Therapeutic angiogenesis is an effective means for tissue salvage in patients with critical limb ischemia. Angiogenesis is defined as a formation of new blood vessels by sprouting of preexisting mature endothelial cells(ECs). In contrast, vasculogenesis is referred to as the creation of primordial blood vessels from endothelial progenitor cells (EPCs) or angioblasts. Neovascular formation in adults has been considered to result exclusively from the former process (i.e., angiogenesis). However, we and other researchers recently identified EPCs in human peripheral blood(PB), and circulating EPCs have been shown to accumulate at active angiogenic sites and to participate in neovascularization, a notion consistent with 'postnatal vasculogenesis'. EPCs in adults originate from bone marrow (BM), and we recently have demonstrated that in vivo implantation of autologous BM-MNCs effectively augmented ischemia-induced neovascularization in animal studies as well as human trial(TACT Trial). Here we summarize recent advances in cell transplantation-mediated therapeutic angiogenesis.

In contrast to conventional assumption that myocytes are never renewed after birth, a growing body of evidence suggests that human cardiac myocytes might divide in myocardial infarction and severe heart failure. Bone marrow cells may also contribute to myocyte regeneration, when injected or mobilized into systemic circulation by cytokines. A clinical study demonstrated that intracoronary administration of autologous bone marrow significantly improved the cardiac function after acute myocardial infarction. No adverse effect was found. Cell therapy using adult stem cells is anticipated to be an effective treatment of heart failure.

In the case of hematopoietic malignancies, direct approach of gene therapy [gene transfer to cancer cells in order to obtain direct therapeutic effects (cell damage)] is difficult, because malignant cells are distributed in the whole body. As for indirect approaches, immuno-gene-therapy is investigated: As a unique approach, suicide gene therapy is applied to donor lymphocyte infusion for relapsed leukemia after bone marrow transplantation. The purpose of gene therapy is to eliminate donor lymphocytes quickly when severe side effects (GVHD) appeared. HSV-TK gene is generally utilized as a suicide gene. Basic studies are conducted to determine whether anti-tumor-angiogenesis therapy is also effective for hematological malignancies. In addition, leukemia development in 2 patients with X-linked severe combined immunodeficiency who underwent hematopoietic stem cell gene therapy is currently a serious problem in the field of gene therapy. In both cases, LMO2 gene was activated through insertional mutagenesis which was caused by retroviral vectormediated gene transfer. This genetic event is considered to be a trigger of T-lymphocytic leukemia development. Further basic studies are needed in terms of safety for stem cell gene therapy.

Breast cancer is sensitive to chemotherapy and endocrine therapy, but the prognosis of advanced or relapsed breast cancer is unsatisfactory. Gene therapy is promising as another useful therapeutic approach for advanced breast cancer. Strategies of gene therapy for breast cancer in ongoing clinical protocols can be divided into four: (1) suppression of oncogenes or transduction of tumor suppressors; (2) enhancement of immunological response to cancer cells; (3) transduction of suicide genes; and (4) protection of bone marrow using drug resistance genes. We have started a clinical study of gene therapy for breast cancer using multidrug resistance gene (MDR1), in which advanced or relapsed breast cancer patients received high dose chemotherapy and autologous peripheral blood stem cell transplantation (PBSCT) with MDR1-transduced hemopoietic cells, and then were treated with docetaxel. Two patients have been treated so far, and in vivo enrichment of MDR1-transduced cells with docetaxel treatment after PBSCT was seen in both cases. Both patients are in complete remission and have no apparent adverse effect from MDR1 gene transduction.

The regenerative researches using human stem cells will be able to solve problems such as incurable diseases and the supply of human organs for transplantation which run short. These researches are also expected to produce business. On the other hand, there are various levels of human stem cells researches from already established to primitive in both safty and ethics. In this situation, a guideline for the human stem cell clinical research will be expected to perform appropriate clinical researches. Then, the Ministry of Health, Labour and Welfare composed a special committee about clinical research using human stem cells in the health, labour and welfare science council, technology section, and will decide upon a guideline on January 29, 2002.

