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(Circulation. 2002;106:1913.)
© 2002 American Heart Association, Inc.

Clinical Investigation and Reports

Repair of Infarcted Myocardium by Autologous Intracoronary Mononuclear Bone Marrow Cell Transplantation in Humans

Bodo E. Strauer, MD; Michael Brehm, MD; Tobias Zeus, MD; Matthias Köstering, MD; Anna Hernandez, PhD; Rüdiger V. Sorg, PhD; Gesine Kögler, PhD; Peter Wernet, MD

From the Department of Medicine, Division of Cardiology (B.E.S., M.B., T.Z., M.K.) and Institute for Transplantation Diagnostics and Cell Therapeutics (A.H., R.V.S., G.K., P.W.), Heinrich-Heine-University of Düsseldorf, Germany.

Correspondence to Professor Dr Bodo E. Strauer, Department of Medicine, Division of Cardiology, Heinrich-Heine-University, Moorenstr 5, 40225 Düsseldorf, Germany. E-mail Strauer{at}med.uni-duesseldorf.de

	Abstract

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Background— Experimental data suggest that bone marrow–derived cells may contribute to the healing of myocardial infarction (MI). For this reason, we analyzed 10 patients who were treated by intracoronary transplantation of autologous, mononuclear bone marrow cells (BMCs) in addition to standard therapy after MI.

Methods and Results— After standard therapy for acute MI, 10 patients were transplanted with autologous mononuclear BMCs via a balloon catheter placed into the infarct-related artery during balloon dilatation (percutaneous transluminal coronary angioplasty). Another 10 patients with acute MI were treated by standard therapy alone. After 3 months of follow-up, the infarct region (determined by left ventriculography) had decreased significantly within the cell therapy group (from 30±13 to 12±7%, P=0.005) and was also significantly smaller compared with the standard therapy group (P=0.04). Likewise, infarction wall movement velocity increased significantly only in the cell therapy group (from 2.0±1.1 to 4.0±2.6 cm/s, P=0.028). Further cardiac examinations (dobutamine stress echocardiography, radionuclide ventriculography, and catheterization of the right heart) were performed for the cell therapy group and showed significant improvement in stroke volume index, left ventricular end-systolic volume and contractility (ratio of systolic pressure and end-systolic volume), and myocardial perfusion of the infarct region.

Conclusions— These results demonstrate for the first time that selective intracoronary transplantation of autologous, mononuclear BMCs is safe and seems to be effective under clinical conditions. The marked therapeutic effect may be attributed to BMC-associated myocardial regeneration and neovascularization.

Key Words: myocardial infarction • cell transplantation, intracoronary • angiogenesis • bone marrow • myogenesis

	Introduction

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Remodeling of the left ventricle after myocardial infarction (MI) represents a major cause of infarct-related heart failure and death. This process depends on acute and chronic transformation of both the necrotic infarct region and the non-necrotic, peri-infarct tissue.^1,2 Despite application of pharmacotherapeutics and mechanical interventions, the cardiomyocytes lost during MI cannot be regenerated. The recent finding that a small population of cardiac muscle cells is able to replicate itself is encouraging but is still consistent with the concept that such regeneration is restricted to viable myocardium.³

In animal experiments, attempts to replace the necrotic zone by transplanting other cells (eg, fetal cardiomyocytes or skeletal myoblasts) have invariably succeeded in reconstituting heart muscle structures, ie, myocardium and coronary vessels. However, these cells fail to integrate structurally and do not display characteristic physiological functions.^4–7 Another approach to reverse myocardial remodeling is to repair myocardial tissue by using bone marrow–derived cells. Bone marrow contains multipotent adult stem cells that show a high capacity for differentiation.^8–10 Experimental studies have shown that bone marrow cells (BMCs) are capable of regenerating infarcted myocardium and inducing myogenesis and angiogenesis; this leads in turn to amelioration of cardiac function in mice and pigs.^11–14 However, procedures based on this phenomenon remain largely uninvestigated in a human clinical setting.

