Bone Marrow Stem Cell Treatment for Myocardial Regeneration

Cell therapy for myocardial regeneration is an exciting new field of medical research that has the potential to revolutionize cardiovascular medicine. Despite significant improvements in emergency treatment, myocardial infarction leads to a net loss of contractile tissue in many patients with coronary artery disease. Often, this is the beginning of a downward spiral towards congestive heart failure and life-threatening arrhythmia. Other than heart transplantation with its obvious limitations, current therapeutic means aim at preventing further episodes of myocardial ischemia and at enabling the organism to survive with a heart that is working at a fraction of its original capacity. They are far from representing a cure. In this situation, it is understandable that cardiac stem cell therapy attracts considerable attention and raises many hopes. In order to adequately judge both the potential benefits and the limitations of cardiac cell therapy, some understanding of the mechanism and the consequences of myocardial infarction and its current treatment concepts is needed.

In the setting of acute myocardial infarction, several studies have shown a functional benefit of intracoronary infusion of bone marrow cells compared with the standard treatment alone,^1-3 but patients with chronic ischemic heart disease and impaired heart function may require a different approach. Therefore, our group developed a protocol for injection of purified CD133⁺ bone marrow stem cells directly into the diseased myocardium at the time of coronary artery bypass surgery. Based on the encouraging results in the first 6 patients⁴, we completed a dose-escalation safety trial and then conducted a controlled study to determine efficacy compared with the standard CABG operation. Figure 1 depicts current strategies of bone marrow stem cell transplantation in the heart.

Bone Marrow Cells and Angiogenesis

During embryonic development the primary vascular plexus is formed by hemangioblasts, stem cells capable of generating both hematopoietic progeny and endothelial cells, in a process termed vasculogenesis. Further blood vessels are generated by both sprouting and non-sprouting angiogenesis, finally leading to the complex functional adult circulatory system⁵. Until recently only two mechanisms of postembryonic vascular remodelling have been recognized. Angiogenesis, the proliferative outgrowth of local capillaries, is one way to reinforce perfusion. Angiogenesis can occur under various conditions, including ischemia. In case of myocardial ischemia due to the occlusion of a coronary artery, preexisting small collateral vessels also bear the capacity to enlarge in a process termed arteriogenesis. It has long been assumed that both mechanisms are mainly due to local proliferation of resident cells.

The advent of cellular therapy of ischemic organ damage has introduced neo-angiogenesis (sometimes also termed vasculogenesis) due to immigrating stem cells and progenitors as a third possible mechanism operative to improved perfusion of the adult damaged heart. Accumulating evidence indicates that immigrating (stem) cells can truly differentiate along endothelial lineage but also provide paracrine support in these three courses of action during regenerative vascular remodelling.^6-8

Putative progenitors for therapeutic angiogenesis have been isolated from adult human peripheral blood based on their expression of CD34, a marker molecule shared by microvascular endothelial cells and hematopoietic stem cells.⁹ The same group provided the proof of concept by transplantation of genetically marked mouse bone marrow into recipient mice that were subsequently subjected to five distinct models of vascular remodelling including myocardial ischemia.¹⁰ In this particular system, transgenic mice constitutively expressing beta-galactosidase under the transcriptional regulation of an endothelial cell-specific promoter were used as donors to replace the bone marrow in the recipient animals. Definitively bone marrow-derived endothelial (progenitor) cells were found in reproductive organ tissues as well as in healing cutaneous wounds one week after punch biopsy. Marrow-derived endothelial progenitor cells were found to incorporate into capillaries among skeletal myocytes in an additional test for peripheral post-ischemic regeneration after hindlimb ischemia, as well as into foci of neovascularization at the border of an infarct after permanent ligation of the anterior descending artery.¹⁰ Most importantly, direct injection of the bone marrow mononuclear cell fraction in rat models of myocardial ischemia increased the capillary density.^11,12 Analysis of the effects of blood and bone marrow mononuclear cell implantation into ischemic myocardium in pigs further revealed that the stem cell effects are not limited to angiogenesis and improved collateral perfusion, but also include the supply of regulatory cytokines.^13,14 However, concerns exist regarding limited efficiency owing to the minute numbers of SC in small sample volumes of non-enriched blood and BM that are delivered intra-myocardially and the risk of foreign tissue differentiation following local stroma cell injections. Kocher et al.¹⁵ circumvented this problem by using positively selected CD34⁺/133⁺ cells from human donors after stem cell mobilization with G-CSF for intravenous injection after permanent ligation of the left anterior descending coronary artery in nude rats, resulting in a five fold increase in the number of capillaries compared to control.

