C. Introduction

      Chronic congestive heart failure represents a major cause of cardiovascular morbidity and mortality in developed countries. It is caused by the loss of functional heart muscle, which is due either to ischemic heart disease or the presence of dysfunctional muscle resulting from a variety of causes, including hypertension, viruses, and idiopathic factors. Following myocardial infarction for example, functional contracting cardiomyocytes are replaced with nonfunctional scar tissue. This ventricular remodeling leads to ventricle dilatation and progressive heart failure which constitute a major clinical problem (Grounds et al., 2002). The remodeling process is characterized by the removal of necrotic cardiomyocytes accompanied by granulation tissue formation with the simultaneous induction of neovascularization in the peri-infarcted bed. The latter is a prerequisite for the survival of surrounding hypertrophied but viable cardiomyocytes and the prevention of further cardiomyocyte loss by apoptosis.

      Several treatments for coronary artery disease are available, which reduce the symptoms and improve the quality of life of CHF patients. They include medical treatments (anticoagulants, b-blocker, angiotensin-converting-enzyme inhibitors, etc), effective percutaneous and surgical revascularization and cardiac pacing systems. Cardiac transplantation remains, however, the ultimate solution for end-stage heart failure. However, the shortage of donor hearts, the complications of immunosuppression, the failure of grafted organs and, not at last, the advanced age of patients suffering from CHF limit the utility of cardiac transplantation significantly.

      Cell therapy as a mean to repair damaged tissues unable to heal is an increasingly attractive concept in modern transplantation medicine. For many clinical situations, i.e. congestive heart failure, Parkinson's disease, diabetes, traumatic injuries (spinal cord) and iatrogenic destruction of the cell (chemotherapy), replacement of lost cells would be the ideal treatment. In many cases, however, the development of cell treatment approaches is hampered by an increasing lack of donors or by the lack of cells suitable for transplantation.

C.1. Seeking stem cells desperately

      Which cells could be used to repair a failing heart and restore its contractile power?

      Two major types of stem cells would eventually fulfill suitable characteristics: 1) embryonic stem cells (ESC), derived from the inner cell mass of the blastocyst, and 2) adult somatic stem cells isolated form several organs (bone marrow, skeletal muscle, brain, etc).

      What is a stem cell? Stem cells are commonly defined as undifferentiated cells. They have the ability to differentiate into virtually all kinds of cell types, a capacity that becomes progressively restricted with development. As shown in Figure I, they have two important characteristics that distinguish them from other types of cells. First, as unspecialized cells, they can proliferate and renew themselves for long periods through cell division. The second is that under certain physiologic or experimental conditions, they can be induced to become cells with special functions. As the matter of fact, they provide a theoretically inexhaustible supply of cells that, depending on type can give rise to some or all body tissues.


Figure I. Definition of a stem cell.

      Stem cells are typically found in the embryo and fetus. In the adult body, they have been identified in various tissue niches, including bone marrow, brain, liver, and skin, as well as in the circulation. They have been termed "adult stem cells". An extremely attractive concept is that adult stem cells could be harvested from a patient, induced to specialize in culture, then incorporated into a tissue construct, and put back into the same individual when repair become necessary, bypassing the need for immunosuppression.

      The experimental donor cells for cellular cardiomyoplasty (CCM), as an alternative treatment of cardiovascular disease, include skeletal myoblasts, bone marrow-derived Mesenchymal stem cells, purified (enriched) haematopoietic stem cell populations and blood and bone marrow-derived endothelial progenitor cells.

C.2. Embryonic stem cells (ESC)

      Pluripotent murine stem cells are derived from two main embryonic sources: ESC from the blastocyst and EG cells from the gonadal ridge of the embryo after gastrulation (Figure II). The successful derivation of murine ESC from the inner cell mass of mouse blastocytes was achieved in 1981 (Martin, 1981), while embryonic germ (EG) cells have been isolated and cultured from primordial germ cells (Stewart et al., 1994).


Figure II. Origin and establishment of pluripotent embryonic stem (ES) and embryonic germ (EG) cell lines from the inner cell mass (ICM) of mouse blastocysts and from primordial germ cells, respectively.

      These cells were shown to be pluripotent, i.e., capable of forming all mature cell phenotypes derived from the three embryonic layers: endoderm, ectoderm, and mesoderm, as shown in Figure III.


Figure III. Tissue derivative of the three embryonic layers.

      In vitro, murine ESC remain undifferentiated when grown in the presence of leukemia inhibitory factor (LIF) and for some cell lines, cultured on murine embryonic fibroblast (MEF) as feeder cells. The mechanism to maintain stem cell as a undifferentiated state is linked to the LIF/Stat3 signaling pathway and the transcription factor Oct-3/4, but it is still unclear how these components works together (Niwa, 2001). When LIF or feeder cells are withdrawn, most types of ESC differentiate spontaneously to form aggregates called embryoid bodies (EBs). Embryoid bodies are comprised of the heterogeneous cells that derived from all three germ layers. These tri-dimensional cell-cell contacts allow the formation of heterogeneous cultures of differentiated cell types including cardiomyocytes, hematopoietic cells, endothelial cells, neurons, skeletal muscle chondrocytes, adipocytes, liver, and pancreatic islets.


Figure IV. The derived ES or EG cells are differentiated in vitro by culturing them via embryo-like aggregates, the embryoid bodies (EBs). After plating of EBs onto adhesive substrates, differentiated cells grow out from the EBs (from Wobus, 2001).

      When murine ESC were cultured into embryoid bodies, some differentiated into a diverse range of cardiomyocyte phenotypes, including ventricular, atrial, sinus nodal, Purkinje, and pacemaker-like cells. These different cardiac phenotypes exhibit developmentally controlled expression of cardiac-specific genes, structural proteins, ion channels, and receptors, and can be distinguished on the basis of their action potentials. A few cell areas on the embryoid bodies display spontaneous contractile activity under light microscope, and are identifiable as ESC-derived cardiomyocytes. Since ESC are pluripotent, considering their use raises the potential risk of teratoma formation. Thus, the purification of differentiated ESC-derived cardiomyocytes from cultures is a key issue. Unfortunately, so far, none of the approaches used on murine ESC can give 100% yield of cells with the required phenotype.

C.3. No stem cells in the heart?

      In the case of the heart, no local stem cells have been so far identified. Cardiomyocytes undergo terminal differentiation soon after birth and are generally considered to irreversibly withdrawn from the cell cycle. Cardiomyocyte DNA synthesis occurs primarily in uteri, with proliferating cells decreasing from 33% at mid-gestation to 2% at birth(MacLellan and Schneider, 2000). Therefore, upon injury, the adult heart results incapable to regenerate the damaged tissue which instead become fibrotic.

      On note is the fact that the group of Piero Anversa was the only one to report that, in humans, some ventricular cardiomyocytes may have the capacity to proliferate or at least to undergo nuclear replication in response to ischemic injury. The dividing myocytes have been identified on the basis of immunohistochemical staining of proliferating nuclear structures such as Ki67 and cell surface expression of specific surface markers CD117. (Itescu et al., 2003). However, this is still a matter of debate and convincing proofs of cell division as a general event are still pending. It remains to be determined where does the dividing cells which homed to the damaged myocardium come from? Are those a resident source of cardiac stem cells, or do they come from a renewable source of circulating bone marrow-derived stem cells? It is not very clear so far (Anversa et al., 2003; Beltrami et al., 2001).

C.4. Cell therapy of the heart

      The cell-based myocardial repair technology "cellular cardiomyoplasty" (CCM), attempt to regenerate functioning muscle in previously infarcted, scarred or dysfunctional myocardial tissue after transplantation of myogenic cells. The use of such a cell therapy approach to replace lost cardiomyocytes with new engraftable ones would represent an invaluable, low-invasiveness technique for treatment of heart failure as an alternative to whole heart transplantation. Replacement and regeneration of functional cardiac muscle after ischemia could be achieved either by stimulating proliferation of endogenous mature cardiomyocytes or by implanting exogenous donor-derived or allogeneic cardiomyocytes. The newly formed cardiomyocytes must integrate precisely into the existing myocardial wall to augment contractile function of the residual myocardium in a synchronized manner and avoid alteration in the electrical condition and syncytial contraction of the heart (Itescu et al., 2003).

