In bone tumor surgery, target of bone tissue engineering technique will be the reconstruction of large bone void after tumor excision. It may be also useful to accelerate the regeneration of processed autologous bone grafts such as irradiated bone. Interconnected porous ceramics bone substitutes have been considered to be useful as a scaffold for bone engineering. We started a clinical trial on bone regeneration to evaluate its safety and efficiency using mesenchymal stem cells derived from autologous bone marrow aspirates and porous hydroxyapatite ceramics as a scaffold. In this study, after osteoblastic differentiation culture for several weeks, cells integrated to porous ceramics are transplanted into the defect after removal of large benign bone tumor.

With regard to the graft materials, cortical bone block, vascularized bone flap and particulate cancellous bone and marrow (PCBM) have been used for the reconstruction of maxillofacial skeleton. Needless to say, the aim of cortical bone block and vascularized bone flap transfer is the transplantation of bony tissue of its own. On the other hand, the main objective of PCBM grafting is the transplant of osteogenic stem cells derived from uncommitted marrow mesenchymal cells. After PCBM grafting, active new bone formation occurs from osteogenic stem cells followed by bone remodeling and replacement of host bone. This process means that PCBM grafting is the method of bone regeneration that is based on in vivo tissue engineering. In this paper, clinical application of PCBM grafting for the reconstruction of maxillofacial skeleton is introduced by showing the repair of maxillary bony defect of cleft lip and palate patients, alveolar ridge augmentation and the reconstruction of large mandibular segmental defects.

Articular cartilage plays pivotal roles in securing smooth joint kinematics and act as a shock absorber, however, it has minimal healing potential. Chondral injury could lead to the development of osteoarthritis (OA) and therefore is a major clinical concern. There have been marrow stimulating technique and osteochondral transplantation explored to promote cartilage repair. In addition, autologous chondrocyte implantation (ACI) has been developed by Peterson and Brittberg and more than 20,000 cases underwent the procedure all over the world. Recent progress in stem cell research has raised the potential application of stem cell therapy to cartilage repair. In this review, potential application of bone marrow or synovial-derived mesenchymal cells to promote cartilage repair would be discussed.

Osteoblasts and chondrocytes are most useful cells for osteogenesis and chondrogenesis, respectively. Mesenchymal stem cells can also be used after in vitro induction or without any treatment. Meanwhile, ES cells, iPS cells and GS cells become alternative cells for osteogenesis and chondrogenesis. Cell sources for the regenerative medicine of bone and cartilage should be determined, based on "benefit" and "risk" balance in clinics.

We have developed an ES cell-based monitoring system for osteogenic differentiation and identified a potent combination of osteogenic genes. ES cells were isolated from mice carrying a transgene expressing GFP driven by the 2.3 kb fragment of rat type I collagen alpha1 promoter. We have screened cDNA libraries and combinations of major osteogenesis-related genes and identified that the combination of constitutively active activin receptor-like kinase 6 (caALK6) and runt-related transcription factor 2 (Runx2) was the minimal unit that induced GFP. These combinations of genes would be a potent and ideal tool for bone regeneration.

While various kinds of fields have been focused on for the researches of regenerative medicine/tissue engineering, the cartilage regenerative medicine has been progressed well. In the present article, the author overviews the present state of cartilage regenerative medicine using various cell sources and scaffolds, and describes the prospects for the spread of clinical indication.

In vitro modification of mesenchymal stem cells and materials via tissue engineering is useful for bone regeneration. In this paper, we briefly summarize our bone tissue engineering approach. It is also known that RNA interference (RNAi) can regulate cell proliferation as well as differentiation cascade. Recently, our study using siRNA demonstrated that bone matrix formation by osteoblastic cells can be inhibited by certain types of siRNA. These findings can show that the proliferation/differentiation of the cells used in regenerative medicine could be regulated by siRNA.

We present a 22-year-old male diagnosed with pro T-acute lymphoblastic leukemia (ALL). His laboratory test showed 181,900/microL of WBC complicated with lymphoadenopathy, pleural effusion, pericardial effusion and hepatosplenomegaly at the onset. Flow cytometry analysis of the leukemic cells showed cCD3+, CD7+, CD2+, CD1a-, CD3-, CD5-, CD4-, CD8-, CD34+, and HLA-DR+ as a pro T-cell phenotype. The patient was treated with induction therapy followed by 3 courses of consolidation therapy and achieved his first complete remission. He underwent up-front stem cell transplantation (SCT) from an HLA-full matched sibling, with early relapse just before transplantation. The conditioning regimen consisted of fludarabine (100 mg/m2) and melphalan (180 mg/m2). He relapsed with an extramedullary mass (gingival, testis, and femoral muscles) 1 year after transplantation. Since bone marrow involvement was not apparent, he received involved field radiation therapy (25.2 Gy/14 frequencies) in each mass. Six months after extramedullary relapse, bone marrow relapse occurred, and the patient died of sepsis due to Pseudomonas aeruginosa during re-induction therapies. Based on the immature T cell phenotype frequently with myeloid markers, a graft-versus- leukemic effect might be expected after allogeneic SCT for Pro T-ALL and a positive indication of SCT for this disease should be considered.

The patient was a 31-year-old woman in whom facial paralysis occurred 1 week after the onset of pyelonephritis. Peripheral facial paralysis, sensorineural deafness, dysarthria, weakness of the four limbs, loss of tendon reflex, and peripheral sensory disturbance of the four limbs were noted during the initial examination. These symptoms were dominant in left side. The number of cells and protein level were increased in the cerebrospinal fluid, and reduced motor nerve action potential was detected with peripheral nerve conduction test. Based on these findings, axonal Guillain-Barré syndrome (GBS) was diagnosed. Left side-dominant bilateral hypoacusia was noted with audiogram, and left side-dominant bilateral prolongation of I wave latency was noted with auditory brainstem response (ABR). The ABR findings indicated that the auditory nerve was impaired on the peripheral side, which may have been associated with GBS. Interestingly, the laterality of the auditory nerve impairment was marked as with other neurological signs, such as paralysis. Since fewer cases of hearing impairment associated with GBS have been reported, further neurotological approaches to GBS are necessary.

