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The isolation of endothelial progenitor cells (EPCs) derived from bone marrow (BM) was an outstanding event in the recognition of ‘de novo vessel formation’ in adults occurring as physiological and pathological responses. The finding that EPCs migrate to sites of neovascularization and differentiate into endothelial cells (ECs) is consistent with “vasculogenesis”, a critical paradigm that is well described for embryonic neovascularization, but proposed recently in adults in which a reservoir of stem or progenitor cells contribute to vascular organogenesis. EPCs have also been considered as therapeutic agents to supply the potent origin of neovascularization under pathological conditions. This review highlights an update of EPC biology as well as their potential use for therapeutic regeneration.

Both gene therapy and cell transplantation are promising approaches for therapeutic angiogenesis. However, gene therapy must overcome biohazard of viral vectors, transfection efficiency, and premature tissue-targeting. Conventional cell therapy is insufficient in some cases because of small cell numbers, poor survival, impaired differentiation, etc. Endothelial progenitor cells (EPCs) play an important role in modulating angiogenesis and vasculogenesis. Here, we present a new concept for hybrid cell-gene therapy using a nonviral vector, gelatin. Genetically-modified EPCs may serve, not only as a tissue-engineering tool to reconstruct the vasculature, but also as a vehicle for gene delivery to injured endothelium. Thus, hybrid cell-gene therapy may be a new therapeutic strategy for the treatment of intractable cardiovascular diseases.

Bone marrow cells are advantageous for exogenous cell transplantation to treat end-stage heart failure regard to autologous source, no ethical issue, capacity to regenerate myocardium, induction of angiogenesis. Based on basic research showing regenerating myocardium using bone marrow, clinical trials in several places were conducted like gold rush in recent years. Endogenous-stem cell therapy may be also a promising strategy. Self-renewal of myocardium may be partly derived from bone marrow and the myocardium itself, which was thought to be a terminally differentiated organ. The past new technologies have been developed and their use expanded despite a lack of concrete evidence regarding their effectiveness. However, we still have a lot of unanswered questions including optimal cell population, cell density, and exact mechanism responsible for the improvement of cardiac dysfunction, fate of fusioned cells, cardiac environmental factors, regulation of proliferation and differentiation of transplanted cells, efficient cell tracking method in human. People involved in this field must be careful as they proceed, as inappropriately designed research might ruin the future of the field of regenerative medicine. Cell-based therapy will continue to expand at a rapid rate over the next decade. Whether the benefits of cell-based therapy are evident in the future remains to be seen.

Myocardial tissue regeneration including isolated cell transplantation and recruitment of bone marrow stem cells via cytokines have now emerged as one of the most promising treatments for patients suffering from severe heart failure. As therapy advances, the challenge to engineer three-dimensional (3-D) myocardial tissue grafts has also started. Tissue engineering has currently been based on the technology using 3-D biodegradable scaffolds as alternatives for extracellular matrix (ECM). However, insufficient cell migration into the scaffolds and inflammatory reaction due to scaffold biodegradation remain problems to be solved. By contrast, we have proposed novel tissue engineering technology layering cell sheets to construct 3-D functional tissues without any artificial biodegradable scaffolds. Electrical and morphological communications are established between layered cardiomyocyte sheets, resulting in simultaneous beating 3-D myocardial tissues both and . Layered cardiomyocyte sheets present long survival and functional improvement in accordance with host growth. For vascularization problems, multi-step transplantation of layered cardiomyocyte sheets overcame the thickness limitation of bio-engineered constructs. Cell sheet technology should have enormous potential for engineering clinically applicable myocardial tissues.

With regards to the therapy for end-stage failing hearts, the heart transplantation is the only effective treatment but is still limited by donor shortage and chronic rejection. Therefore, a novel strategy for myocardial regeneration is desired. Cell transplantation, a new approach for restoring impaired hearts still has several problems. Especially, autologous skeletal myoblasts (Skm) transplantation (Ctx) by injection has been clinically applied. However, its optimal effect and stable delivery methods are undetermined. Tissue implantation provides stable cell delivery with maintained inter-cellular communication and extracellular matrix. We hypothesize that the Skm tissue cardiomyoplasty might be more advantageous in regenerating the impaired heart. LAD ligated hearts (2 weeks) received Skm Ctx either by injection (MI; n=9) or by implanting two engineered monolayer myoblasts sheets (MS; n=9) or non-cellular therapy (Control=C; n=10). After 8 weeks, higher improvement of ejection fraction (%) was measured in MS compared to that of the other groups. Histological comparison revealed greater cellularity and abundant widespread neo-capillaries with a noticeably uniformly thickened wall in MS only. These results demonstrated that engineered skeletal myoblasts sheets regenerated the impaired myocardium, suggesting a promising therapy for severe heart failure.

Pluripotent embryonic stem (ES) cells are potent materials for both regenerative therapeutic approaches and developmental research. Recently, a novel ES cell differentiation system combined with 2-dimensional culture and flowcytometry assisted cell sorting (FACS) has been developed. In this system, cells in the cardiovascular system, that is, endothelial, mural, blood cells and cardiomyocytes can be systematically induced from common progenitor Flk1 (vascular endothelial growth factor receptor-2)-expressing cells. This system can constructively reproduce various stages of cardiovascular development in vitro, such as cell differentiation, diversification, and higher structure formation, providing novel possibilities to elucidate the cellular and molecular mechanisms of cardiovascular development. Cardiovascular cell induction from primate ES cells reveals primate-specific developmental mechanisms. ES cells will also contribute to regenerative medicine not only as a cellular source for transplantation but also for discovery of novel genes and drugs for regeneration. In this review, the significance of ES cell study in basic science and clinical medicine of cardiovascular field is discussed.