As a result of the stem cell mediated angiogenesis, which was attributed to the content of marrow-derived angioblasts, the authors also found an approximately 20% increase of left ventricular ejection fraction and cardiac index together with a reduced severity of ventricular remodeling in human CD34-treated compared to control ischemic animals.¹⁵

Another candidate cell population for the regeneration of ischemic cardiac muscle and vascular endothelium are CD45+ hematopoietic CD34^LOW/- /c-kit⁺, so called side population stem cells with a specific Hoechst 33342 DNA dye efflux pattern.^16,17 Orlic et al.¹⁸ used an alternative method to enrich putative regenerative stem cells for local application by depleting unwanted cell lineages prior to enrichment for the expression of the stem cell factor receptor c-Kit from murine bone marrow. Thus concentrated cells, considered to represent hematopoietic stem cells, were observed to incorporate not only into vascular structures but dominantly led to myocardial regeneration.¹⁸ Subsequent experiments by this group employed mobilization of stem cells by G-CSF prior to experimental myocardial infarction, which also led to a significant increase in vascular density within the scar, a reduction in mortality, and a significant reduction in infarct size.¹⁹

Although the evidence that angiogenesis occurs in ischemic myocardium is convincing, this new therapeutic option also has a potential for serious side effects.²⁰ Most importantly, bone marrow-derived endothelial cells were found as part of the tumor neo-vasculature in experimental colon cancer.¹⁰ This finding might suggest a risk to trigger the growth of silent tumors by systemic use of pro-angiogenic stem cell therapy.

Bone Marow Cells and Myogenesis

While the pro-angiogenic effect of marrow-derived stem cells appears to be well established, stem cell mediated myogenesis remains a matter of debate. The traditional view implies that ischemic damage to the myocardium can only be compensated by hypertrophy, not hyperplasia, of surrounding cardiomyocytes. This dogma has recently been challenged, and intra-myocardial as well as extra-myocardial sources of regenerating contractile cells have been suggested.²¹ Cardiomyocyte proliferation has been described, although only with minute frequency.^22,23 Furthermore, the existence of cardiomyocytes of non-cardiac origin has been suggested by chimerism analyses after transplantation,^24-26 but the biologic relevance of some of these data has been questioned.^27,28

The notion that bone marrow cells can regenerate infarcted myocardium led to great excitement. In their landmark paper, Orlic et al.²⁹ described that injection of genetically labeled murine Lin^NEG/c-kit⁺ stem cells (isolated from mouse bone marrow by depletion of committed cells, and further enriched for expression of c-Kit) led to the formation of new myocardium, occupying two thirds of the infarct region within 9 days. This paper initiated a wave of enthusiasm, but also critical discussion. The data were interpreted to indicate trans-differentiation of adult hematopoietic stem cell by crossing lineage boundaries.³⁰ However, the fact that cells are derived from bone marrow does not necessarily proof that they are hematopoietic in origin, especially in the light of growing knowledge about mesenchymal, non-hematopoietic stem cells within the marrow. The recognition of cell fusion as a common phenomenon in some artificial transplant models for regeneration of ischemic tissue has added to the controversy.^31,32 From the clinicians point of view this was no surprise, since cell fusion is an intrinsic characteristic of contractile cells. Multinucleated skeletal myotubes are a classic example of cell fusion, and cardiomyocytes have long been known to form a large syncytial union.

More serious concerns were produced by two publications, which could not reproduce the promising in vivo trans-differentiation data. Using a modified Lin⁺ depletion protocol for stem cell enrichment in an otherwise similar myocardial ischemia model, Balsam et al.³³ found abundant GFP+ cells in the myocardium after 10 days, which nearly disappeared until day 30. The remaining donor cells lacked cardiac tissue-specific markers, and instead adopted only hematopoietic fates as indicated by the expression of CD45. Murry et al.³⁴ used both cardiomyocyte-restricted and ubiquitously expressed reporter transgenes to follow murine Lin^NEG/c-kit⁺ stem cells after transplantation into healthy and injured mouse hearts, and could not find evidence for relevant differentiation into cardiomyocytes. In defense of the initial paper some have argued that i) the cell isolation protocols were not completely identical, and ii) both groups nevertheless observed some functional improvement in cell-treated hearts. However, it can not been denied that the evidence for myogenesis based on hematopoietic adult stem cells myogenesis is extremely controversial.^21,35-37 Very recently, a direct side-by-side comparison of human CD133⁺ bone marrow cells and human skeletal myoblasts in a myocardial ischemia model in immunoincompetent rats demonstrated similar functional improvement in both groups, although only the myoblasts reached robust engraftment.

