Introduction

Although various therapies for heart failure, such as medication, surgical treatment, transplantation, and mechanical support, have been developed and shown to be effective, questions remain about their longevity as standard treatment. There are still many problems to be solved with standard treatment, such as medical costs, a shortage of donors, and various complications.

On the other hand, myocardial regeneration therapy has emerged as an alternative therapy for heart failure [1–6] and is expected to be a replacement for conventional therapies in the future. With recent advances in molecular biology and new knowledge, such as the existence of cardiac stem cells [7, 8], many researchers in cardiology have started to focus on myocardial regeneration therapy [9–12]. Recently, after several reports that cell transplantation improved cardiac function in experimental models, clinical applications of autologous mesenchymal stem cells (myoblast transplantation) have started.

In this report, we review myocardial regeneration therapy using autologous cells for end-stage heart failure and report recent advances in this field.

Cell therapy

Resident cardiac stem cells (RCSC)

With recent remarkable developments in molecular biology and stem cell research [13–15], RCSC in the heart have been detected by many researchers. This type of cell is considered to be present not only in humans but also in other species of animals, while representative cell markers, such as c-kit [13], Sca-1 [14], and isl-1 [15], have been reported. These cells seem to differ from each other, as those with a particular marker are dissimilar from those with other markers [15]. In 2003, Beltrami et al. [13] reported that they found c-kit positive cells in rat hearts that showed an eternal self-regeneration ability and multi-potency (multi-differentiation). Schneider et al. [14] also reported the presence of Sca-1 positive cells, which they termed RCSC, in damaged areas due to ischemia or ischemic-reperfusion injury that needed oxytocin to differentiate into beating myocytes. These RCSC showed multi-potency and were thought to differentiate into myocytes. Although the presence of RCSC able to differentiate into cardiac myocytes in cardiac tissue has been shown, cardiac tissue regeneration was never considered possible until very recently. Future studies are needed to elucidate the relationship between RCSC and myocardial infarction or the mechanisms of heart failure.

Bone marrow stem cells (BMSC)

BMSC are a source of cell transplantation therapy that have received the most attention in the current era [1–4, 16, 17], with many researchers expecting BMSC to be able to differentiate into myocytes as well as other types of cells. However, as there is no clear definition of BMSC, they are referred to in a variety of ways, such as “mesenchymal stem cells”, “marrow stromal cells”, and “marrow mononuclear cells” [18]. BMSC have the same features as embryonic stem cells and are considered to be an ideal source for cell therapy, because they can be used for autologous cell transplantation. Although the efficacy of myocardial regeneration after transplantation of BMSC has not been clarified, most studies have shown recovery of cardiac function after BMSC transplantation in acute or chronic myocardial infarction animal models, which implies the presence of angiogenesis [19, 20].

A number of clinical applications with BMSC transplantation have been reported, as they are easy and safe to use as a cell source [1–3]. Most clinical trials that used BMSC showed clinical safety and the possibility of future use as a treatment method (Fig. 1). The main efficacy found was relief from heart failure symptoms and improvement of blood flow towards myocytes. Initially, there were many reports with a low degree of accuracy due to a lack of control groups and low numbers of patients. However, recent reports such as the BOOST trial [1], TOPCARE-AMI [2], and ASTAMI [3] have presented more accurate data due to the use of multi-center clinical trials. The BOOST trial reported significant improvement of left ventricular ejection fraction (6.7 %) post-BMSC transplant, while there was no significant improvement in the control group (0.7 % improvement). Most recently, a German multi-center clinical trial called REPAIR-AMI revealed significant improvement of postoperative cardiac function in patients with acute myocardial infarction and showed the efficacy of BMSC therapy as evidence-based medicine [4]. However, Jackson et al. [21] reported that only 0.02 % of BMSC aspirated from bone marrow were able to differentiate into cardiomyocytes, which was not adequate to restore cardiac dysfunction due to myocardial infarction. In light of these findings, it is reasonable to consider that the main mechanism is local improvement of blood flow due to secretion of angiogenic factors (paracrine effect).

Fig. 1
figure 1

Cardiac regenerative therapy using autologous cells

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Skeletal myoblasts

Myoblasts have been shown as a possible cell source for clinical applications through recent advances in research [22, 23]. Satellite cells, found in skeletal muscles and comprising myoblasts, start to differentiate and split for replacement of skeletal muscle when muscle is injured. Marelli et al. [24, 25] targeted this stem cell-like feature of myoblasts and transplanted them into dogs with myocardial infarction, and confirmed that they remained in the myocardium. Murry et al. [26] also reported that autologous myoblasts implanted into a myocardial infarction model formed myotubes, while they were not able to show a connection between host myocardium and implanted myoblasts. On the other hand, Taylor et al. [27] documented functional recovery of cardiac function in cryo-injured rat hearts by autologous myoblasts implantation.