Recent progress in trials of regeneration and gene therapy in endocrine organs, especially in adrenal glands has been reviewed. Gene therapies using adenovirus have been most frequently tested in vivo and in vitro, aiming at improvement of steroidogenesis and suppression of adrenal tumor growth. Although the effects were temporal, promising results have been obtained. Interestingly, adrenocortical tissue was shown to be formed by transplantation of adrenocortical cells and to replace the adrenal functions of adrenalectomized animals. Engineered ES cells stably expressing Ad4BP/SF-1 were shown to be directed toward steroidogenic lineage, suggesting a future possibility of regeneration of adrenal cells.

Insulin has been used generally in treatment of diabetic patients with absolute insulin deficiency since its discovery. However, while normal pancreatic beta-cells continually adjust insulin secretion in response to varying blood glucose levels, insulin administration cannot maintain blood glucose levels within a physiological range that protects from the development of various diabetic complications. It is possible to achieve normoglycemia in absolute insulin insufficiency by transplantation of pancreas or pancreatic islets, but the approach is impractical especially because of the shortage of transplantable pancreases and islets. For this reason, the transplantation of pancreatic beta-cells or islets generated from stem cells has become the more promising therapeutic approach to normoglycemia. In this article, recent progress of regenerative medicine of the pancreas is reviewed.

Hematopoietic stem cell transplantation, a front-runner of regenerative medicine, has been used widely for treatment of leukemia, lymphoma, and severe aplastic anemia. In the past 10 years, more than 14,000 transplants have been performed in Japan by using stem cells from either bone marrow, cord blood, or peripheral blood. Establishment of Japan Bone Marrow Bank and Japan Cord Blood Network has promoted stem cell transplantation from unrelated donors; 4,275 bone marrow and more than 700 cord blood transplantation as of July 31, 2002. There are several problems that remains to be solved, including expansion of donor pool size for both bone marrow and cord blood and financial backup of Japan Bone Marrow Program and Japan Cord Blood Network.

Recent studies revealed that cardiomyocytes can be regenerated from both embryonic- and adult bone marrow-stem cells in animal experiment. Regenerated cardiomyocytes had fetal ventricular phenotype, expressed alpha 1, beta 1, and beta 2-adrenergic, and M2 cholinergic receptors, and revealed spontaneous beating. These cells could be transplanted into the recipient heart, and survive for long period. Clinical application of these cells had some problems at present. Embryonic stem cells had ethical problems, and had a possibility of making teratoma when undifferentiated cells could not be eliminated. Adult stem cells had a difficulty in isolation and culture expansion, since they were not yet well characterized. However, transplantation of regenerated cardiomyocytes would become a future method for the treatment of severe heart failure.

The cornea is a transparent, colorless tissue that functions as the optical surface of the eye. Transparency is vital for useful vision, which can be compromised by disorders to the corneal epithelium, stroma or endothelium. Transplantation of donor corneas is the standard surgical procedure for irreversible damage to the tissue. However, Japan, as well as many countries across the globe, are suffering from a shortage of donor supplies. Recent developments in bioengineering techniques have opened the way for the possible development of regenerated tissue from cultured progenitor cells. An artificial cornea with all the necessary cell components may become available in the near future.

In order to investigate the possibility of the treatment of sensorineural hearing disturbance, experiments were performed using animals. First, the central cochlear pathway in the brain stem to pons was transected in adult rats. Tissue from embryos was transplanted to the lesion site. In 20% of the rats examined, the axons regrew beyond the transected site and regenerated into the denervated side and terminated at the normal targets. The hearing function of animals was also recovered. Those findings contradict the widely held view that the adult mammalian central auditory system cannot be restored following damage. Then, adult rat hippocampus-derived neural stem cells(NSC) were grafted into newborn rat cochlea. Within two to four weeks of grafting to the cochlea, some NSC survived in the cochlear cavity. Some of them had adopted the morphologies and positions of hair cells. This suggests that NSC can adapt to the environment of the cochlea and gives hope for treatment of the damaged cochlea and sensorineural hearing loss.