      To date, many types of cells have been tested as a source of cell therapy for the augmentation of myocardial performance in different experimental models of heart failure. Those include fetal cardiomyocytes (Leor et al., 1996; Li et al., 1996; Reinecke et al., 1999), ESC-derived cardiomyocytes (Min et al., 2002), skeletal myoblasts(Murry et al., 1996; Taylor et al., 1998), immortalized myoblasts(Robinson et al., 1996), fibroblasts(Hutcheson et al., 2000), smooth muscle cells (Li et al., 1999), fibroblasts (Murry et al., 1996), adult cardiac-derived cells(Li et al., 2000), and bone marrow-derived stem cells (Orlic et al., 2001). Different heart models have been used to study the effect of cell engraftment in the heart, e.g. normal heart tissue, cryoinjuried heart tissue, ischemic heart, infarcted scar tissue, dilated cardiomyopathy heart.

C.4.1. ESC into the heart

      Several groups have demonstrated the in vivo feasibility of the intra-cardiac implantation of ESC. Klug et al. were the first to engraft genetically-modified and differentiated murine ESC-derived cardiomyocytes into the left ventricular free wall of mdx mice(Klug et al., 1996). Pure cardiomyocyte cultures were obtained by stable transfecting ESC with a transgene comprised of the a-cardiac myosin heavy chain (MHC) promoter driving a neomycin resistance gene. This construct being expressed only in cardiac cells, in the presence of neomycin (G418) it allowed the only survival of ESC-derived cardiomyocytes. The successful engraftment of donor ESC was confirmed by immunopositivity for dystrophin, and engrafted cells were found to be aligned with the host cardiomyocytes. More recently, Min et al. implanted cardiomyocytes derived from the D3-ESC line into a rat model of ischemic heart. Cardiomyocytes were selected from embryoid bodies by dissecting the spontaneously beating clusters via a sterile micropipette. After transfection with a green fluorescent protein (GFP) marker to identify survival of engrafted ESC, transplantation was performed within 30 minutes after induction of MI, created by ligation of the left anterior descending coronary artery. Under these conditions, the cardiac function was significantly improved 6 weeks after cell transplantation in MI animals compared with the MI control group (Min et al., 2002). However, some areas with undifferentiated cells were still observed, indicating that their extraction from the EBs was poor. Recently, Behfar et al reported that undifferentiated ESC directly implanted into an infarcted heart seems able to commit to the cardiac phenotype. Their study suggested that ESC differentiation require a paracrine pathway in the heart. In vitro, pretreatment of embryonic stem cells with TGFb growth factor members results in embryoid bodies with greater areas of cardiac differentiation (increased beating areas) and normal sarcomeric organization (as revealed by immunostaining). 5 weeks after the In vivo transplantation of undifferentiated ESC carrying the ECFP (cyan fluorescent protein) marker under the control of the cardiac a-actin promoter, they identified ESC-derived blue cardiomyocytes by immunostaining. The left ventricular ejection fraction (measured by echocardiography) was improved in a small number animals (n=3), as compared with control group (Behfar et al., 2002). More recently, Johkura et al. transplanted ESC-derived cardiomyocytes into the retroperitoneum of the adult nude mice. These myocytes proliferated, differentiated and remained viable and contractile for up to 30 days in the ectopic site around large blood vessels. However, contamination of the donor cells with the residual ESC committed to other lineages was likely to occur, even if the embryoid body outgrowths were transplanted after beating cardiomyocytes had appeared. This leaded to the formation of teratoma in the host retroperitoneum (Johkura et al., 2003).

      In summary, the main issues which limit the research and the use of ESC for CCM include difficulties in obtaining pure and sufficient number of ES-derived cardiomyocytes, especially of ventricular- like cardiomyocytes, high risk of teratoma formation and immune rejection.

C.4.2. Human ESC

      Presently, several human ESC lines are available since their first isolation by Thomson in 1998 (Thomson et al., 1998). A registry of them is published by the NIH web site (http://stemcells.nih.gov/registry/). The usage of human ESC as a resource for cell therapy is presently an intensive field of research (Mummery et al., 2003; Nir et al., 2003). At a basic research level, many biological differences should exist between the murine and human ESC and further fundamental studies are required before to investigate the suitability and feasibility of using human ES-derived cardiomyocytes for cell transplantation in humans. From a legal and ethical point of view, research involving human embryonic cells is highly controversial and many countries are reviewing their legislation. Importantly, main ethical issues are raised concerning the derivation of human ESC from human in vitro fertilized embryos, the moral status of the embryo, and the acceptability of using such derived cells for therapeutic purposes. The application of human ESC therapy for the treatment of cardiac diseases in humans is far from being a reality.

C.4.3. Skeletal muscle cells into the heart

      The growth and repair of skeletal muscle is usually initiated by the activation of a population of muscle precursors, called satellite cells. Satellite cells normally lie near the basal lamina of the skeletal muscle fibers and can differentiated into myofibers. Following tissue injury, they are rapidly mobilized, proliferate and fuse, thereby effecting repair and regeneration of the damaged fibers.

      Cardiac and skeletal muscles have a similar sophisticated organization of contractile proteins into sarcomeres and are collectively referred to as striated muscles. Growth of the heart is generally characterized by division of muscle cells during the embryonic stages of life, followed after birth by into a post-mitotic state. The postnatal capacity for cell replication during growth and regeneration of cardiac and skeletal muscle cells is markedly different. Skeletal muscle cells are multinucleated and can readily regenerate from precursor cells (satellite cells / myoblasts), whereas post-natal cardiac muscle is incapable of tissue repair, since cardiac cells exit the cell cycle soon after birth and become post-mitotic (either in vivo or in vitro).

      Autologous skeletal myoblasts appear to be the most well studied and best first generation cells for cardiac repair. This process was pioneered in the 1980s (Sola et al., 1985), and has been applied clinically with varied success. The whole procedure includes extraction of the myoblasts from skeletal muscle, expansion in tissue culture, and injection into the heart muscle. For the success of cell transplantation, the introduced donor cells must be able to survive in their host environment. Intramuscular injection of cultured isolated myoblasts in classical myoblast transfer therapy shows that there is a massive and rapid necrosis of donor myoblasts, with 90% dead within the first hour after injection (Beauchamp et al., 1999). Host natural killer cells appear to play a particularly important role in this rapid death of cultured donor myoblasts.

      In vivo, in various species, such as rabbits and rats, engraftment of skeletal myoblasts were shown colonized injured cardiac tissue(Murry et al., 1996; Pouzet et al., 2000; Taylor et al., 1998). A survival up to 12 weeks after transplantation was observed in normal heart (Reinecke et al., 2002), and up to 18 weeks in cryoinjured myocardium(Reffelmann and Kloner, 2003). An important factor is that skeletal myoblasts are relatively resistant to ischemia and, in contrast to cardiomyocytes (witch injure rapidly within 20 min), they can withstand several hours of severe ischemia without becoming irreversibly injured. Several studies reported improvement of left ventricular performance after myoblast transplantation, e.g. reducing left ventricular dilatation, increasing ex vivo systolic pressures and improving in vivo exercise capacity (Atkins et al., 1999; Jain et al., 2001).

      However, the outcome of engraftment of skeletal myoblasts still remains highly controversial. Some reports claim that transplanted skeletal myoblasts could differentiate and develop into striated cells within the damaged myocardium, thus preventing the progressive ventricular dilatation by improving heart function (Taylor, 2001). On the other hand, some investigators have reported negative results and adverse effects. The systematic investigations by Reinecke et al. did not support the concept of a true transdifferentiation into cardiomyocytes as demonstrated by the lack of a-myosin-heavy-chain, cardiac troponin I, or atrial natriuretic peptide- expression in the grafts. Murry et al. also concluded that skeletal satellite muscle cells differentiate into mature skeletal muscle and do not express cardiac-specific genes after grafting into the heart, lacking any transdifferentiation potential (Reinecke et al., 2002).

      Recently, attempts to use autologous skeletal satellite cells to repair damaged hearts have received considerable attention for the possibility of autotransplantation into human myocardium. The autologous origin would clearly overcomes all problems related to availability, ethics and immunogenicity, key factors for large-scale clinical applicability (Menasche, 2003). Menasche et al. first reported a three-steps protocol in a CHF patient in 2001 (Hagege et al., 2003; Menasche et al., 2003). A muscular biopsy was retrieved from the thigh under local anesthesia and, after enzymatic and mechanical dissociation, the cells were grown for 2-3 weeks in culture, providing at least 400x106cells, 50% of which were myoblasts. These cells are then reimplanted across the post-infarcted scar at the time of coronary bypass graft surgery (Menasche, 2003).

      Several clinical trials are presently running in several countries, with different preliminary results. Some trials reported the presence of arrhythmia and sudden death, which leading to the suggestion to implant AICD´s on the time of procedure. The need of randomized trials is claimed in many conferences and meetings.

