Philippe Menasché

The Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) Trial First Randomized Placebo-Controlled Study of Myoblast Transplantation

Background— Phase I clinical studies have demonstrated the feasibility of implanting autologous skeletal myoblasts in postinfarction scars. However, they have failed to determine whether this procedure was functionally effective and arrhythmogenic. Methods and Results— This multicenter, randomized, placebo-controlled, double-blind study included patients with left ventricular (LV) dysfunction (ejection fraction ≤35%), myocardial infarction, and indication for coronary surgery. Each patient received either cells grown from a skeletal muscle biopsy or a placebo solution injected in and around the scar. All patients received an implantable cardioverter-defibrillator. The primary efficacy end points were the 6-month changes in global and regional LV function assessed by echocardiography. The safety end points comprised a composite index of major cardiac adverse events and ventricular arrhythmias. Ninety-seven patients received myoblasts (400 or 800 million; n=33 and n=34, respectively) or the placebo (n=30). Myoblast transfer did not improve regional or global LV function beyond that seen in control patients. The absolute change in ejection fraction (median [interquartile range]) between 6 months and baseline was 4.4% (0.2; 7.3), 3.4% (−0.3; 12.4), and 5.2% (−4.4; 11.0) in the placebo, low-dose, and high-dose groups, respectively (P=0.95). However, the high-dose cell group demonstrated a significant decrease in LV volumes compared with the placebo group. Despite a higher number of arrhythmic events in the myoblast-treated patients, the 6-month rates of major cardiac adverse events and of ventricular arrhythmias did not differ significantly between the pooled treatment and placebo groups. Conclusions— Myoblast injections combined with coronary surgery in patients with depressed LV function failed to improve echocardiographic heart function. The increased number of early postoperative arrhythmic events after myoblast transplantation, as well as the capability of high-dose injections to revert LV remodeling, warrants further investigation.

Cell-based cardiac repair: reflections at the 10-year point.

Received February 28, 2005; revision received April 25, 2005; accepted April 29, 2005. It has now been more than a decade since the first experiments were performed using cell transplantation for the prevention and treatment of heart failure.1–3 Although the biomedical community was initially somewhat skeptical of this approach, a large body of experimental evidence was amassed showing that injected cells could create new tissue and improve function of the failing heart. This evidence, coupled with the recognized limitations of heart failure treatments and the intuitively appealing concept of “regenerative medicine,” has contributed to a crescendo of activity in cell-based cardiac repair. Given the flurry of clinical trials that are currently under way, we think it is timely to review progress over the past 10 years and provide a critical assessment of where the field stands and where it appears to be headed. Cell-based cardiac repair began with studies of skeletal myoblasts derived from skeletal muscle satellite cells.1–3 Myoblasts were the initial choice because of their availability from autologous or syngeneic sources, their ability to proliferate, and their ability to withstand ischemia better than many cell types. Although it was originally hoped that these cells would transdifferentiate into cardiomyocytes, it is now clear that myoblasts remain stubbornly committed to form mature skeletal muscle in the heart3–5 (with the exception of rare cell fusion events at the graft–host interface6). Skeletal muscle is one of the few cell types in the body that does not normally express gap junction proteins, and hence, structural and physiological studies indicate that skeletal muscle cells do not form electromechanical junctions with cardiomyocytes when engrafted into the heart.7,8 Despite this, numerous studies have shown beneficial effects of skeletal myoblast grafting into the infarcted heart in rodents and large animals.8–13 Cardiomyocytes would …

Transplantation of cardiac-committed mouse embryonic stem cells to infarcted sheep myocardium: a preclinical study

