD133+ group and decreased by 27 ± 6% in controls (p < .01; Fig. 6C; Table 1). We did not find a correlation between presence of cells in the heart 4 weeks after treatment and improvement in FS: 40 ± 19% in cell-positive hearts vs. 48 ± 12% in cell-negative hearts (p = .8).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. Acknowledgements
  10. References

The main new findings of the present study suggest that intravenous delivery of human UCB-derived CD133+ progenitor cells can prevent scar thinning, attenuate systolic dilatation, and improve LV function after MI. The transfused cells were able to migrate, colonize, and survive in the infarcted myocardium. However, the therapeutic effect was independent of transdifferentiation and direct myogenic or angiogenic contribution.

The Rationale for Using UCB CD133+ Progenitor Cells

Human UCB cells are rich in stem and progenitor cells with improved proliferative characteristics [7, 12, 13]. These cells can be easily obtained, can be expanded in vitro [16, 22, 23], have the potential for enhanced self-renewal and angiogenic and myogenic differentiation [7, 23, 24], and can be “banked” for future use. The collection of UCB from as many donors as possible would also increase the likelihood that people from many ethnic groups would be able to find a match. It appears that there is reduced risk of rejection by the recipient's immune system with UCB-derived stem cells [25]. Cord blood progenitor cells are routinely used in patients affected by major hematological disorders as an alternative to bone marrow transplantation for stem cell reconstitution [16, 26]. The use of cord blood may make stem cell transplants available more quickly for patients with severe cases of MI who need the cells as soon as possible.

Murohara et al. [7] have shown that a greater number of angioblast-like EPCs developed from cord blood MNCs than from the same amount of adult peripheral blood EPCs. Transplanted UCB EPCs augmented ischemia-induced neovascularization and regional blood flow in immunodeficient rats in vivo [7]. Pesce et al. [23] have shown that isolated human cord blood CD34+ cells injected into ischemic skeletal muscles give rise to endothelial and skeletal muscle cells in mice [23].

Most recently, three studies have reported the use of human UCB cells to treat MI in rats and mice. Henning et al. [27] injected 1 × 106 human UCB MNCs (subpopulation was not reported) immediately after coronary artery occlusion in Sprague-Dawley rats. Immunosuppression was not administered. Compared with the control, UCB cells reduced infarct size and LV dilatation and improved ejection fraction [27]. The mechanism by which human UCB MNCs reduced infarct size was not defined, and vessel density in the scar was not reported. In another study, Hirata et al. [28] injected 2 × 105 UCB CD34+ cells immediately after ligation of the coronary artery in Wistar rats. The rats were treated with immunosuppression FK 506. In agreement with our findings, 4 weeks after MI, FS was better in UCB-treated rats. Injected cells survived in the scar tissue and improved vessel density, and approximately 1% were incorporated into vessel walls. The authors did not report on immune rejection of the implanted cells. In a most recent report, Ma et al. [29] infused 6 × 106 UCB MNCs (only <1% CD34 cells) in the tail vein of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice 24 hours after MI. Human DNA was detected in marrow, spleen, and liver of both infarcted and noninfarcted mice up to 3 weeks after cell injection. In the heart, however, hDNA was detected in 10 of 19 MI mice but in none of the mice without MI. Infarct size was smaller in cell-treated mice than in untreated hearts. In cell-treated mice, capillary density in the infarct border zone was approximately 20% higher and clusters of UCB-derived cells were detected in the perivascular interstitium. The majority of neovessels appeared to consist of mouse cells. Notably, up to 70% of the cord blood-derived cells in the heart were CD45+. There was no evidence of cardiomyocyte transdifferentiation. Collectively, our findings add strength to the evidence and extend those findings by showing that i.v. delivery of UCB CD133+ cells, 1 week after MI, is feasible and effective.

Mechanism of Therapeutic Effect of UCB CD133+ Cells

Our findings suggest that the benefit is likely a result of factors secreted by the human-derived cells or another type of interaction with the healing infarct rather than a direct mechanical or angiogenic contribution. The idea that progenitor cells may protect the myocardium without directly participating in myocardial repair is supported by several recent works in cardiac [30, 31] and renal disease [32, 33] models.

The present study suggests at least one mechanism that could explain the functional recovery resulting from UCB cell delivery: It might work through prevention of scar thinning by autologous myofibroblast accumulation. By thickening the scar, wall stress is reduced (Laplace law) as is the degree of outward motion of the infarct that occurs during systole (paradoxical systolic bulging) [34, 35]. As a consequence, although the cells did not influence LV diastolic dilatation and mass, there was a lower end-systolic volume in the treated group [34]. This is a significant effect because one of the most important predictors of mortality in patients with MI is the degree of LV systolic dilatation [36].