      There are many important questions remain to be answered for the clinical use of myoblasts. They are related to cell survival, integration, differentiation, and functional effect. E.g. for the long-term benefit of skeletal myoblasts transplantation myocardial injury, cells must be able to survive for years in the heart. The long-term fate of myoblasts is unclear. The effect of transplanted myoblasts on electrical stability of the heart, as they do not form normal electrical junctions with the host, it will increase the risk of arrhythmia. In a phase I human clinical trial, 4 out of 10 cases showed sustained ventricular tachycardia during the early (3 weeks) postoperative period (Menasche et al., 2003). The mechanisms of these arrhythmias might be inhomogeneity in action potential conduction creating reentry pathways. So, how myoblasts electrically integrate into the surrounding myocardium is still a big issue from the clinical point of view. Can myoblasts function as cardiac-like myocytes and thus improve contraction, including adaptation to chronic workload and integration into the host, or can myoblasts just prevent further deterioration of the injured heart? How do we control continuing proliferation after transplantation to avoid undesirable disturbance of local left ventricular geometry? All those questions still need to be answered.

C.4.4. Bone Marrow stem cells

      Bone marrow is a mesodermal-derived tissue and contains haematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), which may both, be derived from a common primitive blast-like cell. The HSC are archetypal stem cells. They have the ability to balance self-renewal against differentiation cell fate decisions They are multipotent, a single stem cell producing at least eight to ten distinct lineages of mature cells. HSC have an extensive proliferative capacity that yields a large number of mature progeny. These cells are rare, comprising only 1/10'000 to 100'000 of total blood cells, and can be obtained from the bone marrow, peripheral blood umbilical cord and fetal liver(Bonnet, 2002; Preston et al., 2003). Krause et al., demonstrated that a single HSC was not only able to repopulate the haematopoietic system in irradiated mice, but also differentiated into lung epithelium, skin, liver and the gastrointestinal tract (Krause et al., 2001).

      Figure V illustrates the transdifferentiation potential observed either in culture or after in vivo injection of cells.


Figure V. Possible pathways of differentiation in adult stem cells. (from Science (Holden and Vogel, 2002))

      Too good to be true? Studies in mice have yielded evidence, now being reassessed, that stem cells from the bone marrow compartment can produce progeny in different organs. Bone marrow, which has several types of stem cells, seems particularly versatile.

      MSCs have the main capacity to differentiate, both in vivo and in vitro, into osteoblast, chondroblasts, and adipocytes when exposed to the appropriate stimuli. Approximately 30% of human marrow aspirate cells adhering to plastic are considered to be MSCs. They are in general more difficult to characterized than the HSC populations. MSCs have been functionally identified in adult murine and human bone marrow by their ability to differentiate to lineages of diverse mesenchymal tissues. Those include bone, cartilage, fat, tendon, and both skeletal and cardiac muscle, which express specific surface markers but lack of haematopoietic lineage markers such as CD34 or CD 45. Adult mouse MSCs in culture were reported to generate spontaneously beating cardiomyocytes(Makino et al., 1999). When MSCs are pre-exposed to 5-azacytidine they are capable of differentiating into skeletal and cardiac muscle phenotypes (Makino et al., 1999). However the reproducibility of this treatment is highly questioned.

      Recently, a number of studies have shown that bone marrow stem cells transplantation is beneficial for myocardial repair/regeneration in different animals and human being. Kamihata et al. injected autologous bone marrow-derived mononuclear cells into myocardial infarcted zone of swine(Kamihata et al., 2001). Three weeks later, regional blood flow and capillary densities were significantly higher, and cardiac function was improved. They concluded that bone marrow implantation may achieve optimal therapeutic angiogenesis through potent angiogenic ligands and cytokines secreted by those cells incorporated into foci of neovascularization. Orlic et al. reported that the injection into female mice of a subgroup of Lin-c-kit+ bone marrow cells from EGFP transgenic male donors at the borderline of an ischemic area resulted in the colonization of the infarcted area. Within 9 days, male EGFP-positive cells had proliferated in situ and expressed protein characteristic of cardiac tissue, including connexin43, thus suggesting intercellular communication (Orlic et al., 2001).

      In human, several trials are ongoing. Perin and Dohmann recently reported that transendocardial injections of autologous mononuclear bone marrow cells in patients with end-stage ischemic heart disease could safely promote neovascularization and improve perfusion and myocardial contractility. 21 patients, all of them with previous myocardial infarction and documented with multivessel disease, were enrolled sequentially, with the first 14 patients assigned to the treatment group and the last 7 patients to the control group. Approximately 4 hours before the cell injection procedure, bone marrow (50ml) was aspirated; mononuclear cells were isolated by density gradient. The electromechanical map was used to identify viable myocardium for treatment; the cells were injected by NOGA catheter in the treatment group. Both of group patients underwent 2-month noninvasive follow-up, and treated patients along underwent a 4-month invasive follow-up. At 2-months, there was a significant reduction in total reversible defect and improvement in global left ventricular function within the two groups on quantitative SPECT analysis. At 4 months, the ejection fraction improved from a baseline of 20% to 29% (p=0.003), concomitantly to a reduction in end-systolic volume (p=0.03) in the treated patients. The limitation of the study includes the small number of patients, the short period of follow-up, and no placebo as control (Perin et al., 2003). Tse et al demonstrated somewhat similar results (Tse et al., 2003). They studied percutaneous delivery (via the Biosense Electromechanical NOGA mapping catheter) of autologous bone-marrow-derived mononuclear cells in eight patients with stable angina. After 3 months of follow-up, there was improvement in myocardial perfusion, regional myocardial wall motion and thickening, but LVEF remained unchanged (Tse et al., 2003). Hamano and Stamm reported two studies with 5 and 6 patients study respectively(Hamano et al., 2001; Stamm et al., 2003), in which both of groups received autologous bone marrow cells at the time of coronary artery bypass grafting, and were followed up for 3 to 12 months. Their results showed an improvement of perfusion of the infarcted myocardium possibly due to neoangiogenesis.

      Presently, bone marrow stem cells provide an interesting and promising option to restore myocardial viability. The transplantation in human study appears feasible, relatively safe and effective, no tumor formation has been scored. However, a major criticism to these preliminary trials is the fact that the number of patients is too small to derive a meaningful efficacy and definitive safety data. Also, no data are available about cell survival following intra-myocardial needle injection, or whether implanted cells did survive and differentiate along the cardiac myocyte and/or endothelial lineage. In addition, some studies in big animals reported that injected cells are very low in number and tend to disappear with time. Further studies are required to better characterize the phenotype and the fate of injected cells.

      Moreover, the in vitro proliferation potential of MSCs is not yet reproducible in order to obtain a suitable number of differentiated and characterized cells. Clearly, cell duplication and reproduction is the first condition to fully recolonize a diseased myocardium and thus improve ventricular function. Another factor is if undifferentiated stem cells are used, a risk is still present of developing other type of tissue(Laham and Oettgen, 2003; Scorsin and Souza, 2001).

      The following three tables (Table I, II, III) are taken from a review by L. Field (Dowell et al., 2003) and represent to date an extensive list of all the cell therapy studies published so far.


Table I. Cardiomyocyte transplantation


Table II. Myoblast transplantation


Table III. Stem cell and stem cell-derived cardiomyocyte transplantation

      References in tables I-III:

      1. R. SoRelle, Myoblast transplant to heart attempted. Circulation 102 (2000), pp.E9030-9031.

      2. B.E. Strauer, M. Brehm, T. Zeus, M. Kostering, A. Hernandez, R.V. Sorg et al., Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 106 (2002), pp. 1913-1918.

      3. M.H. Soonpaa, G.Y. Koh, M.G. Klug and L.J. Field, Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science 264 (1994), pp. 98-101.

      4. G.Y. Koh, M.H. Soonpaa, M.G. Klug, H.P. Pride, B.J. Cooper, D.P. Zipes et al., Stable fetal cardiomyocyte grafts in the hearts of dystrophic mice and dogs. J Clin Invest 96 (1995), pp. 2034-2042.

      5. C.H. Van Meter, Jr., W.C. Claycomb, J.B. Delcarpio, D.M. Smith, H. deGruiter, F. Smart et al., Myoblast transplantation in the porcine model: a potential technique for myocardial repair. J Thorac Cardiovasc Surg 110 (1995), pp. 1442-1448.

      6. J. Leor, M. Patterson, M.J. Quinones, L.H. Kedes and R.A. Kloner, Transplantation of fetal myocardial tissue into the infarcted myocardium of rat. A potential method for repair of infarcted myocardium?. Circulation 94 (1996), pp. II332-336.

