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Intracoronary Transplantation of Progenitor Cells after Acute MI

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

Transcoronary Transplantation of Progenitor Cells after Myocardial Infarction

Birgit Assmus, M.D., Jörg Honold, M.D., Volker Schächinger, M.D., Martina B. Britten, M.D., Ulrich Fischer-Rasokat, M.D., et al.
From the Division of Cardiology and Mo­lecular Cardiology, Department of Medi­cine III (B.A., J.H., V.S., M.B.B., U.F.-R., R.L., C.T., K.P., S.D., A.M.Z.), Division of He­matology, Department of Medicine II (H.M.), and the Department of Diagnos­tic and Interventional Radiology (N.D.A.), Johann Wolfgang Goethe University; and the Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service, Baden–Württem-berg–Hessen (T.T.) — both in Frankfurt, Germany.

N Engl J Med 2006;355:1222-32.

Background

Pilot studies suggest that intracoronary transplantation of progenitor cells derived from bone marrow (BMC) or circulating blood (CPC) may improve left ventricular function after acute myocardial infarction. The effects of cell transplantation in patients with healed myocardial infarction are unknown.

METHODS

After an initial pilot trial involving 17 patients, we randomly assigned, in a controlled crossover study, 75 patients with stable ischemic heart disease who had had a myo­cardial infarction at least 3 months previously to receive either no cell infusion (23 patients) or infusion of CPC (24 patients) or BMC (28 patients) into the patent coro­nary artery supplying the most dyskinetic left ventricular area. The patients in the control group were

  • subsequently randomly assigned to receive CPC or BMC, and
  • the patients who initially received BMC or CPC crossed over to receive CPC or BMC, respectively, at 3 months’ follow-up.

RESULTS

The absolute change in left ventricular ejection fraction was significantly greater among patients receiving BMC (+2.9 percentage points) than among those receiving CPC (−0.4 percentage point, P = 0.003) or no infusion (−1.2 percentage points, P<0.001). The increase in global cardiac function was related to significantly

  • en­hanced regional contractility in the area targeted by intracoronary infusion of BMC.

The crossover phase of the study revealed that intracoronary infusion of BMC was associated with a significant increase in global and regional left ventricular func­tion, regardless of whether patients crossed over from control to BMC or from CPC to BMC.

CONCLUSIONS

Intracoronary infusion of progenitor cells is safe and feasible in patients with healed myocardial infarction. Transplantation of BMC is associated with moderate but significant improvement in the left ventricular ejection fraction after 3 months. (ClinicalTrials.gov number, NCT00289822.)

Introduction

HRONIC HEART FAILURE IS COMMON, and its prevalence continues to increase.1 Ischemic heart disease is the principal cause of heart failure.2 Although myocardial salvage due to early reperfusion therapy has significantly re­duced early mortality rates,3

  • postinfarction heart failure resulting from ventricular remodeling re­mains a problem.4

One possible approach to re­versing postinfarction heart failure is

  • enhance­ment of the regeneration of cardiac myocytes as well as
  • stimulation of neovascularization within the infarcted area.

Initial clinical pilot studies have suggested that

  • intracoronary infusion of pro­genitor cells is feasible and may
  • beneficially af­fect postinfarction remodeling processes in pa­tients with acute myocardial infarction.5-9

However, it is currently unknown whether such a treatment strategy may also be associated with

  • improvements in cardiac function in patients with persistent left ventricular dysfunction due to healed myocardial infarction with established scar formation.

Therefore, in the prospective TOPCARE-CHD (Transplantation of Progenitor Cells and Recovery of LV [Left Ventricular] Function in Patients with Chronic Ischemic Heart Disease) trial, we inves­tigated

  • whether intracoronary infusion of pro­genitor cells into the infarct-related artery at least 3 months after myocardial infarction improves global and regional left ventricular function.

Patient Outcome Criteria

The primary end point of the study was the absolute change in global left ventricular ejection fraction (LVEF) as measured by quantitative left ventricular angiography 3 months after cell infu­sion. Secondary end points included quantitative variables relating to the regional left ventricular function of the target area, as well as left ven­tricular volumes derived from serial left ventric­ular angiograms. In addition, functional status was assessed by NYHA classification. Finally, event-free survival was defined as freedom from death, myocardial infarction, stroke, or rehospi­talization for worsening heart failure. Causes of rehospitalization during follow-up were verified by review of the discharge letters or charts of hospital stays.

DETECTION OF VIABLE MYOCARDIUM

All patients underwent low-dose dobutamine stress echocardiography, combined thallium sin­gle-photon-emission computed tomography and [18F]fluorodeoxyglucose positron-emission tomog­raphy, or both, as previously described.6 It was pos­sible to analyze regional left ventricular viability in 80 patients (87%).

RESULTS

BASELINE CHARACTERISTICS OF THE PATIENTS

A total of 92 patients were enrolled in the study. Of these, 35 patients received BMC as their ini­tial treatment (in phases 1 and 2 of the trial), 34 patients received CPC (in phases 1 and 2), and 23 patients received no intracoronary cell infu­sion (in phase 2, as the control group). Table 1 illustrates that the three groups of patients were well matched.

EFFECTS OF PROGENITOR-CELL INFUSION

Quantitative Characteristics of Left Ventricular Function

Patients with an adverse clinical event (six), sub­total stenosis of the target vessel at follow-up (three), an intraventricular thrombus precluding performance of left ventricular angiography (one), or atrial flutter or fibrillation at follow-up (one) were excluded from the exploratory analysis. In addition, of the 81 eligible patients, left ventricu­lar angiograms could not be quantitatively ana­lyzed in 4 because of inadequate contrast opaci-fication, in 1 because of ventricular extrasystoles, and in 4 because of the patients’ refusal to un­dergo invasive follow-up. Thus, a total of 72 of 81 serial paired left ventricular angiograms were available for quantitative analysis (28 in the BMC group, 26 in the CPC group, and 18 in the control group).