Based on those findings, Menashé et al. [28] performed autologous myoblast implantation in 10 patients with myocardial infarction and undergoing open heart surgery in a French clinical trial. However, they experienced 4 episodes of fatal arrhythmia that required an implantable cardiac defibrillator (ICD). On the other hand, in a clinical trial by Diacrine Co, Ltd. at the Arizona Heart Center, Dib et al. [29] showed that autologous myoblast implantation improved cardiac function and that fewer fatal arrhythmias occurred (2/26) than reported by Menashé. Additional investigations are need to clarify whether the arrhythmias were due to the implanted myoblasts themselves or scar tissue from needle injury.

Recently, a large clinical trial following Menasche’s protocol [5] was conducted by Genzyme Co, Ltd. and Medtronic Co, Ltd. in Europe. This was a multicenter (24 European centers) prospective double-blind randomized trial called myoblast autologous grafting in ischemic cardiomyopathy (MAGIC) (Fig. 2). Their protocol was as follows. Patients who needed CABG were divided into 3 groups after implantation of ICDs. In the high dose group (n = 30) 800 × 106 myoblasts were implanted via 30 separate needle injections, while in the low dose group (n = 33) 400 × 106 myoblasts were implanted in the same manner. The placebo group contained 34 cases. After 6 months, left ventricular end-diastolic volume and end-systolic volume were significantly decreased in the high dose group as compared to the placebo group. Also, those in the low dose group showed reductions in these volumes, though the difference was not significant. On the other hand, there were no significant differences regarding the left ventricular ejection fraction among the 3 groups, and neither local nor whole systolic function was significantly improved. Since there was no significant improvement of local systolic function at the point when the cells were injected (first end-point), this clinical trial was stopped in the early phase while further evaluation continues. On the other hand, Dib et al. [30] are now conducting a Phase II clinical trial approved by the FDA and their results will soon be available.

Fig. 2
figure 2

outline of Magic trial

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In Japan, Termo Co, Ltd. and Genetic Co, Ltd. are about to start a clinical trial using combined therapy with CABG. After 3 years of strict evaluation by the Japanese Ministry of Health and Welfare, a multicenter trial will soon commence.

Myocardial regeneration therapy by autologous cell transplantation at Osaka University

In patients with severe myocardial infarction, myocardium function deteriorates, and proliferation of fibroblasts and fibrosis in the interstitium causes heart failure. After the occurrence of heart failure, the myocardium is damaged and apoptosis occurs. As a result, the number of myocytes decreases and further deterioration occurs because myocytes rarely perform cell division. As already described, cell transplantation was reported as a useful strategy for functional recovery in patients with end-stage heart failure and clinical applications using autologous myoblasts have already started in Europe [1–6]. However, it is difficult to fully recover cardiac function due to a number of problems, such as initial loss of implanted cells up to 70–80 % and the possibility of fatal arrhythmia. A sufficient number of cells is also needed and efficient engraftment of transplanted cells is vital. Also, needle injections have limitations, because they can cause focal inflammation and are not optimal for global cell transplantation.

For clinical application of cardiac cell transplantation therapy, it is very important to maintain the extracellular environment to provide appropriate blood flow and maintain cell function, while vascularization is needed inside the myocardium. In this light, BMSC have vascularization capability, which plays an important role in engrafting transplanted BMSC. We investigated combined cell transplantation of myoblasts and BMSC in MI rat models, and found that combined therapy produced better recovery of cardiac function and thickness of the ventricular wall, while it also inhibited cardiac remodeling (Fig. 3) [31]. We think that this combination therapy provides improved vascularization and engraftment of transplanted cells.

Fig. 3
figure 3

Cardiac regenerative therapy using autologous myoblasts and bone marrow mononuclear cells for patients with ischemic cardiomyopathy