      7. R.K. Li, Z.Q. Jia, R.D. Weisel, D.A. Mickle, J. Zhang, M.K. Mohabeer et al., Cardiomyocyte transplantation improves heart function. Ann Thorac Surg 62 (1996), pp. 654-660 discussion pp. 660-661 .

      8. A.L. Connold, R. Frischknecht and G. Vrbova, A simple method for cardiac surgery in rats. Cell Transplant 5 (1996), pp. 405-409.

      9. S. Gojo, S. Kitamura, W.T. Germeraad, Y. Yoshida, K. Niwaya and K. Kawachi, Ex vivo gene transfer into myocardium using replication-defective retrovirus. Cell Transplant 5 (1996), pp. S81-84.

      10. A.L. Connold, R. Frischknecht, M. Dimitrakos and G. Vrbova, The survival of embryonic cardiomyocytes transplanted into damaged host rat myocardium. J Muscle Res Cell Motil 18 (1997), pp. 63-70.

      11. M. Aoki, R. Morishita, J. Higaki, A. Moriguchi, S. Hayashi, H. Matsushita et al., Survival of grafts of genetically modified cardiac myocytes transfected with FITC-labeled oligodeoxynucleotides and the -galactosidase gene in the noninfarcted area, but not the myocardial infarcted area. Gene Ther 4 (1997), pp. 120-127.

      12. S. Gojo, S. Kitamura, O. Hatano, A. Takakusu, K. Hashimoto, Y. Kanegae et al., Transplantation of genetically marked cardiac muscle cells. J Thorac Cardiovasc Surg 113 (1997), pp. 10-18.

      13. M. Scorsin, A.A. Hagege, F. Marotte, N. Mirochnik, H. Copin, M. Barnoux et al., Does transplantation of cardiomyocytes improve function of infarcted myocardium?. Circulation 96 (1997), pp. II188-193.

      14. R.K. Li, D.A. Mickle, R.D. Weisel, M.K. Mohabeer, J. Zhang, V. Rao et al., Natural history of fetal rat cardiomyocytes transplanted into adult rat myocardial scar tissue. Circulation 96 (1997), pp. II179-186 discussion 186-187 .

      15. Z.Q. Jia, D.A. Mickle, R.D. Weisel, M.K. Mohabeer, F. Merante, V. Rao et al., Transplanted cardiomyocytes survive in scar tissue and improve heart function. Transplant Proc 29 (1997), pp. 2093-2094.

      16. M. Scorsin, A.A. Hagege, I. Dolizy, F. Marotte, N. Mirochnik, H. Copin et al., Can cellular transplantation improve function in doxorubicin-induced heart failure?. Circulation 98 (1998), pp. II151-155 discussion II155-156.

      17. E. Watanabe, D.M. Smith, Jr., J.B. Delcarpio, J. Sun, F.W. Smart, C.H. Van Meter, Jr. et al., Cardiomyocyte transplantation in a porcine myocardial infarction model. Cell Transplant 7 (1998), pp. 239-246.

      18. T. Sakai, R.K. Li, R.D. Weisel, D.A. Mickle, Z.Q. Jia, S. Tomita et al., Fetal cell transplantation: a comparison of three cell types. J Thorac Cardiovasc Surg 118 (1999), pp. 715-724.

      19. H. Reinecke, M. Zhang, T. Bartosek and C.E. Murry, Survival, integration, and differentiation of cardiomyocyte grafts: a study in normal and injured rat hearts. Circulation 100 (1999), pp. 193-202.

      20. R.K. Li, Z.Q. Jia, R.D. Weisel, D.A. Mickle, A. Choi and T.M. Yau, Survival and function of bioengineered cardiac grafts. Circulation 100 (1999), pp. II63-69.

      21. J. Leor, S. Aboulafia-Etzion, A. Dar, L. Shapiro, I.M. Barbash, A. Battler et al., Bioengineered cardiac grafts: a new approach to repair the infarcted myocardium?. Circulation 102 (2000), pp. III56-61.

      22. M. Scorsin, A. Hagege, J.T. Vilquin, M. Fiszman, F. Marotte, J.L. Samuel et al., Comparison of the effects of fetal cardiomyocyte and skeletal myoblast transplantation on postinfarction left ventricular function. J Thorac Cardiovasc Surg 119 (2000), pp. 1169-1175.

      23. T.S. Li, K. Hamano, K. Kajiwara, M. Nishida, N. Zempo and K. Esato, Prolonged survival of xenograft fetal cardiomyocytes by adenovirus-mediated CTLA4-Ig expression. Transplantation 72 (2001), pp. 1983-1985.

      24. S. Etzion, A. Battler, I.M. Barbash, E. Cagnano, P. Zarin, Y. Granot et al., Influence of embryonic cardiomyocyte transplantation on the progression of heart failure in a rat model of extensive myocardial infarction. J Mol Cell Cardiol 33 (2001), pp. 1321-1330.

      25. H. Yokomuro, R.K. Li, D.A. Mickle, R.D. Weisel, S. Verma and T.M. Yau, Transplantation of cryopreserved cardiomyocytes. J Thorac Cardiovasc Surg 121 (2001), pp. 98-107.

      26. Y. Sakakibara, K. Tambara, F. Lu, T. Nishina, G. Sakaguchi, N. Nagaya et al., Combined procedure of surgical repair and cell transplantation for left ventricular aneurysm: an experimental study. Circulation 106 (2002), pp. I193-197.

      27. Y. Sakakibara, K. Tambara, F. Lu, T. Nishina, N. Nagaya, K. Nishimura et al., Cardiomyocyte transplantation does not reverse cardiac remodeling in rats with chronic myocardial infarction. Ann Thorac Surg 74 (2002), pp. 25-30.

      28. Y. Sakakibara, K. Nishimura, K. Tambara, M. Yamamoto, F. Lu, Y. Tabata et al., Prevascularization with gelatin microspheres containing basic fibroblast growth factor enhances the benefits of cardiomyocyte transplantation. J Thorac Cardiovasc Surg 124 (2002), pp. 50-56.

      29. A. Ruhparwar, J. Tebbenjohanns, M. Niehaus, M. Mengel, T. Irtel, T. Kofidis et al., Transplanted fetal cardiomyocytes as cardiac pacemaker. Eur J Cardiothorac Surg 21 (2002), pp. 853-857.

      30. W. Roell, Z.J. Lu, W. Bloch, S. Siedner, K. Tiemann, Y. Xia et al., Cellular cardiomyoplasty improves survival after myocardial injury. Circulation 105 (2002), pp. 2435-2441.

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D. Material and methods

D.1. ESC culture and differentiation

      Mouse ESC CGR8 were cultured in BHK21 medium (GIBCO BRL) supplemented with nonessential amino acids, pyruvate, mercaptoethanol, glutamine, penicillin/streptomycin, 10% fetal calf serum and LIF-conditioned medium in a humidified 5% CO2 atmosphere at 37°C and maintained at < 70% confluency to keep an undifferentiated phenotype(Meyer et al., 2000).


Figure A. Colonies of undifferentiated mouse ESC. a) D3-ES clone growing on mytomycin-inactivated mouse embryonic fibroblasts (MEFs); b) CGR8-ES clone feeder-cell independent (no need of MEFs).Culture and propagation in the presence of LIF.

      The day of the injection procedure, cells were harvested by trypsinization, washed and pelleted, then suspended at a concentration of 2x107/ml in serum-free culture medium. The differentiation of CGR8 cell was performed by the hanging drop method(Maltsev et al., 1994).


Figure B. Cardiac cell differentiation by hanging drop method, and isolated by Percoll gradient centrifugation.

      As illustrated in Figure B, EBs were formed at day 2 in hanging drops (450 cells/20ml) of differentiation medium (BHK21, as described above), containing 20% fetal calf serum and lacking of LIF. After 6 days in suspension culture EBs were plated to gelatin-coated 24-well plates or cover slips (Meyer et al., 2000).

      The beating cardiomyocytes within EBs were counted under phase-contrast microscope at day 8 of differentiation.


Figure C. Sequence of pictures illustrating the Passage from ESC to EBs in suspension then adherent EBs containing beating cardiomyocytes.

D.2. Isolation of ES-derived and neonatal mouse cardiomyocytes

      As illustrated in figure, day-8 EBs containing cardiomyocytes were detached from culture surfaces by incubating them with 0.05% trypsin-EDTA for 1 min at 37°C. Cells were dissociated with 1mg/ml collagenase (CLSII, Wortington), and 0.25% mg/ml pancreatin in a buffer containing (in mmol/L) NaCl 117, HEPES 20, NaH2PO4 1.2, KCl 5.4, Mg SO4 1, glucose 5, pH = 7.35.