Table 2 summarizes the angiographic charac­teristics of the 75 patients included in the ran­domized phase of the study. At baseline, the three groups did not differ with respect to global LVEF, the extent or magnitude of regional left ventricu­lar dysfunction, left ventricular volumes, or stroke volumes.

The absolute change in global LVEF from base­line to 3 months did significantly differ among the three groups of patients. Patients receiving BMC had a significantly larger change in LVEF than patients receiving CPC (P = 0.003) and those in the control group (P<0.001). Similar results were ob­tained when patients from the first two phases of the study (the pilot phase and the randomized phase) were pooled. The results did not differ when patients without evidence of viable myo­cardium before inclusion were analyzed sepa­rately. The change in LVEF was −0.3±3.4 percent­age points in the control group (9 patients), +0.4±3.0 percentage points in the CPC group (18 patients), and +3.7±4.0 percentage points in the BMC group (18 patients) (P = 0.02 for the com­parison with the control group and P = 0.02 for the comparison with the CPC group).

In the subgroup of 35 patients who underwent serial assessment of left ventricular function by MRI, MRI-derived global LVEF increased signifi­cantly, by 4.8±6.0% (P = 0.03) among those receiv­ing BMC (11 patients) and by 2.8±5.2% (P = 0.02) among those receiving CPC (20 patients), where­as no change was observed in 4 control patients (P = 0.14). Thus, MRI-derived assessment of left ventricular function further corroborated the re­sults obtained from the total patient population.

Analysis of regional left ventricular function revealed that BMC treatment significantly in­creased contractility in the center of the left ven­tricular target area (Table 2). Likewise, MRI-derived regional analysis of left ventricular function re­vealed that the number of hypocontractile seg­ments was significantly reduced, from 10.1±3.6 to 8.7±3.6 segments (P = 0.02), and the number of normocontractile segments significantly in­creased, from 3.8±4.5 to 5.4±4.6 segments (P = 0.01), in the BMC group, whereas no significant changes were observed in the CPC group. MRI-derived infarct size, as measured by late enhance­ment volume normalized to left ventricular mass, remained constant both in the CPC group (25± 18% at baseline and 23±14% at 3 months,13 patients) and in the BMC group (20±10% at both time points, 9 patients). Thus, taken together, the data suggest that intracoronary infusion of BMC is associated with significant improvements in global and regional left ventricular contractile function among patients with persistent left ven­tricular dysfunction due to prior myocardial in­farction.

To identify independent predictors of improved global LVEF, a stepwise multivariate regression analysis was performed; it included classic deter­minants of LVEF as well as various baseline characteristics of the three groups (Table 3). The multivariate analysis identified the type of pro­genitor cell infused and the baseline stroke vol­ume as the only statistically significant indepen­dent predictors of LVEF recovery.

Functional Status

The functional status of the patients, as assessed by NYHA classification, improved significantly in the BMC group (from 2.23±0.6 to 1.97±0.7, P = 0.005). It did not improve significantly either in the CPC group (class, 2.16±0.8 at baseline and 1.93±0.8 at 3 months; P = 0.13) or in the control group (class, 1.91±0.7 and 2.09±0.9, respectively; P = 0.27).

RANDOMIZED CROSSOVER PHASE

Of the 24 patients who initially were randomly assigned to CPC infusion, 21 received BMC at the time of their first follow-up examination. Likewise, of the 28 patients who initially were randomly assigned to BMC infusion,

  • 24 received CPC after 3 months.

Of the 23 patients of the control group, 10 patients received CPC and 11 received BMC at their reexamination at 3 months (Fig. 1). As illustrated in Figure 2, regardless of whether patients received BMC as initial treatment, as crossover treatment after CPC infusion, or as crossover treatment after no cell infusion,

  • glob­al LVEF increased significantly after infusion of BMC. In contrast,
  • CPC treatment did not significantly alter LVEF when given either before or after BMC.

Thus, the intrapatient comparison of the dif­ferent treatment strategies not only documents the superiority of intracoronary infusion of BMC over the infusion of CPC for improving global left ventricular function, but also corroborates our findings in the analysis of data according to initial treatment assignment. The

  • preserved im­provement in cardiac function observed among patients who initially received BMC treatment and
  • then crossed over to CPC treatment demon­strates that the initially achieved differences in cardiac function persisted for at least 6 months after intracoronary infusion of BMC.
 Table 1. Baseline Characteristics of the Patients.* (not copied)  

Table 2. Quantitative Variables Pertaining to Left Ventricular Function, as Assessed by Left Ventricular Angiography.*

copy protected

Figure 2. Absolute Change in Quantitative Global Left Ventricular Ejection Fraction (LVEF) during the Crossover Phase of the Trial.

Data at 3 and 6 months are shown for all patients crossing over from BMC to CPC infusion (18 patients), from CPC to BMC infusion
(18 patients), and from no cell infusion to either CPC infusion (10 patients) or BMC infusion (11 patients). I bars represent standard
errors.

Table 3. Stepwise Linear Regression Analysis for Predictors of Improvement in Global Left Ventricular Ejection Fraction.*

Variable Nonstandardized Coefficient B

95% CI for B

P Value

Treatment group

1.49

0.53 to 2.46

0.003
Baseline stroke volume

−0.13

−0.22 to –0.05

0.002
No. of cardiovascular risk factors 0.76
Time since most recent MI 0.48
Concomitant PCI 0.60
Age 0.82
Baseline ejection fraction 0.72
Baseline end-diastolic volume 0.88

* Values are shown only for significant differences. MI denotes myocardial infarc­tion, and PCI percutaneous coronary intervention. For the overall model, the ad­justed R2 was 0.29; P<0.001 by analysis of variance.

 

DISCUSSION

Intrapatient comparison in the crossover phase of the trial rules out the possibility that differences in the patient populations studied may have affected outcomes. However, the mechanisms involved in mediating improved contractile function after intracoronary progenitor-cell infusion are not well understood.