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Okano et al. [32–35] developed a cell-sheet technique for preparing biological grafts, which has since been applied to several diseased organs, such as the heart, eyes, and kidneys, in laboratory and clinical studies. Cell sheets are prepared in special dishes that are coated with a temperature-responsive polymer, poly (N-isopropylacrylamide) (PIPAAm), which changes from being hydrophobic to hydrophilic when the temperature is lowered. This change releases the cell sheets, allowing them to be removed without destroying the cell–cell or cell–extracellular matrix (ECM) interactions within the cell sheets. The greatest advantage of this technique is that cell sheets are made only of cells and the ECM is produced by the cells themselves, without needing an artificial scaffold [36]. Such cell sheets integrate well with native tissues, because the adhesion molecules on their surface have been preserved [37]. We found that myoblast-cell-sheet implantation improved cardiac function and ventricle thickness in an MI rat model (Fig. 4). Furthermore, application of a skeletal myoblast sheet to a dilated cardiomyopathy hamster model resulted in recovery of deteriorated myocardium, along with preservation of alpha- and beta-sarcoglycan expression by the host myocytes and inhibition of fibrosis [38]. We implanted myoblast sheets into 27-week-old DCM hamsters, which were at a moderate heart-failure stage (fractional shortening 16 %), and found preservation of cardiac function and histology along with prolonged survival. In addition, grafting of skeletal myoblast sheets attenuated cardiac remodeling and improved cardiac performance in a pacing-induced heart-failure canine model [39]. These results demonstrate that skeletal myoblast sheets can regenerate deteriorated myocardium caused by coronary artery diseases and DCM in small animal models. Although they indicate that skeletal myoblast sheets have potential as treatment for moderate heart failure, their efficacy for end-stage heart failure is unknown and requires further study.

Fig. 4
figure 4

Development of cardiac regenerative therapy using tissue engineering technology

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Furthermore, we conducted a pre-clinical large animal study using MI porcine models and DCM dog models (Fig. 5), and confirmed the effectiveness of skeletal myoblast sheets [40]. Thereafter, we conducted clinical application of skeletal myoblast implantation after approval from our ethics committee, as noted in the following section.

Fig. 5
figure 5

Autologous skeletal myoblast sheets attenuate cardiac remodeling after myocardial infarction

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Clinical application of skeletal myoblast sheet implantation

In May 2007, we found that implantation of myoblast sheets into a human patient with end-stage heart failure caused by dilated cardiomyopathy (DCM) and under left ventricular assist device (LVAD) support resulted in significant improvement of cardiac performance and was shown to be the means to recovery (Fig. 6) [41]. The patient was a 56-year-old man suffering from idiopathic DCM, who was referred to our hospital while under intra-aortic balloon pumping (IABP) support, oxygenation with a respirator, portable cardiopulmonary bypass, and continuous venovenous hemodiafiltration (CVVHD). On the day of admission to our hospital, he underwent implantation of an extracorporeal pneumatic LVAD (Toyobo, Tokyo, Japan) and a right ventricular assist system (RVAS) with extracorporeal membrane oxygenation (ECMO) using a centrifugal pump. An off-pump examination revealed that the patient could not be weaned from the LVAD. Myoblast-cell-sheet transplantation into human patients was approved by the Ethics Committee and Internal Review Board of Osaka University in July 2006. After receiving informed consent from the patient, an approximately 10-g piece of skeletal muscle was excised from the medial vastus muscle under general anesthesia. Next, isolated autologous myoblasts were seeded onto temperature-responsive culture dishes and 20 autologous myoblast-cell-sheets were transplanted onto the anterior to lateral surface of the dilated heart through a left lateral thoracotomy. Off-pump tests performed at 8 weeks and 3 months after transplantation showed that ejection fraction improved from 26 to 46 %, and the left ventricle dilated dimension (LVDd) was increased from 49 to 53 mm. Three months after myoblast-cell-sheet transplantation, the LVAD was explanted. Following cell-sheet transplantation and LVAD removal, a Holter cardiogram demonstrated that no life-threatening arrhythmia had occurred. This patient is now receiving care in the outpatient clinic and displays no symptoms of heart failure. After this case, we performed the same therapy for 3 other patients. From our results, we hypothesized that the recovery of cardiac function is dependent on the remaining viability of the left ventricle, because the mechanism is primarily a paracrine effect. We also conducted clinical application of autologous skeletal myoblast sheet implantation in patients with ischemic cardiomyopathy without an LVAD. Thus far, 6 patients have undergone this implantation. It is important to evaluate the viability of the remaining myocardium in patients with end-stage heart failure. We plan to assess the safety and efficacy of this therapy by increasing the number of candidates.

Fig. 6
figure 6

Cardiac regenerative therapy using myoblast sheets for patients with LVAD

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Summary

As previously described, it may be possible to use autologous transplantation of BMSC or myoblasts to regenerate damaged myocardium, which causes heart failure. Furthermore, it will eventually be possible to perform regeneration therapy as a routine treatment strategy. In patients with chronic heart failure due to broad myocardial injury such as DCM, it may be more effective to provide treatment with cell sheets, in other words, cardiac tissue-by-tissue engineering, than use of local injection or gene therapy.