      As shown in Figure D, myocytes were separated by centrifugation through a discontinuous Percoll gradient, and collected at the interface of the two layers. Neonatal cardiomyocytes were isolated from ventricles of 2 days-old mouse neonates using a similar method as previously described (Jaconi et al., 2000).


Figure D. Isolation of cardiomyocyte from day-8 EBs by enzymatic dissociation and centrifugation on a discontinuous Percoll Gradient.

D.3. Plasmid construction, stable transfection and isolation of ESC clones

      We designed a mouse ESC clone expressing GFP fused to CD63. This fusion protein allows the expression of green fluorescence associated with membrane and intracellular endosomes. CD63-GFP construct was electroporated into CGR8 cells according to the standard protocol in a Gene Pulser (BioRad) at 240v, 500µF. Stable ESC clones were propagated in the presence of LIF and selected for 10 days using G418 (250µg/ml), and sorted by FACS. In parallel, we also prepared a mouse ESC clone expressing the human Bcl2 gene. Human Bcl2 cDNA inserted into pcDNA3.1(-) at EcoRI and Hind III restriction sites. After selection of stable clones, Bcl2 could be identified by immunohistochemistry using anti-Blc2 monoclonal antibodies (Figure E).


Figure E. ESC stable clones (CGR8) containing the CD63-GFP construct and resistant to neomycin.

D.4. Rat model of myocardial infarction

      Male Sprague-Dawley rats weighing 300-350 grams were initially anesthetized with 4-5% isoflourane in an induction chamber. Following the shaving and weighting, the rat was intubated with a 14-gauge catheter, tracheal ventilation was performed at 70 cycles/min with 2.5-3.0mL tidal volume, room air supplemented with oxygen (Harvard Rodent Ventilator, Model 683, Harvard Apparatus Co, Inc). 1.5-2% isoflourane was maintained for continuous anesthesia.


Figure F. Anestesia chamber and isofluorane delivery system.

      Three electrodes were positioned to record the electrocardiographic tracing (ECG) monitor. The respiration curve was also recorded during all procedure, as indicated in the Figure G.


Figure G. ECG and ventilation setup (left) as installed during the procedure. ECG and ventilation traces recordings (right). An IV line was installed in the left femoral vein if saline infusion is needed.

      A left intercostal thoracotomy was performed under aseptic technique. The forth-intercostal space was opened carefully to avoid accidentally cutting any vessels including the internal mammary artery. The forth and fifth ribs were separated with a small retractor (Harvard Apparatus, France) to explore the heart. The pericardium was removed, the left anterior descending artery and its branch was observed under surgical microscope. A 6-0 polypropylene snare was made passing through the epicardium layer around the origin of the artery between the left atrium and the right pulmonary outflow tract, tying the ligature permanently occluded the artery (Ye et al., 1997).

      The muscle layer and skin were closed with 3-0 suture afterwards. Before the rat woke up completely, extubation was performed and the rat was places in a recovery cage with a supply of oxygen for 30 to 60 minutes.


Figure H. Panels a,b: ligature procedure. After a left intercostal thoracotomy was performed, the fourth intercostal space was opened and the ribs were separated with a small retractor in order to expose the heart. LAD was ligated by a 6-0 polypropylene snare. Panel c: Drawing illustrating that right after LAD ligature, the left ventricular anterior free wall becomes hypokinetic and clearer due to the cyanosis.

D.5. Experimental animal protocol

      We divided the rats into two groups: normal rats (non-MI, n = 24) and MI rats (n= 47). The MI rats were subdivided to acute phase (MI = 1week) and chronic phase (MI = 4 weeks). The undifferentiated murine ESC was implanted to both normal and infarcted rats. Also we implanted cardiomyocytes derived from ESC in a few rats. 1 week or 4 weeks after implantation, the rats were sacrificed and the histologic and pathologic studies were performed. On the each condition, about half of rats received immunosuppression treatment of cyclosporine A. To evaluate the transformation of left ventricular function, the echocardiograph study was performed to the rats with infarction the day before cells grafting and the day before sacrificing respectively (Fig I).


Figure I. Experimental protocol. The cell implantation was performed into two different conditions of the rats: with or without myocardial infarction. About half of rats in each condition treated with immunosuppression of CsA. Echo study was performed for left ventricular function evaluation in MI rats.

D.6. The transplantation Procedure

      Cells transplantation was performed on the normal or 1-week, 4 weeks after myocardial infarction depended on the protocol. Rats were anesthetized, and under sterile technique, the chest was re-opened. The infarcted area was identified visually on the basis of surface scar and wall motion abnormality. As shown in Figure J (left), 25 ml aliquots of cell suspension were injected by means of a 29G insulin syringe into the left ventricular anterior free wall around the scar border. 4 aliquots were injected for each rat for a total of 2x106 cells implanted. Control animals were injected with the same volume of serum free culture medium.


Figure J. Left: Cell transplantation was performed by transmyocardial injection around the infarction border with 4 times 25ml for a total number of cells between 1x106 and 2x106. Right: When required, osmotic minipump (ALZA Corporation) was implanted subcutaneously to deliver CsA continuously.

D.7. Immunosuppression treatment

      To prevent rejection and study the effect of an immunosuppression treatment on ESC fate, groups of rats received immunosupression agent cyclosporin A delivered continuously via an osmotic minipump (ALZA Corporation), as shown in Figure K.


Figure K. Rat with a subcutaneous implantation of an osmotic minipump filled with CsA.

      Alzet mini-osmotic pumps were filled with cyclosporin A (CsA) (Sandimmune, Novartis 50mg/1ml), and was kept overnight at 37°C in PBS before implantation. The CsA release was adjusted at 2.5ml/hour or 10ml/hour and pump were designed for a 7- or 28-days release. The administrated dosage of CsA was calculated as 6-9 mg/Kg/day (Sullivan et al., 2000). After hair shaving and skin cleaning at the site for incision, a hemostat was inserted into the incision to spread the subcutaneous tissue and create a prompt pocket for the pump. The filled pump was implanted subcutaneously and the wound closed with suture. All procedure was performed under sterile circumstances.

D.8. Evaluation of the left ventricular function by echocardiography

      For the evaluation of left ventricular function, transthoracic echocardiogram was performed on the rats after myocardial infarction 1 week or 4 weeks right before implantation (baseline echocardiogram), and 1 week or 4 weeks after implantation, before the sacrifice of the animals. Rats were anesthetized with 4-5% isoflurane in an induction chamber. The chest was shaved, the rats were placed in dorsal decubitus position and intubated for continuous ventilation. 1-2% isoflurane was continuously supplied via a mask(Moises et al., 2000). 3 electrodes were adhered to their paws to record the electrocardiographic tracing simultaneously with the cardiac image identifying the phase of a cardiac cycle.

      Echocardiograms were performed with a commercially available echocardiography system equipped with 7.5 MHz phased-array transducer (Philips-Hewlett-Packard). The transducer was positioned on the left anterior side of the chest (Leor, 2000 #25). At first, longitudinal images of the heart were obtained, including the left ventricle, atrium, the mitral valve and the aorta, followed by the cross-sectional images from the plane of the base to the left ventricular apical region. M-mode tracings were obtained at the level below the tip of the mitral valve leaflets at the level of the papillary muscles (Litwin et al., 1994). All of two-dimensional images, M-mode tracings and Doppler curves were recorded on videotape for later analysis. We calculated the fractional shorting (FS) as a measure of systolic function, according to the M-mode tracing from the cross-sectional view: maximal LV end-diastolic diameter (at the time of maximal cavity dimension), minimal LV end-systolic diameter (at the time of maximum anterior motion of the posterior wall), FS (%) = {(LVEDD-LVESD) / LVEDD} x 100, (Pouzet et al., 2000). All measurements were averaged for 3 consecutive cardiac cycles.

D.9. Histological and immunohistochemistry study

      Either 1 or 4 weeks after cells implantation, the rats were sacrificed with intravenous injection of potassium chloride at the level of the femoral vein, in order to stop the cardiac ventricular contraction on the diastole. Hearts were rapidly excised, the cardiac cavities were rinsed in PBS to removed blood and thrombus, then the hearts were fixed with 10% formalin for 24 hours, and immediately thereafter embedded in paraffin. Blocks were sectioned into 3mm thickness slices and stained with hematoxylin and eosin. Serial sections were immunolabeled with different antibodies against the cell markers and structural cardiac proteins. Upon cell injection, we retrieved the engrafted cells according to the markers carried by the ESC (GFP or human Bcl2), while D3-ESC, which were not genetically modified, were identified only by morphology and the proliferative marker PCNA.