Experimentally, although there is no definitive proof that cardiac myocytes may be regenerated, BMC were shown to contribute to functional re­covery of left ventricular contraction when in­jected into freshly infarcted hearts,13-15 whereas CPC profoundly stimulated ischemia-induced neovascularization.16,17 Both cell types were shown to prevent cardiomyocyte apoptosis and reduce the development of myocardial fibrosis and there­by improve cardiac function after acute myocar­dial infarction.18,19 Indeed, in our TOPCARE-AMI (Transplantation of Progenitor Cells and Regen­eration Enhancement in Acute Myocardial Infarc­tion) studies,6,7,9 intracoronary infusion of CPC was associated with functional improvements similar to those found with the use of BMC im­mediately after myocardial infarction. In the cur­rent study, however, which involved patients who had had a myocardial infarction at least 3 months before therapy,

  • transcoronary adminis­tration of CPC was significantly inferior to administration of BMC in altering global left ven­tricular function.

CPC obtained from patients with chronic ischemic heart disease show pro­found functional impairments,20,21 which might limit their recruitment, after intracoronary infu­sion, into chronically reperfused scar tissue many months or years after myocardial infarction. Thus, additional studies in which larger numbers of functionally enhanced CPC are used will be re­quired to increase the response to intracoronary infusion of CPC.

The magnitude of the improvement after in-tracoronary infusion of BMC, with absolute increases in global LVEF of approximately 2.9 percentage points according to left ventricular angiography and 4.8 percentage points accord­ing to MRI, was modest. However, it should be noted that the improvement in LVEF occurred in the setting of full conventional pharmacologic treatment: more than 90% of the patients were receiving beta-blocker and angiotensin-convert-ing–enzyme inhibitor treatment. Moreover, results from trials of contemporary reperfusion for the treatment of acute myocardial infarction, which is regarded as the most effective treatment strat­egy for improving left ventricular contractile per­formance after ischemic injury, have reported in­creases in global LVEF of 2.8% (in the CADILLAC [Controlled Abciximab and Device Investigation to Lower Late Angioplasty Complications] trial) and 4.1% (in the ADMIRAL [Abciximab before Direct Angioplasty and Stenting in Myocardial Infarction Regarding Acute and Long-Term Fol­low-up] trial).22,23

The number of patients, as well as the dura­tion of follow-up, is not sufficient to address the question of whether the moderate improvement in LVEF associated with one-time intracoronary BMC infusion is associated with reduced mortal­ity and morbidity among patients with heart fail­ure secondary to previous myocardial infarction. We conclude that intracoronary infusion of BMC is associated with persistent improvements in regional and global left ventricular function and improved functional status among patients who have had a myocardial infarction at least 3 months previously. Given the reasonable short-term safety profile of this therapeutic ap­proach, studies on a larger scale are warranted to examine its potential effects on morbidity and mortality among patients with postinfarction heart failure.

REFERENCES (1-8/23)

  1. 2001 Heart and stroke statistical up­date. Dallas: American Heart Association, 2000.
  2. Braunwald E. Cardiovascular medicine at the turn of the millennium: triumphs, concerns, and opportunities. N Engl J Med 1997;337:1360-9.
  3. Lange RA, Hillis LD. Reperfusion ther­apy in acute myocardial infarction. N Engl J Med 2002;346:954-5.
  4. Sutton MG, Sharpe N. Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation 2000;101:2981-8.
  5. Strauer BE, Brehm M, Zeus T, et al. Re­pair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circula­tion 2002;106:1913-8.
  6. Assmus B, Schachinger V, Teupe C, et al. Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myo­cardial Infarction (TOPCARE-AMI). Circu­lation 2002;106:3009-17.
  7. Britten MB, Abolmaali ND, Assmus B, et al. Infarct remodeling after intra-coronary progenitor cell treatment in pa­tients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation 2003;108: 2212-8.
  8. Wollert KC, Meyer GP, Lotz J, et al. In-tracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 2004;364:141-8.

 

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Progenitor Cell Transplant for MI and Cardiogenesis  (Part 1

Author and Curator: Larry H. Bernstein, MD, FCAP
and
Curator: Aviva Lev-Ari, PhD, RN
This article is Part I of a review of three perspectives on stem cell transplantation onto a substantial size of infarcted myocardium to generate cardiogenesis in tissue that is composed of both repair fibroblasts and cardiomyocytes, after essentially nontransmural myocardial infarct.

Progenitor Cell Transplant for MI and Cardiogenesis (Part 1)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/10/28/progenitor-cell-transplant-for-mi-and-cardiogenesis/

Source of Stem Cells to Ameliorate Damage Myocardium (Part 2)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013-10-29/larryhbern/Source_of_Stem_Cells_to_Ameliorate_ Damaged_Myocardium/

An Acellular 3-Dimensional Collagen Scaffold Induces Neo-angiogenesis
 (Part 3)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013-10-29/larryhbern/An_Acellular_3-Dimensional_Collagen_Scaffold _Induces_Neo-angiogenesis/

The same approach is considered for stroke in one of these studies.  These are issues that need to be considered
  1. Adult stem cells
  2. Umbilical cord tissue sourced cells
  3. Sheets of stem cells
  4. Available arterial supply at the margins
  5. Infarct diameter
  6. Depth of ischemic necrosis
  7. Distribution of stroke pressure
  8. Stroke volume
  9. Mean Arterial Pressure (MAP)
  10. Location of infarct
  11. Ratio of myocytes to fibrocytes
  12. Coexisting heart disease and, or
  13. Comorbidities predisposing to cardiovascular disease, hypertension
  14. Inflammatory reaction against the graft

Transplantation of cardiac progenitor cell sheet onto infarcted heart promotes cardiogenesis and improves function

L Zakharova1, D Mastroeni1, N Mutlu1, M Molina1, S Goldman2,3, E Diethrich4, and MA Gaballa1*
1Center for Cardiovascular Research, Banner Sun Health Research Institute, Sun City, AZ; 2Cardiology Section, Southern Arizona VA Health Care System, and 3Department of Internal Medicine, The University of Arizona, Tucson, AZ; and 4Arizona Heart Institute, Phoenix, AZ
Cardiovascular Research (2010) 87, 40–49   http://dx.doi.org/10.1093/cvr/cvq027

Abstract

Aims

Cell-based therapy for myocardial infarction (MI) holds great promise; however, the ideal cell type and delivery system have not been established. Obstacles in the field are the massive cell death after direct injection and the small percentage of surviving cells differentiating into cardiomyocytes. To overcome these challenges we designed a novel study to deliver cardiac progenitor cells as a cell sheet.