BACKGROUND Heart failure develops after myocardial infarction and is a major cause of morbidity and mortality. The ability to direct differentiation of embryonic stem cells (ESC) towards a cardiomyogenic phenotype makes them an attractive therapeutic option for cardiac repair, but species-specific and individual-specific immunological imprinting remains a hurdle. Our aim was to ascertain whether the purported immune privilege of ESC allows for their cross-species engraftment in a clinically relevant large-animal model. METHODS We studied engraftment and differentiation of cardiac-committed mouse ESC in 18 sheep in which a myocardial infarction had been induced; nine controls received medium and nine sheep (five of which were immunosuppressed) received ESC. The gain in myocardial function was measured by echocardiography 1 month after cell transplantation. FINDINGS Cardiac-committed murine ESC engrafted in infarcted myocardium of immunosuppressed and immunocompetent sheep, and differentiated into mature cardiomyocytes that expressed connexins. Colonisation of the scar area by ESC was accompanied by a functional benefit of the damaged myocardium. Left-ventricular ejection fraction deteriorated in the control group by a median of 9.9% (range -20 to 0.3) relative to baseline (p=0.011) whereas in the treated group it improved by 6.6% (-5.7 to 50.8; comparison between groups p=0.002). INTERPRETATION These findings obtained in a clinically relevant large-animal model of heart failure strengthen the potential therapeutic use of ESC to regenerate the severely dysfunctional myocardium and bring additional evidence for an immune privilege of these cells.

A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates

Cell therapy holds promise for tissue regeneration, including in individuals with advanced heart failure. However, treatment of heart disease with bone marrow cells and skeletal muscle progenitors has had only marginal positive benefits in clinical trials, perhaps because adult stem cells have limited plasticity. The identification, among human pluripotent stem cells, of early cardiovascular cell progenitors required for the development of the first cardiac lineage would shed light on human cardiogenesis and might pave the way for cell therapy for cardiac degenerative diseases. Here, we report the isolation of an early population of cardiovascular progenitors, characterized by expression of OCT4, stage-specific embryonic antigen 1 (SSEA-1), and mesoderm posterior 1 (MESP1), derived from human pluripotent stem cells treated with the cardiogenic morphogen BMP2. This progenitor population was multipotential and able to generate cardiomyocytes as well as smooth muscle and endothelial cells. When transplanted into the infarcted myocardium of immunosuppressed nonhuman primates, an SSEA-1+ progenitor population derived from Rhesus embryonic stem cells differentiated into ventricular myocytes and reconstituted 20% of the scar tissue. Notably, primates transplanted with an unpurified population of cardiac-committed cells, which included SSEA-1- cells, developed teratomas in the scar tissue, whereas those transplanted with purified SSEA-1+ cells did not. We therefore believe that the SSEA-1+ progenitors that we have described here have the potential to be used in cardiac regenerative medicine.

Experimental evaluation of Celsior, a new heart preservation solution.

An original heart preservation solution (Celsior) has been developed, the formulation of which has been designed to fulfil two major objectives: (1) to combine the general principles of hypothermic organ preservation with those specific for the myocardium, and (2) to offer the possibility of being used not only as a storage medium but also as a perfusion fluid during initial donor heart arrest, poststorage graft reimplantation and early reperfusion. The major principles addressed by the Celsior formulation include (1) prevention of cell swelling (by mannitol and lactobionate), (2) prevention of by the Celsior formulation include (1) prevention of cell swelling (by mannitol and lactobionate), (2) prevention of oxygen-derived free radical injury (by reduced glutathione, histidine and mannitol), and (3) prevention of contracture by enhancement of energy production (glutamate) and limitation of calcium overload (high magnesium content, slight degree of acidosis). Two experimental preparations were used: The isolated isovolumic buffer-perfused rat heart model and the heterotopic rabbit heart transplantation model. In isolated heart experiments, hearts were arrested with and stored in Celsior for 5 h at 4 degrees C and subsequently reperfused for 1 h. A similar protocol was used in the transplantation experiments except that the total ischemic time was approximately 1 1/2 h longer (corresponding to 6 h of storage followed by the 25 additional minutes of cold ischemia required for graft implantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Human embryonic stem cell-derived cardiac progenitors for severe heart failure treatment: first clinical case report