CD45 and CD68 staining suggests that the implanted human cells differentiated toward a hematopoietic lineage. None of the nine hearts in the cell group showed the presence of myofibroblasts expressing HLA-DR. Thus, the myofibroblasts at the scar tissue did not originate from a human source.

The intention of the present study was not to confirm or refute the ability of the cells to transdifferentiate or fuse with cardiomyocytes or vascular cells. Although a few human cells were incorporated into vascular endothelium (suggesting direct contribution to neovascularization), we could not exclude the possibility of cell fusion. Recent reports support the notion that hematopoietic progenitors can differentiate into cardiomyocytes and endothelial cells [37, 38]. Furthermore, the therapeutic effect on the infarcted myocardium could be independent of direct contribution to cardiac parenchyma, either myocardial or vascular in nature [30]. Other reports have suggested that the therapeutic effects of cells on LV remodeling and function might be independent of implanted cell survival [30, 31], trans-differentiation [15, 30, 31], scar vessel density [31, 39], or infarct size [40].

We are aware of several limitations in our study. Based on our previous study [19], we delivered the cells intravenously 1 week after MI. Homing of the CD133+ progenitor cells into the infarcted heart is allied to the expression of the stem cell chemokine stromal-derived growth factor-1 (SDF-1) in the infarcted tissue and its CXCR4 receptor on human progenitor cells [40, 41]. However, SDF-1 gene expression is highest in the first days after infarction [40, 42]. Thus, it is possible that earlier cell delivery could achieve better migration and therapeutic effect. However, it has been suggested that increased levels of several genes in addition to SDF-1, including those for vascular endothelial growth factor (VEGF), intercellular adhesion molecule-1, vascular cell adhesion molecule-1, stem cell factor (SCF), and hepatocyte growth factor (HGF), might act in concert with SDF-1 to recruit progenitors to the injured heart [43, 44]. We chose to deliver cells 7 days after MI to avoid cell loss due to intense inflammation and washout at the infarct zone. The time of cell delivery, in the present study, might be more relevant to the clinical scenario of cell therapy for MI patients and allow baseline evaluation for myocardial damage and viability as well as donor and recipient HLA matching.

We and other groups described the body distribution of stem cells after systemic i.v. delivery [19, 29, 45, 46]. Heart failure after extensive MI produces congestion and subsequent injury in several organs, including spleen, lungs, liver, kidney, and gut. Tissue damage leads to a dramatic increase in the levels of secreted chemokines, cytokines, and proteolytic enzymes in many organs as part of the regeneration and repair process which have profound impacts on stem cell migration and repopulation [47]. It is possible that hypoxia induced factor-1 (HIF-1) activation and a shift in SDF-1 expression by endothelial cells in other organs during stress influence stem cell homing in different directions [47]. Taken together, the cells distribute and colonize widely to a variety of nonhematopoietic tissues following systemic infusion after MI and may possess the capacity to proliferate within these tissues [46, 47]. The significance of this phenomenon, which also exists in human patients [48], is uncertain and needs further research. Finally, the duration of the beneficial effect on heart is unknown and might be transient [49].

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. Acknowledgements
  10. References

Our study provides encouraging evidence that the use of UCB-derived progenitors can improve LV function in an MI model. The favorable functional effects are probably related to induction of self-repair by autologous myofibroblast accumulation that prevents scar thinning and dyskinesis. The use of UCB stem cells to repair the infarcted myocardium might be of importance to elderly people in whom the availability of autologous, functional stem cells is limited. Improving methods for stem cell expansion, storage, and induction of immune tolerance and enhancing homing and colonization would increase the prospect of applying UCB cells in the treatment of elderly and frail MI patients.

Table Table 1.. Results of two-dimensional echocardiography study
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Figure Figure 1.. Representative fluorescence-activated cell sorting (FACS) analysis after MiniMACS purification of umbilical cord blood (UCB) cells. The eluted cells were double-stained with nonspecific isotype controls (left) or with both phycoerythrin (PE)–anti-CD133 and fluorescein isothiocyanate (FITC)–anti-CD34 antibodies (right). The percentage of cells in each quadrant is indicated. Overall, the purity of CD133+ cells was more than 95%.