Methods and results

Cell sheets composed of rat or human cardiac progenitor cells (cardiospheres), and cardiac stromal cells were transplanted onto the infarcted myocardium after coronary artery ligation in rats. Three weeks later, transplanted cells survived, proliferated, and differentiated into cardiomyocytes (14.6 ± 4.7%). Cell sheet transplantation suppressed cardiac wall thinning and increased capillary density (194 ± 20 vs. 97 ± 24 per mm2, P < 0.05) compared with the untreated MI. Cell migration from the sheet was observed along the necrotic trails within the infarcted area. The migrated cells were located in the vicinity of stromal-derived factor (SDF-1) released from the injured myocardium, and about 20% of these cells expressed CXCR4, suggesting that the SDF-1/CXCR4 axis plays, at least, a role in cell migration. Transplantation of cell sheets resulted in a preservation of cardiac contractile function after MI, as was shown by a greater ejection fraction and lower left ventricular end diastolic pressure compared with untreated MI.

Conclusion

The scaffold-free cardiosphere-derived cell sheet approach seeks to efficiently deliver cells and increase cell survival.These transplanted cells effectively rescue myocardium function after infarction by promoting not only neovascular-ization but also inducing a significant level of cardiomyogenesis
Keywords  Myocardial infarction • Cardiac progenitor cells • Cardiospheres • Cardiac regeneration • Contractility

Introduction

Despite advances in cardiac treatment after myocardial infarction (MI), congestive heart failure remains the number one killer world-wide. MI results in an irreversible loss of functional cardiomyocytes followed by scar tissue formation. To date, heart transplant remains the gold standard for treatment of end-stage heart failure, a procedure which will always be limited by the availability of a donor heart. Hence, developing a new form of therapy is vital.
A number of adult non-cardiac progenitor cells have been tested for myocardial regeneration, including skeletal myoblasts,1 bone-marrow2, and endothelial progenitor cells.3,4 In addition, several cardiac resident stem cell populations have been characterized based on the expression of stem cell marker proteins.5–8 Among these, the c-Kit+ population has been reported to promote myocardial repair.5,9 Recently, an ex vivo method to expand cardiac-derived progenitor cells from human myocardial biopsies and murine hearts was developed.10 Using this approach, undifferentiated cells (or cardiospheres) grow as self-adherent clusters from postnatal atrium or ventricular biopsy specimens.11
To date, the most common technique for cell delivery is direct injection into the infarcted myocardium.12 This approach is inefficient because more than 90% of the delivered cells die by apoptosis and only a small number of the survived cells differentiated into cardiomyocytes.13 An alternative approach to cell delivery is a biodegradable scaffold-based engineered tissue.14,15 This approach has the clear advantage in creating tissue patches of different shapes and sizes and in creating a beating heart by decellularization technology.16 Advances are being made to overcome the issue of small patch thickness and to minimize possible toxicity of the degraded substances from the scaffold.15 Recently, scaffold-free cell sheets were created from fibroblasts, mesenchymal cells, or neonatal myocytes.17,18 Transplantation of these sheets resulted in a limited improvement in cardiac function due to induced neovascularization and angiogenesis through secretion of angiogenic factors.17–19 However, few of those progenitor cells have differentiated into cardiomyocytes.17 The need to improve cardiac contractile function suggests focusing on cells with higher potential to differentiate to cardiomyocytes with an improved delivery method.
In the present study, we report a cell-based therapeutic strategy that surpasses limitation inherent in previously used methodologies. We have created a scaffold-free sheet composed of cardiac progenitor cells (cardiospheres) incorporated into a layer of cardiac stromal cells. The progenitor cells survived when transplanted as a cell sheet onto the infarcted area, improved cardiac contractile functions, and supported recovery of damaged myocardium by promoting not only vascularization but also a significant level of cardiomyogenesis. We also showed that cells from a sheet can be recruited to the site of injury driven, at least partially, by the stromal-derived factor (SDF-1) gradient.

Methods

Detailed methods are provided in the Supplementary Methods

Animals

Three-month-old Sprague Dawley male rats were used. Rats were randomly placed into four groups:
(1) sham-operated rats, n = 12;
(2) MI, n = 12;
(3) MI treated with rat sheet, n = 10; and
(4) MI treated with human sheet, n = 10.

Myocardial infarction

MI was created by the ligation of the left coronary artery.20 Animals were intubated and ventilated using a small animal ventilator (Harvard Apparatus). A left thoracotomy was performed via the third intercostal rib, and the left coronary artery was ligated. The extent of infarct was verified by measuring the area at risk: heart was perfused with PBS containing 4 mg/mL Evans Blue as previously described by our laboratory.20 The area at risk was estimated by recording the size of the under-perfused (pale-colored) area of myocardium (see Supplementary material online, Figure S1). Only animals with an area at risk >30% were used in the present study. Post-mortem infarct size was measured using triphenyl tetrazolium chloride staining as previously described by our laboratory.20