An investigation of one patient receiving autologous skeletal myoblasts into a postinfarction scar during coronary artery bypass grafting revealed improvement of contraction and viability 5 months afterward.¹⁵ Autologous mononuclear BMCs transplanted in a similar surgical setting showed long-term improvement of myocardial perfusion in 3 of 5 patients and no change in 2 patients.¹⁶ However, such studies entail a surgical approach and are therefore associated with well-known perioperative risks. Moreover, this surgical procedure cannot be used with MI. We therefore looked for a nonsurgical, safer mode for transplanting autologous cells into postinfarction tissue. A pilot study from our group demonstrated that intracoronary transplantation of autologous mononuclear BMCs 6 days after MI was associated with a marked decrease in infarct area and an increase in left ventricular (LV) function after 3 and 6 months of follow-up.¹⁷ To confirm these results and validate this promising new therapy for MI, we established a clinical trial involving 20 patients for comparing the safety and bioefficacy of autologous BMC transplantation. All 20 patients underwent standard therapy, and 10 patients received additional intracoronary cell transplantation. All 20 patients were followed up for 3 months.

	Methods

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Patient Population
All 20 patients had suffered transmural infarction according to World Health Organization criteria with the involvement of the left anterior descending coronary artery (n=4), left circumflex coronary artery (n=3), or right coronary artery (n=13). Mean duration of infarct pain was 12±10 hours before invasive diagnostics and therapy. Patients had to be <70 years old and were excluded if one of the following criteria were met: screening >72 hours after infarction, cardiac shock, severe comorbidity, alcohol or drug dependency, or excessive travel distance to the study center.

After right and left heart catheterization, coronary angiography, and left ventriculography, mechanical treatment was initiated with recanalization of the infarct-related artery by balloon angioplasty (n=20) and subsequent stent implantation (n=19). All patients were monitored in our intensive care unit, and no arrhythmogenic events or hemodynamic impairments were recorded in either patient group.

All 20 patients were briefed in detail about the procedure of BMC transplantation. Informed consent was obtained from 10 patients, who formed the cell therapy group, whereas 10 patients who refused additional cell therapy served as controls. The local ethics committee of the Heinrich-Heine-University, Düsseldorf, approved the study protocol. All procedures conformed to institutional guidelines.

Before taking part in rehabilitation programs, all patients left the hospital with standard medication consisting of acetylsalicylic acid, an ACE inhibitor, a ß-blocker, and a statin.

Bone Marrow Aspiration, Isolation, and Cultivation
Seven (±2) days after acute coronary angiography, bone marrow (40 mL) was aspirated under local anesthesia from ilium of cell therapy patients (n=10). Mononuclear BMCs were isolated by Ficoll density separation on Lymphocyte Separation Medium (BioWhittaker) before the erythrocytes were lysed with H₂O. For overnight cultivation, 1x10⁶ BMCs/mL were placed in Teflon bags (Vuelife, Cell Genix) and cultivated in X-Vivo 15 Medium (BioWhittaker) supplemented with 2% heat-inactivated autologous plasma. The next day, BMCs were harvested and washed 3 times with heparinized saline before final resuspension in heparinized saline. Viability was 93±3%. Heparinization and filtration (cell strainer, FALCON) was carried out to prevent cell clotting and microembolization during intracoronary transplantation. The mean number of mononuclear cells harvested after overnight culture was 2.8x10⁷; this consisted of 0.65±0.4% AC133-positive cells and 2.1±0.28% CD34-positive cells. All microbiological tests of the clinically used cell preparations proved negative. As a viability and quality ex vivo control, 1x10⁵ cells grown in H5100 medium (Stem Cell Technology) were found to be able to generate mesenchymal cells in culture.

Intracoronary Transplantation of BMCs
Five to nine days after onset of acute infarction, cells were directly transplanted into the infarcted zone (Figure 1). This was accomplished with the use of a balloon catheter, which was placed within the infarct-related artery. After exact positioning of the balloon at the site of the former infarct-vessel occlusion, percutaneous transluminal coronary angioplasty (PTCA) was performed 6 to 7 times for 2 to 4 minutes each. During this time, intracoronary cell transplantation via the balloon catheter was performed, using 6 to 7 fractional high-pressure infusions of 2 to 3 mL cell suspension, each of which contained 1.5 to 4x10⁶ mononuclear cells. PTCA thoroughly prevented the backflow of cells and at the same time produced a stop-flow beyond the site of the balloon inflation to facilitate high-pressure infusion of cells into the infarcted zone. Thus, prolonged contact time for cellular migration was allowed.¹⁸

View larger version (83K): [in this window] [in a new window]   Figure 1. Procedure of cell transplantation into infarcted myocardium in humans. a, The balloon catheter enters the infarct-related artery and is placed above the border zone of the infarction. It is then inflated and the cell suspension is infused at high pressure under stop-flow conditions. b, In this way, cells are transplanted into the infarcted zone via the infarct-related vasculature (red dots). Cells infiltrate the infarcted zone. Blue and white arrows suggest the possible route of migration. c, A supply of blood flow exists within the infarcted zone.35 The cells are therefore able to reach both the border and the infarcted zone.