D.9.1. Antibodies

      All the used antibodies are summarized in the following table :

Antibody Antigen Recognized Dilution Source
Ab6556 GFP 1:400 Abcom UK
MF-20 Sarcomere myosin 1:400 DSHB, Iowa, USA
PC10 PCNA 1:100 DAKO
CXN-6 Connexin-43 1:200 SIGMA
EA-53 a-actinin (sarcomeric) 1:200 SIGMA
M0887 BCL-2 1:100 DAKO

      Anti-Bcl-2 antibody is a monoclonal mouse antibody specific for human Bcl-2 oncoprotein (DAKO, M0887). Monoclonal anti- a-actinin (sarcomeric, clone EA-53, mouse ascites fluid, Sigma) is specific for a-skeletal muscle actinin and a-cardiac muscle actinin. It stains Z lines and dots in stress fibers of myotubes in skeletal and cardiac muscle, but not in non-sarcomeric muscle elements (connective tissue, epithelium, nerves, and smooth muscle). Monoclonal anti-PCNA (proliferating cell nuclear antigen) antibody (clone PC-10, DAKO) reacts with PCNA, a useful index of cell proliferation. Anti-myosin antibody MF 20 (DSHB, Developmental Studies Hybridoma Bank, University of Iowa, USA) is an IgG2b monoclonal antibody, which reacts with all sarcomeric myosin. Anti-connexin-43 antibody (clone cxn-6, Sigma) is a mouse IgM isotype monoclonal antibody, which reacts with the gap junction protein connexin 43 (Cx43). Anti-GFP antibody (Abcam, ab6556) is a rabbit polyclonal to GFP (affinity purified) antibody.

D.9.2. Immunohistochemistry

      For immunohistochemical studies, tissue slices were serially rehydrated in 100%, 95%, and 70% ethanol after deparaffinization with xylol. Endogenous peroxidase in the sample was blocked and the antigens were retrieved on a microwave for 15 minutes in pH=6.0, 10mM TP citrate solution. Non-specific binding were blocked with 5% BSA/PBS solution for another 15 minutes. The slices were stained with following primary antibodies at room temperature for 1 hour: anti-Bcl-2 (1:100), anti-a-actinin (1:200), anti-PCNA (1:100), anti-GFP (1:400), anti-sarcomere myosin (1:400), and anti-Cx43 (1:200). Then the slices were incubated for 30 minutes at room temperature with one of the following biotinylated secondary antibodies diluted 1:100 to 1:200 in 5% BSA/PBS: Biotinylated anti-rabbit IgG (H+L), affinity purified (VECTOR, BA-1000), Biotinylated anti-mouse IgM (mu-chain specific) affinity purified (VECTOR, BA-2020), Biotinylated anti-mouse IgG (H+L) affinity purified and rat adsorbed. This was followed by another 30 minutes incubation in avidin-biotin solution (Vectastin ABC Kit). All the incubations were preceded by through rinsing with PBS. The reaction products were visualized with 3,3'-Diaminobenzidine tetrahydrochloride Dehydrate and 3% hydrogen peroxide. Then the slices were washed with distilled water; counter staining was performed with hemotoxyline for 1 minute. Control secondary antibodies were processed simultaneously using the same protocol, except that the dilution solution was devoid of primary antibodies. The tissue was observed, photographed and digitally saved on a ZEISS Axioskop 2 Plus photomicroscope.

D.10. Statistics Analysis

      All values are presented as means ± S.E. Difference between two groups was compared by unpaired Student's t-test. A level of p < 0.05 was considered as significant.

E. Results

E.1. Grafting undifferentiated ESC in normal (non-infarcted) heart

      We first established optimal conditions for the ESC implantation into the ventricle wall of normal rat hearts (n=24). We inspected the fate of the cells 1 week or 4 weeks later. 10/24 rats were treated with CsA. We tested different kind of ESC lines, such as the feeder-cell dependent cell lines D3 and feeder cell-independent cell line CGR8 (wild type of containing Bcl2 or GFP marker). As depicted in Figure 1, H&E staining revealed an consistent granulation tissue in the control rats while 33% (1/3) of the CsA-treated animals displayed, in addition to granulation, the growth of teratoma after 1 week. This percentage increased to 100% (6/6) after 4 weeks. At this time point, the tumors were highly proliferative and some even bigger that the ventricle diameter, as indicated by the positive PCNA staining (see Fig.2b and Fig. 3b). The ESC marker Bcl-2 (as well as GFP, not shown) was also strongly positive in the teratoma area (see Figure 2c for Bcl2-positive tumors), indicating the ESC derivation. In contrast, in the absence of immunosuppression, no teratoma was found even after 4 weeks (0/13) (Fig. 2d and Fig. 3 c) and no cell proliferation or granulation was still present at this later time (Fig 2e and Fig. 3d).

      The tumors were identified as teratoma with characteristics of tissues originating from the ectoderm (skin), mesoderm (cartilage, muscle) and endoderm (epithelium of bronchi and of pancreas) (see Figure 4).

E.2. Rat model of myocardial infarction (MI)

      Large, reproducible lesions were induced in rat heart by coronary artery ligation, as illustrated in Figure 5. In the left panel, the explanted heart, 4 weeks after the coronary ligature presented a large infarcted area (dotted line) with a collapsed left ventricular wall due to the extremely reduced thickness. Before sacrifice, the infarcted area was clearly identifiable by echocardiography as shown in the right panel of Figure 5. Using a parasternal short axis view it was possible to estimate the akinetic territory going from 12 to 4 o'clock, as indicated by the dotted line. In this example, the impairment of the left ventricular ejection fraction (LVEF) was visually estimated at 25%.


panel: macroscopical view of the explanted heart. Right panel: echocardiographic picture of the same heart using a parasternal short axis view.

      Figure 6 illustrates heart sections produced from normal rats (panels a,b,c), acute MI rats (1 week, panels d,e,f) and chronic MI rats (4 weeks, panels g,h,i). Upon H&E staining, an acute progressive tissue inflammation was observed after 1 week of MI (Fig 6d,e,f), as compared with normal heart (Fig. 6a,b,c). One could observe neutrophilic infiltration, fragmentation and smudging of the muscle fibers and coagulative necrosis (Fig.6 e, f). 4 weeks from the MI, almost no granulation tissue was seen; instead collagen deposition and myocardial scar were present.

      As displayed in Figure 7, we first compared normal heart with infarcted hearts 1 week after MI. At this time point, one could see an acute and progressive tissue inflammation on left ventricular wall. Pericarditis was present. The tissue pathology of heart sections revealed by H&E staining or by immunohistochemistry indicated i) a strong cell proliferation in the infarcted area (increased PCNA staining over control), ii) disorganized cardiac fibers with an abnormal a-actinin and MHC distribution in the sarcomeres (fuzzy staining instead of z-lines and A-bands respectively), iii) partial loss of Cx43 at the gap junctions between fibers.

      The same type of abnormal pathology but exacerbated was observed after 4 weeks. At this time the ventricular wall become very thin and completely fibrotic (Figure 5, left panel and Figure 6 g, h, i). Only scar tissue was visible with non contracting, dead wavy fibers.

E.3. Engraftment of undifferentiated ESC in acute phase of MI (1 week)

      Dead cells or infiltrated leukocytes releases lysosome content, which result in further cellular autolysis or heterolysis, followed by coagulated necrosis. Enzymatic digestion of cells and protein denaturation is then removed by phagocytosis. We engrafted undifferentiated ESC at this time point to study the influence of the inflammation on cell proliferation and differentiation. In fact lot of cytokines are released by inflammatory cells, which are though to modify substantially cell fate as well as tissue repair.

      We compared 20 rats implanted with undifferentiated ESC and distributed in 2 groups, each comprising animals treated and non-treated with CsA, as summarized by the following scheme:


Figure 6 : [Illustration non disponible]

      In MI rats after 1 week post-implantation the histology indicated an inflammatory reaction still underway. Infiltrated mononuclear cells together with ingrowth fibroblasts and capillaries formed a granulation tissue in the infarcted area, similarly to control conditions showed in Figure 6 and 7 (MI but no cells engrafted). Animals injected with buffer only displayed a tissue morphopathology identical to non injected animals at same stages (n=4).

