A 53-year-old woman with acute myeloid leukemia (M2; normal karyotype) in first remission underwent the nonmyeloablative allogeneic peripheral blood stem cell transplantation from her HLA-identical brother, with conditioning consisting of fludarabine and low dose total body irradiation (2Gy). Karyotype analysis of bone marrow on day 28 after the recovery of the hematopoiesis showed 46 XY (20/20). However, pancytopenia progressed from day 130 and the patient became transfusion dependent. Because of the hypoplastic bone marrow and the high ratio (81%) of recipient cells among the peripheral T-cells, she was diagnosed as the late graft failure. Cyclophosphamide was added to the conditioning and the second transplant was performed using the same donor's cryopreserved stem cells. Hematopoiesis recovered and the complete chimerism in T-cells was confirmed on day 28. Although the transplant dose of the CD34 and CD3 positive cells was the same between the two transplantation, the patient suffering from the late graft failure obtained the stable engraftment after the second transplant with more immunosuppressive conditioning regimen.

We have a new therapeutic modality, regenerative medicine, for patients with severe ischemic heart disease. Growth factor administration and cell transplantation are available. Therapeutic angiogenesis with bone marrow cell transplantation has been used clinically with favorable results. Basic fibroblast growth factor slow-release administration has recently started to be used clinically as another angiogenic therapy. It is more potent when combined with a donor artery and omentum (Bio-CABG). Myogenic cell transplantation is in clinical trials aimed at myocardial regeneration. However, it remains unresolved how transplanted myoblasts improve cardiac function and how we can prevent fatal arrhythmia. Many are researching cardiac stem cells and embryonic stem cells as candidates for myocardial regeneration. Recently, the paracrine effects of transplanted mesenchymal stem cells in the ischemic heart have been reported to contribute to improved cardiac function. Therefore, growth factors and cytokines may play an important role in the regeneration process induced by transplanted cells. We combined cell transplanstation with growth factor administration as well as reconstructive surgery for dilated left ventricle, which yielded excellent results. Our integrated strategy may result in the maximal benefits to patients in the future.

Regeneration therapy has currently emerged as one of the promising treatments for the patients suffering from severe heart dysfunction. Direct transplantation of isolated myoblasts or bone marrow mononuclear cells, and recruitment of stem cells from bone marrow by G-CSF administration have been already clinically performed. As further advanced therapy, research on fabricating three-dimensional (3-D) cardiac grafts by tissue engineering technology has also now begun. Most popular approach of tissue engineering is using 3-D biodegradable scaffolds as alternatives of extracellular matrix. By contrast, we have successfully fabricated macroscopically beating myocardial tissue by layering cell sheets which are harvested from intelligent culture dishes grafted with temperature-responsive polymer in nano-scale. These regenerative medicines should rescue many patients.

We describe a 56-year-old woman with chronic myeloid leukemia (CML) and a past history of stroke, who underwent nonmyeloablative hematopoietic stem cell transplantation (NST) with conditioning consisting of fludarabine and cyclophosphamide. The regimen related toxicity was minimal and patient did not require transfusions of any blood products nor did she have any infections after the NST Since mixed chimerism was observed in both lymphocytes (70% were donor type) and granulocytes (none were donor type) at 56 days after NST, donor lymphocyte infusion (DLI) was performed on day 68 and then immunosuppressant therapy was discontinued. DLI resulted in graft versus leukemia (GVL) effect, causing pancytopenia and bone marrow aplasia. A second hematopoietic stem cell transplantation was performed without conditioning on day 157, and complete donor type hematopoiesis and molecular remission of CML were achieved. Although engraftment of donor hematopoietic stem cells was not obtained after the first transplantation, donor lymphocytes were engrafted by nonmyeloablative conditioning and immunosuppression. That is, the same result might have been achieved even if the patient had received transfusion of only donor lymphocyte subsets in the first step. Based on this case report, a potential cell therapy is proposed composed of the preceding donor lymphocyte infusion alone, which induces GVL effects, and subsequent donor hematopoietic stem cell transplantation.

Increasing enthusiasm in the field of stem cell research is raising the hope of novel cell replacement therapies for Parkinson's disease (PD), but it also raises both scientific and ethical concerns. In most cases, dopaminergic cells are transplanted ectopically into the striatum instead of the substantia nigra. If the main mechanism underlying any observed functional recovery with these cell replacement therapies is restoration of dopaminergic neurotransmission, then viral vector-mediated gene delivery of dopamine-synthesizing enzymes is a more straight forward approach. The development of a recombinant adeno-associated viral (AAV) vector is making gene therapy for PD a feasible therapeutic option in the clinical arena. Efficient and long-term expression of genes for dopamine-synthesizing enzymes in the striatum restored local dopamine production and allowed behavioral recovery in animal models of PD. A clinical trial to evaluate the safety and efficacy of AAV vector-mediated gene transfer of aromatic L-amino acid decarboxylase, an enzyme that converts L-dopa to dopamine, is underway. With this strategy patients would still need to take L-dopa to control their PD symptoms, however, dopamine production could be regulated by altering the dose of L-dopa. Another AAV vector-based clinical trial is also ongoing in which the subthalamic nucleus is transduced to produce inhibitory transmitters.