Tissue engineered grafts based on polymeric or acellular xenogeneric matrices have been widely studied, and found to have greater durability and functionality with growth potential and less immunogenicity than current bioprostheses. On the other hand, there are still several problems to be solved such as degradation control of biodegradable polymeric scaffolds and unwanted transfer of unknown animal related infectious diseases. In this chapter, our novel tissue processing of decellularization named PowerGraft by ultrahigh pressure treatment for safe tissue transplantation is reported. Porcine heart valves were isolated under sterile conditions and treated by cold isostatic pressing (CIP) at 4°C for disruption of donor cells. The cell debris was then washed out in PBS under microwave irradiation at 4°C. The tissues were completely cell free when they were treated by a CIP of 980 MPa (10,000 atm) for 10 min. There was no porcine endogeneous retrovirus (PERV) detected in the treated tissue. There were no significant changes in biomechanical properties of breaking strength and elastic modulus. From the in vitro incubation test, the tissues were disinfected when CIP was applied to the tissues contaminated by normal bacteria floras. The endothelial cells were well seeded on the acellular bioscaffold by the roller and circulation culture systems sequentially. This PowerGraft processing may provide a more durable and safe bioscaffold for tissue transplantation.

Autologous tubular tissues as small caliber vascular prostheses were created in vivo using tissue engineering. We named them “Biotubes”. The six kinds of polymeric rods made of polyethylene (PE), poly-fluoroacetate (PFA), poly-methyl methacrylate (PMMA), segmented poly-urethane (PU), polyvinyl chloride (PVC) and silicone (Si) as a mold were embedded in the dorsal skin of six of New Zealand White rabbits. Biotubes were formed after 1 month by fibrous tissue encapsulation around the polymeric implant except PFA. None of the Biotubes were ruptured when a hydrostatic pressure was applied up to 200 mmHg. The wall thickness of the Biotubes ranged from 50 to 200 µm depending on the implant materials in the order PFA<PVC<PMMA <PU<PE. The tissue mostly consisted of fibroblasts and collagen-rich extracellular matrices. The tissue created by Si rod was relatively firm and inelastic and the one created by PMMA was relatively soft. For PMMA, PE and PVC the stiffness parameter (β value; one of the indexes for compliance) of the Biotubes was similar to those of the human coronary, femoral and carotid arteries, respectively. Biotubes, autologous tubular tissues, can be applied for use as small caliber vessels and are ideal prostheses because of avoidance of immunological rejection.

Materials commonly used to repair complex cardiac defects in cardiovascular surgery lack growth potential and have other unwanted side-effects. We designed and tested a biodegradable scaffold seeded with bone marrow cells (BMCs) that avoids these problems.

We aspirated BMCs from human subjects during surgery and isolated mononuclear bone marrow cells (MN-BMCs) using the Ficoll technique. These cells were seeded onto a tubular or sheet-shaped scaffold and implanted as an autograft. Post-operative evaluations in human subjects were performed using computed tomography, magnetic resonance imaging and cardiac catheterization.

Since September 2001, we have performed 43 procedures in 42 patients using tissue-engineered materials. There was one mortality case for heart failure at 2 months after the operation. One case required re-operation for an R-L shunt in the atrium after a lateral tunnel procedure at 2 months after the operation. Stenosis of the tissue engineered graft was observed in 2 patients who underwent a total cavo-pulomonary connection (TCPC). Mild stenosis was observed in 2 patients after a pulmonary artery stenosis repair, and stenosis of a superior vena cava repair was observed in one case. The other surgical interventions had excellent outcomes.

These results provide direct evidence that the utilization of BMCs enable the establishment of tissue-engineered vascular autografts (TEVAs), and are especially useful for children who require biocompatibility and growth potential materials in cardiovascular surgery.

Atherosclerosis is responsible for more than half of all deaths in western countries. Numerous studies have reported that exuberant accumulation of smooth muscle cells play a principal role in the pathogenesis of vascular diseases. It has been assumed that smooth muscle cells derived from the adjacent medial layer migrate, proliferate and synthesize extracellular matrix. Although much effort has been devoted, targeting migration and proliferation of medial smooth muscle cells, no effective therapy to prevent occlusive vascular remodeling has been established. Recently, we reported that bone marrow cells substantially contribute to the pathogenesis of vascular diseases, in models of post-angioplasty restenosis, graft vasculopathy and hyperlipidemia-induced atherosclerosis. It was suggested that bone marrow cells may have the potential to give rise to vascular progenitor cells that home in the damaged vessels and differentiate into smooth muscle cells or endothelial cells, thereby contributing to vascular repair, remodeling, and lesion formation. This article overviews recent findings on circulating vascular precursors and describes potential therapeutic strategies for vascular diseases, targeting mobilization, homing, differentiation and proliferation of circulating progenitor cells.