Our own studies underline the angiogenic capacity of CD133⁺ stem cells from adult human bone marrow and cord blood in a Scid-mouse myocardial infarction model³⁸. Moreover both cell preparations had beneficial effect on postinfarction mortality and apoptosis. Only adult bone marrow preparations contained a higher c-kit population and caused cardiac functional restoration in echocardiography. These findings underscore our limited understanding of how stem cells can elicit an improvement of heart function.

In contrast, the myogenic potential of stroma cell-derived mesenchymal stem cells is much better documented. Stroma cells are usually isolated based on their ability to adhere to plastic, not by selection for expression of certain surface markers. Their number in primary marrow aspirates is low, but they readily multiply for numerous cycles in culture, without apparent genotypic and phenotypic changes. Several years ago, Wakitani et al.³⁹ reported the in vitro development of myogenic cells from rat bone marrow mesenchymal stem cells exposed to the DNA-demethylating agent 5-azacytidine, and Makino et al.⁴⁰ isolated a cardiomyogenic cell line from murine bone marrow stromal cells that were treated with 5-azacytidine and screened for spontaneous beating. Those cells connected with adjoining cells, formed myotube-like structures, and beat spontaneously and synchronously. They expressed various cardiomyocytes-specific proteins, had a cardiomyocyte-like ultrastructure, and generated several types of sinus node-like and ventricular cell-like action potentials. When isogenic marrow stromal cells are implanted in rat hearts, they appear to become integrated in cardiac myofibers, assume the histologic phenotype of cardiomyocytes, express connexins, and form gap junctions with native cardiomyocytes.^41,42 Again, epigenetic modification with 5-azacytidine is believed to facilitate differentiation towards a cardiomyocyte phenotype in vivo.⁴³ Human mesenchymal stem cells derived from the marrow of volunteers have also been injected in hearts of immunodeficient mice, and again it was observed that they assume cardiomyocyte morphology and express various cardiomyocyte-specific proteins.⁴⁴

Under different cultivation conditions, mesenchymal stem cells readily assume an osteoblast, chondrocyte, or adipocyte phenotype. In fact, preclinical research on regeneration of skeletal components is much more advanced than that on cardiovascular applications. It is therefore no surprise that, when unmodified mesenchymal stem cells are implanted the heart, they may form islets resembling chondrogenic or osteogenic tissue.

To date, there is very little, if any, information on stroma cell surface markers that might be helpful in identifying subpopulations with a particular potential for myogenic differentiation. It is therefore still unclear whether unmodified stroma cells that were expanded in vitro following simple isolation by plastic adherence will ultimately be useful in clinical protocols, whether a certain pro-myogenic subpopulation will be identified, or whether epigenetic re-programming prior to implantation will be necessary for functionally relevant myocardial regeneration in humans.

Combination of (Stem) Cell Treatment with CABG Surgery

CABG patients were among the first to be included in clinical trails of cell therapy for myocardial regeneration. The most obvious reason is that the infarcted myocardium can be readily accessed during the operation, a unique opportunity to delivery cells in the centre or the border zone of the infarcted tissue by rather simple means.

Bone Marrow Mononuclear Cells

Probably the simplest approach to myocardial cell therapy in the clinical setting is the transfer of bone marrow mononuclear cells into the myocardium. The proponents of this approach argue that by using unmodified marrow or unselected mononuclear cells, the 'ideal' cell for myocardial regeneration, which has not yet been identified, is not lost during the preparation process. Conversely, opponents argue that the vast majority of the bone marrow mononuclear cells are blood cells of all lineages and their immediate progenitors, while only few cells formally meet the stem cell criteria. Whether the local concentration of relevant stem- or progenitor cells will surpass the hypothetical threshold for induction of regeneration processes remains unclear. Indubitably, marrow mononuclear cells can be easily collected and prepared during a standard CABG operation, which is an obvious and important logistic advantage.