AIMS Comparative studies suggest that stem cells committed to a cardiac lineage are more effective for improving heart function than those featuring an extra-cardiac phenotype. We have therefore developed a population of human embryonic stem cell (ESC)-derived cardiac progenitor cells. METHODS AND RESULTS Undifferentiated human ESCs (I6 line) were amplified and cardiac-committed by exposure to bone morphogenetic protein-2 and a fibroblast growth factor receptor inhibitor. Cells responding to these cardio-instructive cues express the cardiac transcription factor Isl-1 and the stage-specific embryonic antigen SSEA-1 which was then used to purify them by immunomagnetic sorting. The Isl-1(+) SSEA-1(+) cells were then embedded into a fibrin scaffold which was surgically delivered onto the infarct area in a 68-year-old patient suffering from severe heart failure [New York Heart Association [NYHA] functional Class III; left ventricular ejection fraction (LVEF): 26%]. A coronary artery bypass was performed concomitantly in a non-infarcted area. The implanted cells featured a high degree of purity (99% were SSEA-1(+)), had lost the expression of Sox-2 and Nanog, taken as markers for pluripotency, and strongly expressed Isl-1. The intraoperative delivery of the patch was expeditious. The post-operative course was uncomplicated either. After 3 months, the patient is symptomatically improved (NYHA functional Class I; LVEF: 36%) and a new-onset contractility is echocardiographically evident in the previously akinetic cell/patch-treated, non-revascularized area. There have been no complications such as arrhythmias, tumour formation, or immunosuppression-related adverse events. CONCLUSION This observation demonstrates the feasibility of generating a clinical-grade population of human ESC-derived cardiac progenitors and combining it within a tissue-engineered construct. While any conclusion pertaining to efficacy would be meaningless, the patients functional outcome yet provides an encouraging hint. Beyond this case, the platform that has been set could be useful for generating different ESC-derived lineage-specific progenies.

Differentiation In Vivo of Cardiac Committed Human Embryonic Stem Cells in Postmyocardial Infarcted Rats

Human embryonic stem (HES) cells can give rise to cardiomyocytes in vitro. However, whether undifferentiated HES cells also feature a myocardial regenerative capacity after in vivo engraftment has not been established yet. We compared two HES cell lines (HUES‐1 and I6) that were specified toward a cardiac lineage by exposure to bone morphogenetic protein‐2 (BMP2) and SU5402, a fibroblast growth factor receptor inhibitor. Real‐time polymerase chain reaction (PCR) revealed that the cardiogenic inductive factor turned on expression of mesodermal and cardiac genes (Tbx6, Isl1, FoxH1, Nkx2.5, Mef2c, and α‐actin). Thirty immunosuppressed rats underwent coronary artery ligation and, 2 weeks later, were randomized and received in‐scar injections of either culture medium (controls) or BMP2 (±SU5402)‐treated HES cells. After 2 months, human cells were detected by anti‐human lamin immunostaining, and their cardiomyocytic differentiation was evidenced by their expression of cardiac markers by reverse transcription‐PCR and immunofluorescence using an anti‐β myosin antibody. No teratoma was observed in hearts or any other organ of the body. The ability of cardiac‐specified HES cells to differentiate along the cardiomyogenic pathway following transplantation into infarcted myocardium raises the hope that these cells might become effective candidates for myocardial regeneration.

Ischemic preconditioning in cardiac surgery: A word of caution

OBJECTIVE Ischemic preconditioning is now established as an effective means of reducing infarct size. However, it remains uncertain whether preconditioning can improve the myocardial protection afforded by cardioplegia. The present study was designed to address this issue. METHODS After the institution of cardiopulmonary bypass, 10 patients were preconditioned with 3 minutes of aortic crossclamping followed by 2 minutes of reperfusion before the onset of retrograde continuous warm cardioplegic arrest. Ten case-matched patients served as controls. Three blood samples were drawn simultaneously from the radial artery and the coronary sinus before bypass, at the end of the 5-minute preconditioning protocol or after 5 minutes of bypass in control patients, and at the end of cardioplegic arrest. These samples were assayed for creatine kinase MB isoenzyme and lactate. Right atrial biopsy specimens taken at the same time points were processed by Northern blotting for the expression of messenger ribonucleic acid of both c-fos and heat shock protein 70. RESULTS At the end of arrest, the release of creatine kinase MB from the myocardium was markedly greater in preconditioned patients than in the controls. The transmyocardial lactate gradient was shifted toward production in the preconditioned group (+0.22 +/- 0.13 mmol/L) and toward extraction in the control group (-0.06 +/- 0.21 mmol/L). Molecular biology data did not suggest a protective effect of preconditioning. There were no clinical adverse events related to preconditioning. CONCLUSIONS Preconditioning does not enhance cardioplegic protection and might even be deleterious. These results do not dismiss its use in cardiac operations. They rather emphasize the need for identifying pharmacologic mediators that could safely and effectively duplicate the cardioprotective effects of ischemic preconditioning.