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Figure Figure 2.. Detection of human cells in rat hearts 1 month after cell infusion. (A): FISH of UCB CD133+ progenitor cells. The cells were derived from a pool of UCB samples from female and male newborns and were processed for two-color FISH and subsequent nuclear staining with 4,6-diamidino-2-phenylindole (DAPI). Hybridization to X and Y chromosome probes is detected by green and orange fluorescent signals, respectively. Original magnification: × 1000. (B): Cytospin slide of UCB cells processed for HLA-DR immunostaining. More than 90% of the human CD133+ cells are stained brown (positive). Original magnification: ×400. (C): FISH in myocardial frozen section indicating the presence of human cells. Cells were identified by positive FISH signals for the human X (arrows) chromosome. The rat cells do not express hybridization signals and are stained by the nonspecific DAPI nuclear stain only. Original magnification: ×1000. (D): FISH staining in heart frozen section. Inset shows phase-contrast image of myocardial tissue in the subendocardium area (original magnification: ×400). Higher magnification of FISH signals for X chromosome (arrows) identified two human cells near adjacent cardiomyocytes (C). Original magnification: ×1000. (E): Micrograph of human cell cluster detached from the host myocardial tissue into LV cavity. The engrafted human cells did not integrate with the host tissue. Positive cells exhibiting the same green signal pattern of XX. Original magnification: ×1000. (F): Micrograph of the infarcted heart from cell-treated animal. Sections stained with antibody to HLA-DR. Positive brown cells (arrows) are identified near vessel (v) walls at the border of infarct zone (S) and viable myocardium (H). Original magnification: ×400. Abbreviations: FISH, fluorescent in situ hybridization; UCB, umbilical cord blood; LV, left ventricular.

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Figure Figure 3.. Detection of human cells in rat hearts 1 month after cell infusion. (A, B): Sections stained with antibody to hematopoietic stem cell and endothelial progenitor cell marker CD34. (A): Micrograph shows positive CD34 brown staining confined to endothelial cells at vessel walls (thick arrows). A few positive cells were located between cardiomyocytes at the extracellular matrix (thin arrows; magnification: ×400). (B): Control heart section stained with antibody to CD34 exhibits negative staining. (C, D): Sections stained with antibody to monocyte/macrophage antigen CD68. (C): The shape, localization, morphology, and positive CD68 staining indicate that some cells differentiate into monocytes/macrophages (arrows). Original magnification: ×400. (D): Control sections stained with antibody to CD68 appear negative. Original magnification: ×400. (E, F): Sections stained with antibody to leukocyte common antigen-CD45. (E): Cluster of CD45 positive cells is observed in cell-treated heart (brown cells) and define hematopoietic cells. Arrows point to cell transplant near vessels. Original magnification: ×400. (F): Expression of CD45 is not observed in control heart sections. Original magnification: ×400.

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Figure Figure 4.. Low-power view (×100) of representative sections of scar tissue costained with antibody to α-SMA and HLA-DR. (A): Scar is thicker in cell-treated heart. Myofibroblasts, stained brown (positive) for α-SMA, significantly populated the scar tissue. Original magnification: ×100. (B): In contrast, myofibroblasts in the control heart are limited to subendocardium. Original magnification: ×100. Serial sections are used for colocalization of α-SMA and HLA-DR staining in the treated heart. (C): Sections stained with antibody to myofibroblast marker α-SMA appear brown and define myofibroblast accumulation in the scar. (D): Colocalization of HLA-DR in sequential section shows that the myofibroblasts are HLA-DR–negative and therefore are not from a human source. Original magnification: ×100. Abbreviations: SMA, smooth muscle actin; LV, left ventricular.

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Figure Figure 5.. Detection of human cells in other organs by HLA-DR staining. One month after cell infusion, immunostaining for HLA-DR identified human cells (brown staining) in the following: (A): Spleen. (B): Liver. (C): A few cells in the lungs (arrows). (D): Small intestine (arrow). (E): Rare positive cells in the bone marrow (arrow). Original magnification: ×400.

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Figure Figure 6.. Functional effects of CD133+ progenitor cells 1 month after cell or phosphate-buffered saline (PBS) infusion. By two-dimensional echocardiography, cell therapy improved significantly. (A): Left ventricular (LV) fractional shortening (FS) from baseline (before infusion) vs. 1 month after infusion. (B): LV diastolic anterior wall thickness from baseline (before infusion) vs. 1 month after infusion. (C): LV systolic anterior wall thickness from baseline (before infusion) vs. 1 month after infusion. In contrast, control hearts experienced significant deterioration in those functional parameters. Change (%) in baseline parameter was calculated as [(follow-up parameter-baseline parameter)/baseline parameter] × 100.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. Acknowledgements
  10. References

This work was partially supported by Shlezak Fund, Tel-Aviv University. The magnetic stem cell separation process was supported by a supply grant from Miltenyi Biotec. This sponsor had no role in study design, collection, analysis, interpretation of data, or on the decision to submit this paper for publication. We thank Patricia Benjamin for performing the echocardiograpy studies on rats.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosures
  9. Acknowledgements
  10. References
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