Isolation of cardiosphere-forming cells

Cardiospheres were generated as described10 from atrial tissues obtained from:
(1) human atrial resection samples obtained from patients (aged from 53 to 73 years old) undergoing cardiac bypass surgery at Arizonam Heart Hospital (Phoenix, AZ) in compliance with Institutional Review Board protocol (n = 10),
(2) 3-month-old SD rats (n = 10). Briefly, tissues were cut into 1–2 mm3 pieces and tissue fragments were cultured ‘as explants’ in a complete explants medium for 4 weeks (Supplementary Methods).
Cell sheet preparation, labelling, handling, and transplantation
Cardiosphere-forming cells (CFCs) combined with cardiac stromal cells were seeded on double-coated plates (poly-L-lysine and collagen type IV from human placenta) in cardiosphere growing medium (Supplementary Methods). The sheets created from the same cell donors were divided into two groups,
one for transplantation and the other for characterization by immunostaining and RT–PCR (Supplementary Methods).
Prior to transplantation, rat cell sheets were labelled with 2 mM 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine, DiI, for tracking transplanted cells in rat host myocardium (Molecular Probes, Eugene, OR). Sheets created using human cells were transplanted unlabelled. Sheets were gently peeled off the collagen-coated plate and folded twice to form four layers. The entire sheet with 200 ml of media was
  • gently aspirated into the pipette tip,
  • transferred to the supporting polycarbonate filter (Costar) and
  • spread off by adding media drops on the sheet (Figure 2A).
Polycarbonate filter was used as a flexible mechanical support for cell sheet to facilitate handling during the transplantation. Immediately after LAD occlusion, the cell sheet was transplanted onto the infarcted area, allowed to adhere to the ventricle for 5–7 min, and the filter was removed before closing the chest (Figure 2A).

Cardiac function

Three weeks after MI, closed-chest in vivo cardiac function was measured using a Millar pressure conductance catheter system (Millar Instruments, Houston, TX) (Supplementary Methods).

Cell sheet survival, engraftment, and cell migration

Rat host myocardium and cell sheet composition after transplantation were characterized by immunostaining (Supplementary Methods). Rat-originated cells were traced by DiI, while human-originated cells were identified by immunostaining with anti-human nuclei or human lamin antibodies.
  1. To assess sheet-originated cardiomyocytes within the host myocardium, the number of cells positive for both human nuclei and myosin heavy chain (MHC) (human sheet); or both DiI and MHC (rat sheet) were counted.
  2. To assess sheet-originated capillaries within the rat host myocardium, the number of cells positive for both human nuclei and von Willebrand factor (vWf) (human sheet); or both DiI and vWf (rat sheet) were counted. Cells were counted in five microscopic fields within cell sheet and area of infarct (n = 5). The number of cells expressing specific markers was normalized to the total number of cells determined by 40,6-diamidino-2-phenylindole staining of the nuclei DNA.
  3. To assess the survival of transplanted cells, sections were stained with Ki-67 antibody followed by fluorescent detection and caspase 3 primary antibodies followed by DAB detection (Supplementary Methods).
  4. To evaluate human sheet engraftment, sections were stained with human lamin antibody followed by fluorescent detection (Supplementary Methods).
  5. Rat host inflammatory response to the transplanted human cell sheet 21 days after transplantation was evaluated by counting tissue mononuclear phagocytes and neutrophils (Supplementary Methods).

Imaging

Images were captured using Olympus IX70 confocal microscope (Olympus Corp, Tokyo, Japan) equipped with argon and krypton lasers or Olympus IX-51 epifluorescence microscope using excitation/emission maximum filters: 490/520 nm, 570 /595 nm, and 355 /465 nm. Images were processed using DP2-BSW software (Olympus Corp).

Statistics

All data are represented as mean ± SE Significance (P < 0.05) was deter-mined using ANOVA (StatView).

Results

Generation of cardiospheres

Cardiospheres were generated from atrial tissue explants. After 7–14 days in culture, a layer of stromal cells arose from the attached explants (Supplementary material online, Figure S2a). CFCs, small phase-bright single cells, emerged from explants and bedded down on the stromal cell layer (Supplementary material online, Figure S2b).
  • After 4 weeks, single CFCs, as well as cardiospheres (spherical colonies generated from CFCs) were observed (Supplementary material online, Figure S2c).
Cellular characteristics of cardiospheres in vitro
Immunocytochemical analysis of dissociated cardiospheres revealed that
  • 30% of cells were c-Kitþ indicating that the CFCs maintain multi-potency. About
  • 22 and 28% of cells expressed a, b-MHC and cardiac troponin I, respectively.
These cells represent an immature cardiomyocyte population because they were smaller (10–15 pm in length vs. 60–80 pm for mature cardiomyocytes) and no organized structure of MHC was detected. Furthermore
  • 17% of the cells expressed a-smooth muscle actin (SMA) and
  • 6% were positive for vimentin,
    • both are mesenchymal cell markers (Supplementary material online, Figure S3a and b).
  • Less then 5% of cells were positive for endothelial cell marker; vWf.
Cell characteristics of human cardiospheres are similar to those from rat tissues (Supplementary material online, Figure S3c).
Cardiospheres were further characterized based on the expression of c-Kit antigen. RT–PCR analysis was performed on both c-Kitþ and c-Kit2 subsets isolated from re-suspended cardiospheres. KDR, kinase domain protein receptor, was recently identified as a marker for cardiovascular lineage progenitors in differentiating embryonic stem cells.21 Here, we found that
  • the c-Kitþ cells were also Nkx2.5 and GATA4-positive, but were low or negative for KDR (Supplementary material online, Figure S3d). In contrast,
  • c-Kit2 cells strongly expressed KDR and GATA4, but were negative for Nkx2.5.
  • Both c-Kitþ and c-Kit2 subsets did not express Isl1, a marker for multipotent secondary heart field progenitors.22
Characteristics of cell sheet prior to transplantation
The cell sheet is a layer of cardiac stromal cells in which the cardiospheres were incorporated at a frequency of 21 ± 0.5 spheres per 100,000 viable cells (Figure 1A). The average diameter of cardiospheres within a sheet was 0.13 ± 0.02 mm and their average area was 0.2 ± 0.06 mm2 (Figure 1A). After sheets were peeled off the plate, it exhibited a heterogeneous thickness ranging from 0.05– 0.1 mm (n 1/4 10), H&E staining (Figure1B) and Masson’s Trichrome staining (Figure 1C) of the sheet sections revealed tissue-like organized structures composed of muscle tissue intertwined with streaks of collagen with no necrotic core. Based on the immunostaining results, sheet compiled of several cell types including
  • SMAþ cardiac stromal cells (50%),
  • MHCþ cardiomyocytes (20%), and
  • vWfþ endothelial cells (10%) (Figure 1D and E).
  • 15% of the sheet-forming cells were c-Kitþ suggesting the cells multipotency (Figure 1E).
  • Cells within the sheet expressed gap-junction protein C43, an indicator of electromechanical coupling between cells (Figure 1D).
  • 40% of cells were positive for the proliferation marker Ki-67 suggesting an active cell cycle state (Figure 1D, middle panel).
Human sheet expressed genes
  1. known to be upregulated in undifferentiated cardiovascular progenitors such as c-Kit and KDR;
  2. cardiac transcription factors Nkx2.5 and GATA4; genes related to adhesion, cell homing, and
  3. migration such as ICAM (intercellular adhesion molecule), CXCR4 (receptor for SDF-1), and
  4. matrix metalloprotease 2 (MMP2).
No expression of Isl1 was detected in human sheet (Figure 1F).
sheet transplant on MI_Image_2
Figure 1 Cell sheet characteristics. (A) Fully formed cell sheet. Arrow indicates integrated cardiosphere. (B) H&E staining; pink colour (arrowhead) indicates cytosol and blue (arrows) indicates nuclear stain. Note that there is no necrotic core within the cell sheet. (C) Masson’s Trichrome staining of sheet section. Arrowhead indicates collagen deposition within the sheet. (D and E) Sheet sections were labelled with antibodies against following markers: (D) vWf (green), Ki-67 (green), C43 (green); (E) c-Kit (green), MHC (red), SMA (red) as indicated on top of each panel. Nuclei were labelled with blue fluorescence of 40,6-diamidino-2-phenylindole (DAPI). (F) Gene expression analysis of the cell sheet. Scale bars, 200 pm (A) or 50 pm (B–E).