      In contrast, 40% (2/5) of the rats treated with CsA formed a tumor after 1 week, while 100% (8/8) presented a highly proliferating teratoma after 4 weeks (strong PCNA staining), respectively. Figure 8 shows the heart sections corresponding to this last condition (i.e. T=4 weeks). Strikingly, in contrast to normal rats (no MI), some areas of the teratoma contained cells positive for MHC, a-actinin and Cx43, indicating a partial differentiation of ESC into muscle cells and fibers.

      As illustrated in Figure 9, 4 weeks after implantation, only a scar tissue was visible in the absence of CsA and no teratoma was observed. Moreover no engrafted could be identified: both Bcl-2 and GFP staining were negative. Also, all types of the cardiomyocytes specific staining, cardiac muscle gap protein (Cx43), sarcomere myosin (MF20), sarcomere actinin (a-actinin) were negative on the area of MI (Fig. 9), which suggest that without CsA, no cardiomyocyte differentiation in MI area could be identified.

E.4. Grafting of undifferentiated ESC in chronic phase of MI (4 weeks)

      Histology evaluation of the infarcted area after 4 weeks ligation confirmed the presence of transmural fibrous tissue at the apex of left ventricle (total rats = 18). Comparing with infarcted area after 1-week ligation, pathologic changes after 4-weeks ligation comprised a fibrotic repairing tissue instead of necrosis. There was no more granulation tissue, but a rich vascular network and collagen deposition. Left ventricular cavity is more dilated globular (Figure 6). The same type of morphology was observed in CsA-immunosuppressed rats injected with undifferentiated ESC. 20% (1/5) of these animals developed a tumoral mass 1 week later, a percentage reaching 100% (3/3) after 4 weeks. These masses contained tissue from the three different germ layers, typical of teratoma. They were positive for the human Bcl2 marker, confirming their origin from injected ESC. Animals injected with culture medium or injected with ESC but without CsA-immunosuppression did not present any teratoma or identifiable ESC in the grafting area. Bcl-2 and PCNA staining were both negative.

E. 5. Grafting of ESC-derived CMC in MI rats.

      CMC was derived from Bcl2-ESC after Percoll gradient isolation of beating EBs at day 8, were injected into 1week MI rats (n=5). All the rats received CsA and were sacrificed for the pathological examination 4 weeks later. Figure 10 shows that in 2 out of 5 animal hearts, implanted CMC formed aggregates with positive staining for Bcl2 and PCNA. Strikingly, these aggregates resulted positive for MHC and a-actinin Figure 11A. However, 3 out of 5 did not show any teratoma formation, as reported in Figure 11B. In those cases, there was no positive staining for PCNA or MHC-positive ES-derived cells.


Figure 11. Immunohistochemistry analysis of MI rat hearts implanted with ES-derived cardiomyocytes 4 weeks after coronary ligature. A) Teratoma formation with extensive positive labeling for MHC and a-actinin. B) Same conditions as in A, but in 3 out of 5, no teratoma was found.

E.6. Effect of time and immunosuppression on teratoma formation

      In our study however, independently of the type of cardiac injury (acute or chronic MI), the injected ESC had the tendency of differentiate to teratoma in vivo, displaying different tissue types but myocytes. The teratoma formation was exclusively dependent on the immunosuppression treatment and the length of transplantation. Figure 12 summarizes the data on the frequency of teratoma formation as a function of time (weeks of transplantation), normal rats being compared to MI 1 week and 4 weeks. Clearly, in all of these three of conditions, non-immunosuppressed rats never developed teratoma, indicating an active removal of non-autologous cells from the immune system.


Figure 12. Effect of time and immunosuppression on the occurrence of teratoma. Normal rats were compared to MI rats (1 week and 4 weeks). After transplantation of undifferentiated ESC, teratoma formation was dependent on the administration of CsA as well as on the duration of cell implantation, but not the type of ESC, nor on the presence of a cardiac injury.

E.7. The evaluation of left ventricular function by echocardiogram

      The echocardiogram was performed before and after ESC transplantation. Cardiac parameters were collected. Figure 16 show the two-dimensional imaging and M-mode tracing of echocardiogram. Cardiac function of MI rat was much worse compared with non-MI rat (control), which was identified by the echocardiograph: segmental abnormality of LV wall motion, global decrease of LV contractility, a change in shape of LV cavity. When tumor was formed (which may growth towards or outwards LV chamber), it could be identified from both 2-D and M-mode imaging, but it will influence on the judgment of the LV wall motion and LV function (Figure 13).

      Cardiac function was assessed according to the value measured on the M-mode tracing.

      Table 1 showed the difference between the cardiac parameters such as: heart rate, (LVEDDafterT - LVEDDbeforeT), (LVESDafter T - LVESDafter T), and (FSafter T - FSafter T).

      When ESC were transplanted into acute or chronic phase of MI (1 week or 4 weeks) under immunosuppression, comparison of delta values between those groups measured 4 weeks after transplantation did not give any significant difference.

      Ischemia-induced inflammation present at an early MI stage (up to 1 week) did not influence heart functions.

      Table 1. Comparison MI=1 week vs. MI=4weeks (delta of cardiac parameters (afterT - before T)), in the presence of CsA. Effect 4 weeks after cell transplantation (n=11).

  No CsA with CsA P
Heart rate (bpm) 11 ± 45 29 ± 13 0.396
LVEED (mm) 3.7 ± 2.6 2.2 ± 1.3 0.497
LVESD (mm) 2.2 ± 1.5 11.4 ± 0.9 0.610
FS (%) 3.3 ± 1.9 2.3 ± 4.4 1.000

      Also in our study, immunosuppression treatment did not affect the changes of cardiac parameters (table 2). Under the same condition, i.e. 1 week after LAD ligature, ESC transplantation for a duration of 4 weeks, there was no significant difference on the cardiac delta value) whether CsA was administrated or not.

      Table 2. Effect of CsA treatment in MI=1 week (delta of parameters), 4 weeks after transplantation (n=12).

Figure 13 : [Illustration non disponible]
  No CsA with CsA P
Heart rate (bpm) 11 ± 45 29 ± 13 0.396
LVEED (mm) 3.7 ± 2.6 2.2 ± 1.3 0.497
LVESD (mm) 2.2 ± 1.5 11.4 ± 0.9 0.610
FS (%) 3.3 ± 1.9 2.3 ± 4.4 1.000

      We could not finally conclude with the data, because of the presence of teratoma caused to many artifacts to the measurement of the different parameters.

F. Discussion

      The major aim of this study was to investigate whether an infarcted heart could instruct undifferentiated mouse ESC to acquire a cardiac phenotype in situ in order to improve cardiac function in a rat model of myocardial infarction in the rat. We studied different type of murine ESC lines engrafted into rat model of myocardial infarction. The main findings of this study indicate that:

  1. Undifferentiated murine ESC implanted into normal rat hearts developed into teratoma if the animals were immunosuppressed with CsA. The morbidity increased with time from 20% to 100% at 4 weeks post-implantation. In contrast, in the absence of immunosuppression, an extended granulation tissue was first observed and, thereafter, injected ESC could not be retrieved, being most probably suppressed by the immune system.
  2. The teratoma derived from the implanted ESC since those were positive for Bcl2 and GFP ESC markers. They were not related to the type of ESC lines used, since different cell lines, e.g., feeder cell-dependent D3 and feeder cell-independent CGR8-Bcl2 and CGR8-EFFP gave similar results.
  3. The type of cardiac injury (acute or chronic myocardial infarction) did not influence the teratoma formation. Both infarcted and non-infarcted hearts could develop teratoma after grafting of undifferentiated ESC when accompanied with immunosuppression.
  4. When ESC-derived CMC were implanted in MI hearts we observed cells positive for cardiac markers but not organized into mature fibers. The growth of other tissue types was also observed.
  5. Left ventricular functions were not modified by the transplantation of undifferentiated ESC or ESC-derived CMC. Because of teratoma formation, it was impossible to observe a reliable change (either improvement or worsening) of the cardiac functions.

      That undifferentiated ESC have the ability to differentiate to all types of tissue upon implantation it is a notion well established. However, the possibility to commit to the right phenotype depending to which niche or organ they are exposed, this is definitely a new and attractive concept and deserves further exploration. As first reported by Puceat et al, the engraftment of murine ESC into chronic infarcted rat hearts leaded to their differentiation into cardiomyocytes without the need of immunosuppression. This work proposed that the heart might exert a TGFb-mediated paracrine action on the implanted cells, especially under conditions in which the heart undergoes a lesion such as MI. Under these conditions in fact, an increased production of TGFb has been reported in the literature. This could favor the mesoderm layer development within ESC and commit them to cardiomyocytes. In addition, Puceat et al demonstrated in vitro that ESC treated with TGFb expressed increased amounts of cardiac transcription factors, and more cells acquired a cardiac phenotype(Behfar et al., 2002). It is therefore likely that cardiac injury could induce cardiac repair and thus cardiomyocyte differentiation through TGFb signaling.