Functional Assessment of Hemodynamics
After 3 months, all 20 patients were followed up by left heart catheterization, left ventriculography, and coronary angiography. Ejection fraction, infarct region, and regional wall movement of the infarcted zone during ejection were determined by left ventriculography. Ejection fraction was measured with Quantcor software (Siemens). To quantify infarction wall movement velocity, 5 axes were placed perpendicular to the long axis in the main akinetic or dyskinetic segment of the ventricular wall. Relative systolic and diastolic lengths were measured, and the mean difference was divided by the systolic duration (in seconds). To quantify the infarct region, the centerline method according to Sheehan was used.¹⁹ All hemodynamic investigations were obtained by two independent observers.

In the cell therapy group before and 3 months after cell transplantation, additional examinations for measuring hemodynamics and myocardial perfusion included dobutamine stress echocardiography, radionuclide ventriculography, catheterization of the right heart, and stress-redistribution-reinjection ²⁰¹thallium scintigraphy. The contractility index P_syst/ESV was calculated by dividing LV systolic pressure (P_syst) by end-systolic volume (ESV). Perfusion defect was calculated by scintigraphic bull’s-eye technique. Each examination was performed according to standard protocols.

There were no complications or side effects determined in any patient throughout the diagnostic or therapeutic procedure or within the 3-month follow-up period.

Statistical Analysis
All data are presented as mean±SD. Statistical significance was accepted when P was <0.05. Discrete variables were compared as rates, and comparisons were made by ² analysis. Intra-individual comparison of baseline versus follow-up continuous variables was performed with a paired t test. Comparison of nonparametric data between the two groups was performed with Wilcoxon test and Mann-Whitney test. Statistical analysis was performed with SPSS for Windows (version 10.1).

	Results

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Clinical data between the two groups did not differ significantly. The range of creatinine kinase levels was slightly but not significantly higher in the standard therapy group than it was in the cell therapy group (Table 1).

View this table: [in this window] [in a new window]   TABLE 1. Baseline Characteristics of the Patients

Comparison of the 2 groups 3 months after cell or standard therapy showed several significant differences in LV dynamics, according to the global and regional analysis of left ventriculogram. The infarct region as a percentage of hypokinetic, akinetic, or dyskinetic segments of the circumference of the left ventricle decreased significantly in the cell therapy group (from 30±13 to 12±7%, P= 0.005). It was also significantly smaller compared with the standard therapy group after 3 months (P=0.04). Within the standard therapy group, only a statistically nonsignificant decrease from 25±8 to 20±11% could be seen. Wall movement velocity over the infarct region rose significantly in the cell therapy group (from 2.0±1.1 to 4.0±2.6 cm/s, P=0.028) but not in the standard therapy group (from 1.8±1.3 to 2.3±1.6 cm/s, P=NS). No significant difference was observed between the two groups. Ejection fraction increased in both groups, albeit nonsignificantly (from 57±8 to 62± 10% in the cell therapy group and from 60±7 to 64±7% in the standard therapy group) (Table 2).

View this table: [in this window] [in a new window]   TABLE 2. Comparison of Cell Therapy and Standard Therapy Groups

Further significant improvement could also be seen on additional analysis of the cell therapy group alone. Perfusion defect was considerably decreased by 26% in the cell therapy group (from 174±99 to 128±71 cm², P=0.016, assessed by ²⁰¹thallium scintigraphy) (Figure 2). Parallel to the reduction in perfusion defect, improvement (Table 3) could also be seen in:

View larger version (132K): [in this window] [in a new window]   Figure 2. Improved myocardial perfusion of infarcted anterior wall 3 months after intracoronary cell transplantation subsequent to an acute anterior wall infarction detected by 201thallium scintigraphy. The images on the left (A, D, sagittal) and in the middle (B, E) show the long axis, whereas those on the right (C, F, frontal) show the short axis of the heart. Initially the anterior wall, with green-colored apical and anterior regions, had reduced myocardial perfusion (A, B, C). Three months after cell transplantation the same anterior wall, now yellow in color, revealed a significant improvement in myocardial perfusion (D, E, F). All illustrations depict the exercise phase.

View this table: [in this window] [in a new window]   TABLE 3. Cardiac Function Analysis at 3-Month Follow-Up

1. Cardiac function, as revealed by increase in stroke volume index (from 49±7 to 56±7 mL/m², P=0.010) and ejection fraction (from 51±14 to 53±13%, P=NS).
   