Cell sheet survival and proliferation

Two approaches were used to track transplanted cells in the host myocardium.
  • rat cell sheets were labelled with red fluorescent dye, DiI, prior to the transplantation.
  • the sheet created from human cells (human sheet) were identified in rat host myocardium by immunostaining with human nuclei antibodies.
DiI-labelling together with trichrome staining showed engraftment of the cardiosphere-derived cell sheet to the infarcted myocardium (Figure 2B–D). In vivo sheets grew into a stratum with heterogeneous thickness ranging from 0.1–0.5 mm over native tissue. The percentage of Ki-67þ cells within the sheet was 37.5 ± 6.5 (Figure 2F) whereas host tissue was mostly negative (except for the vasculature).
To assess the viability of transplanted cells, the heart sections were stained with the apoptosis marker, caspase 3. A low level of caspase 3 was detected within the sheet, suggesting that the majority of transplanted cells survived after transplantation (Figure 2G).
sheet transplant on MI_Image_3
Figure 2 Transplantation and growth of cell sheet after transplantation.
(A) Sheet transplantation onto infarcted heart. Detached cell sheet on six-well plate (left); cell sheet folded on filter (middle); and transplanted onto left ventricle (right). Scale bar 2 mm. DiI-labelled cell sheets grafted above MI area at day 3
(B) and day 21
(C) after transplantation.
(D) LV section of untreated MI rat at day 21 showing no significant red fluorescence background.
Bottom row (B–D) demonstrates the enlargement of box-selected area of corresponding top panels.
(E) Similar sections stained with Masson’s Trichrome. Section of rat (F) or human (G) sheet treated rat at day 21 after MI.
(F) Section was stained with antibody against Ki-67 (green). Cell sheet was pre-labelled with DiI (red). Nuclei stained with blue fluorescence of DAPI.
(G) Section was double stained with human nuclei (blue) and caspase 3 (brown, arrows) antibodies and counterstained with eosin.
Asterisks (**) indicate cell sheet area. Scale bars 200 mm (B–D, top row), 100 mm (B–D, bottom row, and E) or 50 mm (F, G).
Identification of inflammatory response
Twenty-one days after transplantation of human cell sheet, inflammatory response of rat host was examined. Transplantation of human sheet on infarcted rats reduced the number of mononuclear phagocytes (ED1-like positive cells) compared with untreated MI control (Supplementary material online, Figure S4a–e and l). In addition, the number of neutrophils was similar in both control untreated MI and sheet-treated sections (Supplementary material online, Figure S4f–k and m). These data suggest that at 21 days post transplantation, human cell sheet was not associated with significant infiltration of host immune cells.