HIV encephalopathy is one of the complexified viral diseases. In the infected brains, HIV-infection is restricted in macrophages and microglia although its damage extends to neurons and oligodendrocytes. Accumulating evidences have suggested that many viral and host factors are involved in this disease. However, its precise mechanism is still unsolved. To examine the mechanism of the disease, we developed an HIV-1-infected human cell-transplanted mouse model and TNF-related apoptosis-inducing ligand was identified as a neurotoxic host factors in HIV-infected brain. Next, we examined the neurotoxic host factors using co-culture system with macrophage-tropic HIV-1-infected macrophages as followings: Target brain cells are murine neuron/glia mixed culture, murine neurospehre-forming culture and rat brain hippocampus slice culture. In these systems, neurons and neural stem cells were preferentially damaged. On the other hand, we also identified two anti-HIV genes, CD 14 and CD63 (dN), which inhibit HIV-1-induced cytotoxicity using a lentiviral screening system. Because they express on monocyte or activated macrophage and microglia, these results suggest that CXCR4-using HIV-1 cannnot expand inside of brain. We also extended the screening system to identify the host factors which protect against HIV-1-induced encephalopathy. Our study will contibute to development of new therapeutic strategy for HIV encephalopathy as well as other CNS diseases.

In the dystrophin-deficient mdx mice, an animal model of Duchenne muscular dystrophy (DMD), damaged skeletal muscles are efficiently regenerated and thus the animals thrive. The phenotypic differences between DMD patients and mdx mice suggest the existence of factors that modulate the muscle wasting in the mdx mice. To identify these factors, we searched for mRNAs affected by the mdx mutation using cDNA microarrays with newly established skeletal muscle cell lines derived from mdx and normal mice. We found that genes encoding thymosin beta4, frizzled related protein 2 (FRP2), and regeneration-associated muscle protease (RAMP) are up-regulated in skeletal muscle of mdx mice. Thymosin beta4 was induced in both regenerating muscle fibers and inflammatory cells after muscle injury. It stimulated migration and chemotaxis of myoblasts. FRP2 was dramatically induced upon muscle injury. RNA interference-mediated knockdown of FRP2 mRNA in myoblasts resulted in a massive cell death. Thus FRP2 may enhance the survival rate of myoblasts in the regenerative regions. RAMP mRNA was specifically induced in the regenerating areas of injured skeletal muscle. Expression of RAMP and FRP2 was much lower in individual muscle cell lines derived from biopsy specimens from several DMD patients compared to in a normal muscle cell line. Above results suggest that thymosin beta4, FRP2, and RAMP may play roles in the regeneration of skeletal muscle and that down-regulation of these molecules could be involved in the progression of DMD in humans.

Chronic degenerative diseases and traumatic injuries are responsible for a decline in neuronal function. Cell transplantation is one of the strategies with potential for treatment of such neural disorders, and many kinds of cells including embryonic stem cells and neural stem cells have been considered as candidates for transplantation therapy. Bone marrow stromal cells (MSCs) have great potential as therapeutic agents since they are easy to isolate and can be expanded from patients without serious ethical and technical problems. We found a method for the highly efficient and specific induction of functional neurons, skeletal muscle cells and Schwann cells from both rat and human MSCs. Induced neurons and Schwann cells were transplanted to animal models of Parkinson's disease, stroke, peripheral nerve injury, and spinal cord injury, resulting in the successful integration of transplanted cells and improvement in behavior of transplanted animals. Induced skeletal muscle cells differentiate into muscle fibers upon transplantation into degenerated muscles of rats and mdx-nude mice. The induced population contained Pax7-positive cells that contribute to subsequent regeneration of muscle upon repetitive damage without additional transplantation of cells. Here we focus on the respective potentials of MSC-derived cells and discuss the possibility of clinical application in neurodegenerative and muscle degenerative diseases.

Neural progenitor cells, including neural stem cells (NSCs), are an important potential graft material for cell therapeutics of damaged spinal cord. Here we used as a source of graft material a NSC-enriched population derived from human fetal spinal cord (Embryonic week 8-9) and expanded in vitro by neurosphere formation. NSCs labeled with BrdU (TP) or culture medium (CON) were transplanted into the adult marmoset spinal cord after contusion injury at C5 level. Grafted NSCs survived and migrated up to 7 mm far from the lesion epicenter. Double-staining with TuJ1 for neuron, GFAP for astrocyte, or CNPase for oligodendrocyte and BrdU revealed that grafted NSCs differentiated into neurons and oligodendrocytes 8 weeks after transplantation. More neurofilaments were observed in TP than those of CON. Furthermore, behavioral assessment of forelimb muscle strength using bar grip test and amount of spontaneous motor activity using infrared-rays monitoring revealed that the grafted NSCs significantly increased both of them compared to those of CON. These results indicate that in vitro expanded NSCs derived from human fetal spinal cord are useful sources for the therapeutics of spinal cord injury in primates.

Neural stem cells (NSCs) ar self-renewing, multipotential progenitor cells. A single NSC can give rise to a wide variety of CNS cells, including neurons, astrocytes, and oligodendrocytes. Because of these characteristics, there is an increasing interest in NSCs and neural progenitor cells, both from a basic developmental biology perspective and from a clinical one that is aimed at developing therapeutic applications for the damaged brain. Current research into the nature of the NSCs present in the CNS includes the study of the extracellular factors and signal transduction cascades involved in their differentiation and maintenance, their population dynamics, and their localization in the embryonic and adult brain. These lines of research, combined with other studies intended to permit the prospective identification and isolation of NSCs, and their induction into particular neuronal phenotypes--which will be introduced in my talk--should lead to the development of feasible strategies for manipulating NSC cells in situ to treat the damaged brain and spinal cord injury.