2. Cardiac geometry, as shown by decreases in both end-diastolic (from 158±20 to 143±30 mL, P=NS) and end-systolic volume (from 82±26 to 67±21 mL, P=0.011). Radionuclide ventriculography was used to acquire the data.
   
3. Contractility as evaluated by an increase in the velocity of circumferential fiber shortening (from 20.5±4.2 to 24.4±7.7 mm/s, P=NS, assessed by stress echocardiography) and by a marked increase in the ratio of systolic pressure to end-systolic volume (from 1.81±1.44 to 2.27±1.72 mm Hg/mL, P=0.005).
   

	Discussion

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The present report describes the first clinical trial of intracoronary, autologous, mononuclear BMC transplantation for improving heart function and myocardial perfusion in patients after acute MI. The results demonstrate that transplanted autologous BMCs may lead to repair of infarcted tissue when applied during the immediate postinfarction period. These results also show that the intracoronary approach of BMC transplantation seems to represent a novel and effective therapeutic procedure for concentrating and/or depositing infused cells within the region of interest.

Neogenesis of both cardiomyocytes and coronary capillaries with some functional improvement has been shown recently by several investigators using bone marrow–derived cells in experimental infarction.^{11–14,18,20–23} Moreover, transendothelial migration from the coronary capillaries and incorporation of cells into heart muscle has been observed experimentally.^3,12,24–26 Until now, clinical data only existed for the cell therapy of surgically treated chronic ischemic heart disease.^15,16 Our aim was to transform the encouraging results from animal models to a safe clinical setting. The most crucial questions we had to address while designing and realizing this trial were: (1) What cell population should we deliver? (2) Which application method is the most efficient? (3) When should the cells be transplanted?

In recent years, several laboratories have shown that environmentally dictated changes of fate (transdetermination) are not restricted to stem cells but may also involve progenitor cells at different steps of a given differentiation pathway (transdifferentiation). Moreover, mesenchymal stem cells may represent an ideal cell source for treating different diseases.²⁷ Adult, mononuclear BMCs contain such stem and progenitor cells (1%), eg, mesodermal progenitor cells, hematopoietic progenitor cells, and endothelial progenitor cells. In several animal infarction models it has been shown that: (1) Bone marrow hemangioblasts contribute to the formation of new vessels; (2) bone marrow hematopoietic stem cells differentiate into cardiomyocytes, endothelium, and smooth muscle cells^8–13; (3) BMCs give rise to mesodermal progenitor cells that differentiate to endothelial cells²⁸; and (4) endothelial progenitors can transdifferentiate into beating cardiomyocytes.²⁹ Thus, several different fractions of mononuclear BMCs may contribute to the regeneration of necrotic myocardium and vessels. In order to utilize this large and perhaps heterogeneous regenerative potential, we decided to use all mononuclear cells from the bone marrow aspirate as a whole, rather than a subpopulation. No further expansion was performed because experimental data have revealed a dramatic decline in the homing capacity of in vitro amplified hematopoietic stem or progenitor cells.³⁰

The second question was how to deliver the cells most efficiently. When given intravenously, only a very small fraction of infused cells can reach the infarct region after the following injection: assuming a normal coronary blood flow of 80 mL/min per 100 g of LV weight, a quantity of 160 mL per left ventricle (assuming a regular LV mass of 200 g) will flow per minute.^31,32 This corresponds to only about 3% of cardiac output (assuming a cardiac output of 5000 mL/min).³¹ Therefore, intravenous application would require many circulation passages to enable infused cells to come into contact with the infarct-related artery. Throughout this long circulation and recirculation time, homing of cells to other organs could considerably reduce the numbers of cells dedicated to cell repair in the infarcted zone. Thus, supplying the entire complement of cells by intracoronary administration obviously seems to be advantageous for the tissue repair of infarcted heart muscle and may also be superior to intraventricular injection,³³ because all cells are able to flow through the infarcted and peri-infarcted tissue during the immediate first passage. Accordingly, by this intracoronary procedure the infarct tissue and the peri-infarct zone can be enriched with the maximum available amount of cells at all times.

As stem cells differentiate into more mature types of progenitor cells, it is thought that a special microenvironment in so-called niches regulates cell activity by providing specific combinations of cytokines and by establishing direct cellular contact. For successful long-term engraftment, at least some stem cells have to reach their niches, a process referred to as homing. Mouse experiments have shown that significant numbers of BMCs appear in liver, spleen, and bone marrow after intravenous injection.³⁴ To offer the BMCs the best chance of finding their niche within the myocardium, a selective intracoronary delivery route was chosen. Presumably, therefore, fewer cells were lost by extraction toward organs of secondary interest by this first pass–like effect. To facilitate transendothelial passage and migration into the infarcted zone, cells were infused by high-pressure injection directly into the necrotic area, and the balloon was kept inflated for 2 to 3 minutes; the cells were not washed away immediately under these conditions.