Cell sheet engraftment and migration

Development of new vasculature was determined in cardiac tissue sections by co-localization of DiI labelling and vWf staining (Figure 3C). Three weeks after transplantation, the capillary density of ischaemic myocardium in the sheet-treated group significantly increased compared with MI animals (194 ± 20 vs. 97 ± 24 per mm2, P < 0.05, Figure 3A and B). The capillaries originated from the sheet ranged in diameter from 10 to 40 jim (n 1/4 30). A gradient in capillary density was observed with higher density in the sheet area which was decreased towards underlying infarcted myocardium. Mature blood vessels were identified within the sheet area and in the underlying myocardium in close proximity to the sheet evident by vWf and SMA double staining (Figure 3D).
sheet transplant on MI_Image_4
Figure 3 Neovascularization of infarcted wall. (A) Frozen tissue sections stained with vWf antibody (green). LV section of control (sham), infarcted (MI), and MI treated with cell sheet (sheet) rats. Scale bar, 100 jim. (B) Capillary density decreased in the MI compared with sham (*P < 0.05) and improved after cell sheet treatment (#P < 0.05). (C) Neovascularization within cell sheet area was recognized by co-localization of DiI- (red) and vWf (green) staining. Scale bar 100 jim. (D) Mature blood vessels (arrows) were identified by co-localization of SMA (red) and vWf (green) staining. Scale bar 50 jim.
Furthermore, 3 weeks after transplantation, a large number of labelled human nuclei positive or DiI-labelled cells were detected deep within the infarcted area indicating cell migration from the epicardial surface to the infarct (Figure 4A, B, and D). Minor or no migration was detected when the cell sheet was transplanted onto non-infarcted myocardium, sham control (Figure 4C). To evaluate engraftment of sheet-originated cells, sections were labelled with anti-human nuclear lamin antibody. Quantification of engraftment was performed using two approaches: fluorescence intensity and cell counting. Fluorescence intensity of the signal was analysed and compared for different areas of myocardium (Figure 4E–J). Since the transplanted sheets are created by human cells and are stained with human nuclear lamin-labelled with green fluorescence, the signal intensity of the sheet is set to 100% (100% of cells are lamin-positive). Myocardial area with no or limited number of labelled cells had the lowest level of fluorescence signal (13%, or 3.2 ± 1.4% of total number of cells), while
  1. the area where the cell migrated from the sheet to the infarcted myocardium had higher signal intensity (47%, or 11.9 ± 1.7% of total number of cells), indicating a higher number of sheet-originated cells are engrafted in the infarcted area.) (Figure 4K and L).
  2. Migrated cells were positive for KDR (Supplementary material online, Figure S5).
sheet transplant on MI_Image_5
Figure 4 Engraftment quantification of cells migrated from the sheet into the infarcted area of MI. Animals were treated with rat (A) or human (B–F) sheets. Cardiomyocytes were labelled with MHC antibody (A, green or B, red). Rat sheet-originated cells were identified with DiI-labelling, red (A). Arrows indicate the track of migrating cells. Human sheet-originated cells were identified by immunostaining with human nuclei antibody followed by secondary antibodies conjugated with either Alexa 488 (B, E and F, green) or AP (C, D, blue). No migration was detected when the cell sheet was transplanted onto non-infarcted myocardium (C). Heart sections were counterstained with eosin, pink (C–D). Higher magnification of area selected in the box is presented (D, right). Immunofluorescence of sheet (green) grafted to the myocardium surface (E) or cells migrated to the infarction area (F). Fluorescence profiles acrossthe cell sheet itself(G, box 1), area underlying cell sheet (I, box 2) and infarction areawith migrated cells (F, box 3). Mean fluorescence intensityofthe grafted human (K) cells was determined by outlining the region of interest (ROI) and subtracting the background fluorescence for the same region. Fluorescence intensity was normalized to the area of ROI (ii 1/4 6). (L) Percent engraftment was defined as number of lamin-positive cells divided by total number of cells per ROI. ‘M’, myocardium,’S’ sheet, ‘I’ infarction. Scale bars 100 mm (A–C, D, left, E and F), or 50 mm (D, right).
To elucidate a possible mechanism of cell migration, sections were stained to detect SDF1 and its unique receptor CXCR4. The migration patterns of cells from the sheet coincided with SDF-1 expression. Within 3 days after MI, SDF-1 was expressed in the injured myocardium (Figure 5A). At 3 weeks after MI and sheet transplantation, SDF-1 was co-localized with the migrated labelled cells (Figure 5B). PCR analysis revealed CXCR4 expression in cell sheet before transplantation (Figure 1F). However, after transplantation only a fraction of migrated cells expressed CXCR4 (Figure 5C).
sheet transplant on MI_Image_6
Figure 5 Migration of sheet-originated cells into the infarcted area. Confocal images of MI animals treated with sheets from rats (A and B) or human (C). SDF1 (green) was detected at border zone of the infarct at day 3 (A) and day 21 (B). Rat sheet-originated cells were identified with DiI-labelling (red). Note co-localization of DiI-positive sheet-originated cells with SDF1 at 21 days after MI (B). Human cells were identified by immunostaining with human nuclei antibody, red, (C). Note human cells that migrated to the area of infarct express CXCR4 (green) (C). Scale bar, 200 mm (A, B) or 50 mm (C). ‘M’, myocardium, ‘S’ sheet, ‘I’ infarct.

3.7 Cardiac regeneration

The differentiation of migrating cells into cardiomyocytes was evident by the co-localization of MHC staining with either human nuclei (Figure 6A) or DiI (Figure 6B and C). In contrast to the immature cardiomyocyte-like cells within the pre-transplanted cell sheet, the migrated and newly differentiated cells within the myocardium were about 30–50 mm in size and co-expressed C43 (see Supplementary material online, Figure S6). Cardiomyogenesis within the infarcted myocardium was observed in the sheets created from either rat or human cells.
sheet transplant on MI_Image_6
Figure 6 Cardiac regeneration. Sections of MI animals treated with human (A) or rat (B, C) sheets. Human sheet was identified by immunostaining with human nuclei antibody (green). Section was double-stained with MHC (red) antibody. Newly formed cardiomyocytes was identified by co-localization of human nuclei and MHC (yellow, arrow). (B) Rat sheet-originated cells were identified by DiI labelling (red). Section was double-stained with MHC (green) antibody. Newly formed cardiomyocytes were detected by co-localization of DiI with MHC (yellow, arrows). (C) Higher magnification of area selected in the boxes (B). Scale bars 200 mm (B), or 20 mm (A, C). ‘M’, myocardium, ‘S’ sheet, ‘I’ infarct.

Cell sheet improved cardiac contractile function and retarded LV remodelling after MI

Closed-chest in vivo cardiac function was derived from left ventricle (LV) pressure–volume loops (PV loops), which were measured using a solid-state Millar conductance catheter system. MI resulted in a characteristic decline in LV systolic parameters and an increase in diastolic parameters (Table 1). Cell sheet treatment improved both systolic and diastolic parameters (Table 1). Specifically, load-dependent parameters of systolic function: ejection fraction (EF), dP/dTmax, and cardiac index (CI) were decreased in MI rats and increased towards sham control with the cell sheet treatment (Table 1). Diastolic function parameters, dP/dTmin, relaxation constant (Tau), EDV, and EDP were increased in the MI rats and returned towards sham control parameters after sheet treatment (Table 1). However, load-independent systolic function, Emax, was decreased after MI. Treatment with human sheet improved Emax, while treatment with rat sheet had no effect (Table 1). Treatment with either rat or human sheets retarded LV remodelling; as such that it increased the ratio of anteriolateral wall thickness/LV inner diameter (t/Di) and wall thickness/LV outer diameter (t/Do) (see Supplementary material online, Table S3). However, human sheets appear to further improve LV remodelling compared with rat sheets as indicated by increased ratio of wall thickness to ventricular diameter and decreased both EDV and EDP (Table 1 and see Supplementary material online, Table S3).
Table 1 Hemodynamic parameters
Table 1. hemodynamic parameters