Recovery from central nervous system damage in adult mammals is hindered by their limited ability to replace lost cells and damaged myelin, and reestablish functional neural connections. However, recent progresses in stem cell biology are making it feasible to induce the regeneration of injured axons after spinal cord injury. Transplantation of in vitro expanded neural stem cells into rat spinal cord 9 days after contusion injury induced their differentiation into neurons and oligodendrocytes, and the functional recovery of skilled forelimb movement. It was partly because the microenvironment within the injured spinal cord at 9 days after injury was more favorable for grafted neural stem cells in terms of their survival and differentiation.

Neural stem cells(NSCs) are multipotential progenitor cells that can generate neurons, astrocytes, and oligodendrocytes, the three major cell types in the central nervous system. Due to their self-renewal activities, NSCs can proliferate in an undifferentiated state in vitro, allowing them to be expanded mitotically and harvested in bulk. Recent advances in stem cell biology have led us to investigate methods for the regenerative manipulation of the damaged CNS. However, there is much that is still not known about regulatory mechanisms of the differentiation and self-renewal of NSCs. In this article, we review some of the basic notions regarding the extracellular factors and signal transduction cascades involved in the differentiation and maintenance of NSCs.

In the context of minimally invasive surgery, the next logical step is to provide a biological replacement for missing tissue without the need for a harvesting operation. Tissue engineering is defined as the fabrication of living parts for the body from cells in the laboratory. Donor cells such as stem cell or cultivated, differentiated cells are seeded on a appropriately configurated scaffold replicating extracellular matrix. Growth factors are added to the in-vitro system to encourage stem cell proliferation. The engineered structure is then transplanted to the recipient. In this article, the regeneration strategies for tooth and periodontal tissue using tissue engineering concepts. Culture expanded mesenchymal stem cell is useful for periodontal tissue and tooth regeneration with bioabsorbable matrix such as b-TCP and collagen fiber. In the near future, these regenerated tissue will be accepted in clinical situations.

Huge bone defects that occur as a result of severe trauma or bone and oft tissue tumor are frequently difficult to achieve adequate union. Autogenous or allogeneic bone grafting and reconstruction using biomaterials are the traditional method used to repair bone defects, but it is not easy to reconstruct the structure and strength. Articular cartilage has very limited intrinsic healing capacity. Although numerous attempts to repair full-thickness articular cartilage defects have been conducted, no methods have successfully regenerated long-lasting hyaline cartilage. One of the most promising procedures for bone and cartilage repair is tissue engineering. New techniques of cell therapy for the treatment of bone and cartilage lesions are currently developed. This paper will summarize the current concepts of cell therapy for bone and cartilage repair and its future application.

Biomaterial is one of the most important factors to be developed in regenerative medicine. In this paper, the biomaterials essential to regenerative medicine were described. Biomaterials in regenerative medicine are classified into 5 categories; 1) Biomaterials for cell culture, 2) Biomaterials for cell inducement, 3) Biomaterials for scaffold, 4) Biomaterials for immuno-isolation, 5) Biomaterials assist to regenerative medicine. As for 1), temperature responsible gel that can take out the cultured cell sheet without use protein breakdown enzyme, is mainly introduced. Micro-photolithography to make a micro patterning for cell inducement, kinds of materials for scaffold, isolated membrane and micro capsules, carrier for cell growth factors are mentioned in 2) to 5).

It is confident that mammalian somatic cell and embryonic stem cell can form whole individuals if they are transferred to enucleated oocytes at M phase. However, a large proportion of clones were stillborn or died within several months due to various reasons. Morphological abnormalities have also been observed in clones. The exact reasons for such abnormalities are not clear, but genetic or epigenetic modification in donor cells and insufficient reprogramming of nuclei due to improper nuclear transfer technique are considered. In this review, we discuss the present status of animal cloning and reprogramming mechanism of nuclei.

In vitro differentiation of embryonic stem cells is expected as a useful means to prepare target cells for transplantation therapy. If normal and functional cells can be induced to differentiate only by adding a specific growth factor, it should be a safer and simpler method. Some of the examples showed, however, that it is necessary to follow an actual differentiation process taking place in embryogenesis to induce a specific cell lineage, so that it is difficult to selectively obtain target cells by using a single growth factor. Yet, it is also suggested that cell specification may be controlled by a specific growth factor or its combination in a simplified situation such as a single cell culture in a serum-free medium.