The time point for delivery was chosen as 7 to 8 days after infarction onset for the following reasons:

1. In dogs, infarcted territory becomes rich in capillaries and contains enlarged, pericyte-poor "mother vessels" and endothelial bridges 7 days after myocardial ischemia and reperfusion. Twenty-eight days later, a significant muscular vessel wall has already formed.³⁵ Thus, with such timing, cells may be able to reach the worst damaged parts and at the same time salvage tissue. Transendothelial cell migration may also be enhanced because an adequate muscular coat is not yet formed.
   
2. Until now, only one animal study has attempted to determine the optimum time for cardiomyocyte transplantation to maximize myocardial function after LV injury. Adult rat hearts were cryoinjured and fetal rat cardiomyocytes were transplanted immediately, 2 weeks later, and 4 weeks later. The authors discussed the inflammatory process, which is strongest in the first days after infarction, as being responsible for the negative results after immediate cell transplantation, and they assumed that the best results seen after 2 weeks may have been due to transplantation before scar expansion.³⁶ Until now, however, no systematic experiments have been performed with BMCs to correlate the results of transplantation with the length of such a time delay.
   
3. Another important variable is the inflammatory response in MI, which seems to be a superbly orchestrated interaction of cells, cytokines, growth factors and extracellular matrix proteins mediating myocardial repair. In the first 48 hours, debridement and formation of a fibrin-based provisional matrix predominates before a healing phase ensues.^37–40 Moreover, vascular endothelial growth factor is at its peak concentration 7 days after MI, and the decline of adhesion molecules (intercellular adhesion molecules, vascular cell adhesion molecules) does not take place before days 3 to 4 after MI. We assumed that transplantation of mononuclear BMCs within the "hot" phase of post-MI inflammation might lead them to take part in the inflammation cascade rather than the formation of functional myocardium and vessels.
   

Taking all of this into account, we can conclude that cell transplantation within the first 5 days after acute infarction is not possible for logistical reasons and is not advisable because of the inflammatory process. On the other hand, transplantation 2 weeks after infarction scar formation seems to reduce the benefit of cell transplantation. Although the ideal time point for transplantation remains to be defined, it is most likely between days 7 and 14 after the onset of MI, as in the present study.

This trial was designed as a phase I safety and feasibility trial, meaning that no control group is necessarily required. However, to validate the results, we correlated them with those obtained from 10 patients who refused to get additional cell therapy and thus received standard therapy alone. We are aware of the fact that such a comparison does not reach the power of a randomly allocated, blinded control group. However, the significant improvement with regard to infarct region, hemodynamics (stroke volume index), cardiac geometry (LV end-systolic volume), and contractility (P_syst/ESV and infarction wall movement velocity) did confirm a positive effect of the additional cell therapy because the changes observed in the standard therapy group failed to reach significance.

Another important factor for interpreting the results is time interval between onset of symptoms and revascularization of the infarct-related artery by angioplasty; this represents a crucial determinant of LV recovery. For patients with acute MI, it has been shown that if the time interval is >4 hours, no significant changes in ejection fraction, regional wall motion, or ESV are observed after 6-month follow-up by echocardiography and angiography.⁴¹ None of our 20 patients was treated by angioplasty within 4 hours after onset of symptoms. Our average time interval was 12±10 hours. Thus, PTCA-induced improvement of LV function can be nearly excluded; indeed, the only mild and nonsignificant changes within the standard therapy group are consistent with the above-mentioned data.⁴¹ In contrast, the cell therapy group showed considerable and significant improvement in the same parameters, which may be attributed to BMC-mediated coronary angioneogenesis and cardiomyoneogenesis.

These results show that transplantation of autologous BMCs, as well as the intracoronary approach, represent a novel and effective therapeutic procedure for the repair of infarcted myocardium. For this method of therapy, no ethical problems exist, and no side effects were observed at any point of time. The therapeutic benefit for the patient’s heart seems to prevail. However, further experimental studies, controlled prospective clinical trials, and variations of cell preparations are required to define the role of this new approach for the therapy of acute MI in humans.

Received August 2, 2002; accepted August 2, 2002.

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