Discussion

The majority of the cardiac progenitor cells delivered using our scaffold-free cell sheet survived after transplantation onto the infarcted heart. A significant percentage of transplanted cells migrated from the cell sheet to the site of infarction and differentiated into car-diomyocytes and vasculature leading to improving cardiac contractile function and retarding LV remodelling. Thus, delivery of cardiac progenitor cells together with cardiac mesenchymal cells in a form of scaffold-free cell sheet is an effective approach for cardiac regeneration after MI.
Consistent with previous studies,5,11 here we showed that cardio-spheres are composed of multipotent precursors, which have the capacity to differentiate to cardiomyocytes and other cardiac cell types. When we fractioned cardiospheres based on c-Kit expression, we identified two subsets: Kitþ /KDR2/low/Nkx2.5þ and Kit2/KDRþ/ Nkx2.52(Supplementary material online, Figure S3d), which are likely reflecting cardiac and vascular progenitors.20
In the present study, delivery of cardiac progenitor cells as a cell sheet facilitates cell survival after transplantation. Necrotic cores, commonly observed in tissue engineered patches,23,24 are absent in cardiosphere sheets prior to transplantation (Figure 1B and C). Poor cell survival is caused by multiple processes such as: ischemia from the lack of vasculature and anoikis due to cell detachment from sub-strate.25 A possible mechanism of cell survival within the sheet is the induction of neo-vessels soon after transplantation due to the presence of endothelial cells within the sheet before transplantation (Figure 10). The cell sheet continued to grow in vivo (Figure 2B and C), suppressed cardiac wall thinning, and prevented LV remodelling at 21 days after transplantation (see Supplementary material online, Table S3). This maybe due to the induction of neovascularization (Figure 3), which may prevents ischemia-induced cell death (Figure 2G). Another likely mechanism of cell survival is that the cells within the scaffold-free sheet maintained cell-to-cell adhesion16 as shown by ICAM expression (Figure 1F). The cells also exhibit C43-positive junctions (Figure 10, see Supplementary material online, Figure S6), which may facilitate electromechanical coupling between the transplanted cells and the native myocardium.
We observed cell migration from the sheet to the infarcted myocardium (Figure 4A and B, E and F), which may be facilitated by the strong expression of MMP2 in the cell sheet (Figure 1F). Although, the mechanism of cardiac progenitor cell migration remains unclear, previous observations showed that SDF-1 is upregulated after MI and plays a role in bone-marrow and cardiac stem cell migration.26,27 Our data suggest that SDF-1-CXCR4 axis plays, at least in part, a role in cardiac progenitor cell migration from cell sheet to the infarcted myocardium. This conclusion is based on the following observations: (1) cell sheet expresses CXCR4 prior to transplantation (Figure 1F), (2) migrated cells are located in the vicinity of SDF-1 release (Figure 5A and B), and (3) about 20% of migrated cells expressed CXCR4. Note, not all the migrated cells expressed CXCR4 suggesting other mechanisms are involved in cell migration (Figure 5C).
Here we report that implanting cardiosphere-generated cell sheet onto infarcted myocardium not only improved vascularization but also promoted cardiogenesis within the infarcted area (Figure 6). A larger number of newly formed cardiomyocytes were found deep within the infarct compared with the cell sheet periphery. Notably the transplantation of the cell sheet resulted in a significant improvement of the cardiac contractile function after MI, as was shown by an increase of EF and decrease of LV end diastolic pressure (Table 1).
The beneficial effect of cell sheet is, in part, due to the presence of a large number of activated cardiac mesenchymal stromal cells (myofibroblasts) within the sheet. Myofibroblasts are known to provide a mechanical support for grafted cells, facilitating contraction28 and to induce neovascularization through the release of cytokines.17 In addition, mesenchymal cells are uniquely immunotolerant. In xenograft models unmatched mesenchymal cells transplanted to the heart of immunocompetent rats were shown to suppress host immune response29 presumably due to inhibition of T-cell activation.30 Consistently with previous study from our laboratory,31 here, we demonstrated host tolerance to the cell sheet 21 days after MI. Finally, phase II and III clinical trials are currently undergoing in which allogeneic MSCs are used to treat MI in patients (Osiris Therapeutic, Inc.).
In summary, our results show that cardiac progenitor cells can be delivered as a cell sheet, composed of a layer of cardiac stromal cells impregnated with cardiospheres. After transplantation, cells from the cell sheet migrated to the infarct, partially driven by SDF-1 gradient, and differentiated into cardiomyocytes and vasculature. Transplantation of cell sheet was associated with prevention of LV remodelling, reconstitution of cardiac mass, reversal of wall thinning, and significant improvement in cardiac contractile function after MI. Our data also suggest that strategies, which utilize undigested cells, intact cell–cell interactions, and combined cell types such as our scaffold-free cell sheet should be considered in designing effective cell therapy.

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Iwasaki H, Kawamoto A, Ishikawa M, Oyamada A, Nakamori S, Nishimura H et al. Dose-dependent contribution of CD34-positive cell transplantation to concurrent vasculogenesis and cardiomyogenesis for functional regenerative recovery after myocardial infarction. Circulation 2006;113:1311–1325.
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Oh H, Bradfute SB, Gallardo TD, Nakamura T, Gaussin V, Mishina Y et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci USA 2003;100:12313–12318.
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Pfister O, Mouquet F, Jain M, Summer R, Helmes M, Fine A et al. CD31- but Not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation. Circ Res 2005;97:52–61.
Dawn B, Stein AB, Urbanek K, Rota M, Whang B, Rastaldo R et al. Cardiac stem cells delivered intravascularly traverse the vessel barrier, regenerate infarcted myocardium, and improve cardiac function. Proc Natl Acad Sci USA 2005;102:3766–3771.

 

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