      Upon injury, several organs are able to release factors that activate repairing mechanisms and induce resident stem cells, partially committed but not fully differentiated, to further progress into their fate and replace damaged or dead cells with new units. Well-known examples are the bone marrow, the skin, the liver and the skeletal muscle. This does not seem to be the case of the brain that, despite possessing a population of neural stem cells, does not seem to be able to activate enough brain stem cells upon a major injury or cell loss (ex. brain stroke, Parkinson's or Alzheimer's diseases) (Cao et al., 2002). It is therefore conceivable that an healthy organ may not be capable to locally release cues able to prime the implanted cells, while a damaged tissue may be activated to release important molecules for both the recruitment of resident adult stem cells when available, or distant stem cells from other compartments (i.e. the bone marrow). The identification of those factors will therefore be fundamental to optimally initiate in vitro a suited differentiation that could pursued in situ. One could also envisage to genetically inducing the implanted cells to transiently secrete factors favoring a neovascularization, for example via the insertion of VEGF (vascular endothelial growth factor) transgene under the control of an inducible promoter. This would allow an optimal integration in the recipient organ an avoid long-term deleterious effects of an uncontrolled long-term secretion(Springer et al., 1998; Suzuki et al., 2001; Yang et al., 2002).

      The timing of cell transplantation post-injury is like to play an important role because it related to donor cell survival, homing and/or post transplantation proliferation. At early stages of MI, the vascular and cellular responses of both acute and chronic inflammation reaction are mediated by chemical factors derived from plasma or cells and triggered by inflammatory stimuli such as cytokines. This should possibly affect donor cell survival. At later stages of MI, tissue repairing had occurred; it was characterized by the replacement of injured/dead cells by connective tissue, fibrosis, thus leaving a permanent scar. Hence, this situation may reduce the positive effect of cell therapy. In our study, we compared ESC implanted at both of stages (acute and chronic phase), and the results did not provide differences in cell survival or morphology between different implantation timings.

      A key point is surely the need of an immunosuppression treatment when mouse-to-rat transplantation is envisaged. In our study, we used CsA as immunosuppressive agent, continuously delivered by osmotic minipump, to avoid host versus donor graft rejection.

      In our experimental conditions, we could not reproduce the data from Puceat et al, which did not observe any ESC rejection by the host in the absence of immunosuppression. In contrast, we showed that the cells did not survive if the immune system was not depressed by CsA. Soon or later the engrafted cells turned into tumors, as compared to control conditions.

      Only one study reported the occurrence of teratoma when undifferentiated murine ESC were directly injected into the knee joint or the subcutaneous space on the back of SCID mice(Wakitani et al., 2003). The tumors grew and destroyed the joints.

      Impede the rejection of the implanted cells by the recipient remains a barrier to be solved. In fact, immunosuppressive drugs are associated with many highly unpleasant side effects and such a treatment would not represent an optimally acceptable option. Interestingly, ESC seems to express less immune-related cell surface proteins (e.g., class I products of the major histocompatibility complex) (O'Shea, 1999). The group of Benvenisty et al. (Drukker et al., 2002) addressed the graft rejection issue of cells derived from hESC by showing that both undifferentiated or differentiated hESC express no MHC-II proteins or HLA-G and very low levels of MHC class I (MHC-I) proteins on their surface. MHC-I molecules, however, may be dramatically and rapidly induced by treating the cells with interferons. If a similar phenomenon will be observed after transplantation, allogeneic human ESC might be rejected by cytotoxic T lymphocytes.

      It is likely that the problem of rejection of grafted human ES-derived cells could be overcome (or at least minimized) by establishing 'histocompatibility banks' of hESC with completely HLA-typed ESC clones derived using Good Manufacture Practice (GMP) protocols. Ideally, if large numbers of cell lines from genetically diverse populations can be maintained, this would provide isotype-matching cells for virtually any patient. Other possibilities include means of reducing or abolishing cell immunogenicity. ESC, unlike adult cells, can be easily modified genetically by, for example, inserting immunosuppressive molecules such as Fas ligand, or removing immunoactive proteins such as B7 antigens (Walker et al., 1997). Alternatively, one could delete the foreign major histocompatibility complex (MHC) genes or insert genes coding for the recipient's MHC(Westphal and Leder, 1997). Ultimately, neoplastic growth or immunopathology could be suppressed by introducing into ESC before implantation suicide genes that permit their ablation in case of misbehavior. For example, herpes thymidine kinase sensitizes mouse ESC to destruction by the guanosine analog gancyclovir (Fareed and Moolten, 2002).

      Total immunocompatability of tissue engineered from human ESC (Cibelli et al., 2002; Lanza et al., 1999a; Lanza et al., 1999b; Solter and Gearhart, 1999) could be theoretically obtained by somatic nuclear transfer (also defined as therapeutical cloning). This procedure uses the transfer of a somatic cell nucleus from an individual into an enucleated oocyte(Wilmut et al., 1997; Wilmut et al., 1998). Such an oocyte would then undergo embryonic development to the blastocyst stage prior to the isolation from the inner cell mass of hESC that would be genetically matched to the tissues of the nucleus donor. So far, one group has claimed the derivation by nuclear transfer of a human embryo up to a six-cell stage only(Cibelli et al., 2002), but the success of this result is still questioned. Clearly, this procedure of somatic nuclear transfer is still highly problematic from an ethical and practical point of view.

      The implantation of pure population of cells correctly committed to the suitable phenotype, i.e. the cardiac one, seems to be a critical issue that remain to be solved.

      Up to date, several studies have reported ESC-derived CMC engrafted into host myocardium.

      Li et al (1996) has reported successfully transplanted cardiomyocytes isolated from fetal rat heart into cryoinjured myocardial scar tissue, and improved heart function. Min et al (Min et al., 2002) showed that the implantation in rats of clusters of beating mouse cardiomyocytes dissected from EBs and labelled with GFP within 30 minutes after induction of myocardial infarction resulted in stable intramyocardial grafts and improve cardiac function in postinfarcted failing hearts. These authors did not discuss about immunoreactivity, rejection and teratoma formation, although some area of undifferentiated cells were observed in the graft. We performed the same type of experiments and we observed that engrafted CMC clusters neither terminally do not differentiate, nor they organized into connected myofibers.

G. Conclusions

      The clinical application of cell therapy in CHF requires a source of grafting cells providing the following properties: i) the possibility to derive them easily; ii) the availability to propagate to such a big mount of cell number; iii) the possibility to survive in the micro-circumstance of the heart and to integrate with the host cell; vi) finally it should restore and improve the function of a damaged heart.

      Successful use of any stem cell-based therapy will eventually depend on our ability to isolate specific cell types in large numbers that will differentiate to a fully functional state, as well as on the challenging demonstration of their in vivo function. To this end, it is crucial to pursue basic research on human ESC biology to ensure better understanding of basic principles, what genes and proteins are essential for hES developmental progression. This comprehension does not only specifically involve the transplantation of ESC-derived cells but will also help the development of new therapeutic strategies to improve the transdifferentiation and expansion of adult stem cells such as umbilical cord blood cells or bone marrow cells. Many hurdles (not only technical but also ethical) have to be cleared before the research reaches a point where clinical trials can begin.

      Although ESC would bring several advantages for cell therapy strategies due to their great differentiation potential, availability and expansion capacity in culture, a great attention has to be paid to problems of growth control. The development of teratoma has to be avoided before to envisage their use for transplantation. The use of ESC as a resource of cell therapy of CHF remain a promising idea, but there is still a long way to go from basic research to the clinic application, carefully attention must be paid to avoid deleterious effects.

H. Implementation and future experiments

      The present goals are to:

  1. Improve the in vitro differentiation of ESC into cardiomyocytes using specific growth factors.
  2. Optimize the methods for the isolation of ESC-derived cardiomyocytes in order to obtain a population as pure as possible and avoid contamination with undifferentiated cells or cells committed to other lineages.
  3. Develop new methods of cell engraftment. In particular we aim at the production of 3D-cardiac tissue in vitro containing cardiomyocytes. Those would probably result more appropriate in order to achieve a better engraftment.
  4. To develop imaging tools such as magnetic resonance imaging (MRI) in order to monitor the implanted cells in vivo after a labeling with nanoparticles or iron oxide. MRI will allow to follow cell fate in vivo and measure cardiac functions in a non invasive way.

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