The first such report came from Yamaguchi University, Japan. Hamano and colleagues described 5 patients who underwent CABG simultaneously bone marrow collection from the iliac crest.⁴⁵ The mononuclear cell fraction was prepared using a commercially available apheresis system, and between 5 and 22 injections of 5x10⁷ to 1x10⁸ cells were performed in the ischemic myocardium that was not directly revascularized by bypass grafting. In 3 of those 5 patients, improved perfusion of the cell-treated tissue was noted postoperatively. No complications such as arrhythmia or local calcification were noted, but no statement was made with respect to LV function.

In a similar trial, Galinanes and colleagues from Leicester University, UK, collected marrow by sternal bone aspirate at the time of CABG surgery.⁴⁶ This was diluted with autologous serum and injected into LV scar tissue. Postoperatively, regional contractility in LV wall segments that did or did not receive marrow cells was assessed by dobutamine stress echocardiography, and only the segmental wall motion score of the areas injected with bone marrow and receiving a bypass graft in combination improved upon dobutamine stress. Most likely, many more patients have been subjected to similar treatment protocols elsewhere, but very little or no information as to the functional outcome is available. Most importantly, no controlled trial has so far demonstrated the superiority of CABG and mononuclear cell injection over CABG alone.

Bone marrow stem cells: Our own group has focused on the intramyocardial injection of purified hematopoietic bone marrow stem cells since 2001.⁴⁷ We chose not to simply inject an unmodified mononuclear bone marrow cell suspension, because the large number of leukocytes and their progenitors may primarily induce local inflammation, rendering the actual stem cell effects insignificant. Instead, we prepare a purified stem cell suspension using clinically approved methods. Two monoclonal antibodies are currently available for clinical selection of bone marrow stem cells, anti-CD34 and anti- CD133. Approximately 60-70% of the CD34⁺ bone marrow cells co-express the CD133 antigen, and 70- 80% of the CD133⁺ cells are CD34⁺ as well. The CD133⁺ bone marrow cell population contains a small proportion of clonogenic cells, which have a very high potential to induce neoangiogenesis.⁴⁸ Furthermore, there is accumulating evidence that the CD133⁺/CD34^- subpopulation includes multipotent stem cells with a significant potential for differentiation into mesenchymal and other non-hematopoietic lineages. Between 2001 and 2003, we conducted a formal phase-I safety and feasibility trial in 15 patients, including a dose-escalation protocol. Since 2003, an open-label controlled phase-II trial is being undertaken, that will eventually include 100 patients. Fifty patients will undergo CABG & intramyocardial stem cell delivery, and 50 patients with comparable characteristics will have CABG alone.

The inclusion criteria were defined as follows: 1. documented transmural myocardial infarction more than 10 days and less than 3 months prior to admission for surgery; 2. presence of a localized area of akinetic LV wall without paradoxical systolic movement that corresponded with the infarct localization; 3. the infarct area should not be amenable to surgical or interventional revascularization; 4. elective CABG indicated to bypass stenoses or occlusions of coronary arteries other than the infarct vessel; 5. Absence of severe concomitant disease (i.e. terminal renal failure, malignoma, debilitating neurological disease). Patients who underwent emergency operation for unstable angina, reoperations, concomitant valve procedures, or had a history of significant ventricular arrhythmia are excluded. In our experience, it proved rather time-consuming to recruit patients who met the inclusion criteria (approximately 10 patients per year), probably because the modern rapid catheter interventions in acute myocardial infarction prevent the development of completely akinetic LV wall areas in many patients.⁴⁹

Outlook

Based on the existing experience, it seems to be justified to conclude that transplantation of autologous bone marrow cells in the infarct border zone can be safely performed in patients with ischemic heart disease. Whether neoangiogenesis, neomyogenesis, or both occur in the human situation remains unclear at this point, and carefully designed controlled studies are needed to further determine the efficacy of clinical cell transplantation for ischemic heart disease. It may well be that relevant myocardial regeneration already can be induced by using adult stem/progenitor cells that have not been expanded and modified ex vivo. However, given the tremendous amount of data demonstrating functional benefits in large animal models, careful clinical studies are needed to clarify clinical safety and efficacy for every cell population. Therefore the clinical introduction of such treatment will need both patience and public support before established treatment protocols are available. The positive initial clinical experience with intramyocardial bone marrow stem cell transplantation is raising hope for further regenerative cardiac stem cell therapies.