Efficacy of Chordal Cutting to Relieve Chronic Persistent Ischemic Mitral Regurgitation

Background—Mitral regurgitation (MR) conveys adverse prognosis in ischemic heart disease. Leaflet closure is restricted by tethering to displaced papillary muscles, and is, therefore, incompletely treated by annular reduction. In an acute ischemic model, we reduced such MR by cutting a limited number of critically positioned chordae to the leaflet base that most restrict closure but are not required to prevent prolapse. Whether this is effective without prolapse, recurrent MR, or left ventricular (LV) failure in chronic persistent ischemic MR, despite greater LV remodeling, remains to be established. Therefore, we studied 7 sheep with chronic inferobasal infarcts known to produce progressive MR over 2 months. In all of those sheep, after a mean of 4.1 months, the 2 central basal (intermediate) chordae were cut at the chronic ischemic MR stage. 3-Dimensional echo quantified MR, LV function, and valve geometry. Five other sheep were followed for a mean of 7.8±1.2 months after inferobasal infarction with chordal cutting. Results—All 7 of the sheep with chronic ischemic MR (increased from 1.4±0.4 to 11.1±0.5 mL/beat, regurgitant fraction=39.0±4.2%, P <0.0001) showed anterior leaflet angulation at the basal chord insertion. Although end-systolic volume had doubled, cutting the 2 central basal chordae significantly decreased the MR to baseline (P <0.0001) without prolapse or decline in EF (41.1±1.5% to 42.6±1.6%, P =not significant [NS]). The five sheep with long-term follow-up showed no prolapse or MR, and no significant post-infarct decrease in LV ejection fraction (EF; 38.9±2.4% to 41.4±1.2%, P =NS). Conclusion—Cutting a minimum number of basal (intermediate) chordae can improve coaptation and reduce chronic persistent ischemic MR without impairing LVEF. No adverse effects were noted long-term after chordal cutting at the time of infarction.

Composite cell sheets: a further step toward safe and effective myocardial regeneration by cardiac progenitors derived from embryonic stem cells.

Background— The safety and efficacy of myocardial regeneration using embryonic stem cells are limited by the risk of teratoma and the high rate of cell death. Methods and Results— To address these issues, we developed a composite construct made of a sheet of adipose tissue–derived stroma cells and embryonic stem cell–derived cardiac progenitors. Ten Rhesus monkeys underwent a transient coronary artery occlusion followed, 2 weeks later, by the open-chest delivery of the composite cell sheet over the infarcted area or a sham operation. The sheet was made of adipose tissue–derived stroma cells grown from a biopsy of autologous adipose tissue and cultured onto temperature-responsive dishes. Allogeneic Rhesus embryonic stem cells were committed to a cardiac lineage and immunomagnetically sorted to yield SSEA-1+ cardiac progenitors, which were then deposited onto the cell sheet. Cyclosporine was given for 2 months until the animals were euthanized. Preimplantation studies showed that the SSEA-1+ progenitors expressed cardiac markers and had lost pluripotency. After 2 months, there was no teratoma in any of the 5 cell-treated monkeys. Analysis of >1500 histological sections showed that the SSEA-1+ cardiac progenitors had differentiated into cardiomyocytes, as evidenced by immunofluorescence and real-time polymerase chain reaction. There were also a robust engraftment of autologous adipose tissue–derived stroma cells and increased angiogenesis compared with the sham animals. Conclusions— These data collected in a clinically relevant nonhuman primate model show that developmentally restricted SSEA-1+ cardiac progenitors appear to be safe and highlight the benefit of the epicardial delivery of a construct harboring cells with a cardiomyogenic differentiation potential and cells providing them the necessary trophic support.