Recovery of lost brain function is an important issue in medical studies because neurons of the central nervous system (CNS) have poor potential for regeneration. Since few CNS diseases can be treated completely by medicines, regenerative therapy by using stem cells should be studied as a new type of therapeutic intervention. The efficacy of cell replacement therapy in Parkinson's disease has been well investigated. Several studies on fetal tissue transplantation have revealed that quantity and purity of transplanted cells are necessary for recovery of symptoms. SFEB (Serum-free floating culture of embryoid body-like aggregates) method is capable of inducing multi-potential CNS progenitors that can be steered to differentiate into region-specific tissues. On the basis of the existing knowledge of embryology, we have succeeded in the generating of various types of neurons such as telencephalic, cerebeller (Purkinje and granule cells), retinal (photoreceptor cells) and hypothalamic neurons. Application of this culture method to human ES (hES) cells is necessary for clinical purpose: however, poor survival of hES cells in SFEB culture might limit the possibility of using these cells for future medical applications. We found that a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, markedly diminished the dissociation-induced apoptosis of hES cells and enabled the cells to form aggregates in SFEB culture. For both mouse and human ES cells, SFEB culture is a favorable method that can generate large amounts of region-specific neurons. However, stem cell-based therapy continues to face several obstacles. It is important that researchers in the basic sciences and clinical medicine should discuss these problems together to overcome both scientific and ethical issues related to stem cells.

We identified glypican-3 (GPC3), as a novel oncofetal antigen, overexpressed specifically in hepatocellular carcinoma (HCC) and melanoma in humans by utilizing genome-wide cDNA microarray analyses of HCC tissues and normal fetal and adult tissues. We also found that GPC3 is a novel tumor marker for HCC and melanoma, and that the pre-immunization of BALB/c mice with dendritic cells pulsed with the H-2K(d)-restricted mouse GPC3 298-306 (EYILSLEEL) peptide prevented the growth of tumor expressing mouse GPC3. Because of similarities in the binding peptide motifs between H-2K(d) and HLA-A24 (A(*)2402), the H-2K(d)-restricted GPC3 298-306 peptide thus seemed to be useful for the immunotherapy of HLA-A24(+) patients with HCC and melanoma. We investigated whether the GPC3 298-306 peptide could induce GPC3 reactive CTLs from the peripheral blood mononuclear cells (PBMCs) of HLA-A24 (A(*)2402)(+) HCC patients. In addition, we used HLA-A2.1 (HHD) transgenic mice (Tgm) to identify the HLA-A2 (A(*)0201)-restricted GPC3 epitopes to expand the applications of GPC3 based immunotherapy to the HLA-A2(+) HCC patients. We found that the GPC3 144-152 (FVGEFFTDV) peptide could induce peptide-reactive CTLs in HLA-A2.1 (HHD) Tgm without inducing autoimmunity. In 5 out of 8 HLA-A2(+) GPC3(+) HCC patients, the GPC3 144-152 peptide-reactive CTLs were generated from PBMCs by in vitro stimulation with the peptide and the GPC3 298-306 peptide-reactive CTLs were also generated from PBMCs in 4 of 6 HLA-A24(+) GPC3(+) HCC patients. The inoculation of these CTLs reduced the human HCC tumor mass implanted into NOD/SCID mice. We have recently started a phase I clinical trial of GPC3 peptide vaccine-based immunotherapy of patients with advanced HCC. We have also succeeded in inhibition of growth of tumors expressing mouse GPC3 by immunization of mice with dendritic cells differentiated in vitro from mouse embryonic stem cells and pulsed with the GPC3 peptides. Our study raises the possibility that these GPC3 peptides may therefore be applicable to cancer immunotherapy for a large number of patients with HCC and melanoma.

Wilm's tumor gene WT1, which has an oncogenic function, is expressed in various kinds of hematological malignancies and solid cancers. WT1 antibodies at higher titers and WT1-specific cytotoxic T lymphocytes (CTLs) at higher frequencies were detected in cancer patients than in healthy donors, indicating that WT1 protein was immunogenic. Furthermore, WT1-specific immune responses are considered to be involved with Graft versus Leukemia effect in the context of hematopoietic stem cell transplantation. These findings provided us with the rationale for cancer immunotherapy targeting WT1. Clinical trials of WT1 peptide vaccination for cancer patients were started, and WT1 vaccination-driven immunological responses and clinical responses, including reduction of leukemic cells, reduction of M-protein amount in myeloma, and shrinkage of solid cancer, were observed. Further enhancement of efficacy of WT1 peptide vaccine can be expected by co-administration of WT1-specific helper peptide or anti-cancer chemotherapy agent. WT1 peptide vaccination in the setting of MRD (minimal residual disease) may prolong "progression-free survival time", or decrease "relapse rate".

Cyclic thrombocytopenia is a rare disorder with periodic changes of the platelet count. We experienced a patient with cyclic thrombocytopenia and Sjögren syndrome (SS), and studied the mechanism of the cyclic changes of the platelet count. The patient, a 75-year-old woman, was referred to our hospital because of dry mouth, dry eyes, and severe thrombocytopenia. Her platelet count varied from 0.2 x 10(4)/microl to 22.7 x 10(4)/microl in 14-28-day cycles. Anti-SS-A/Ro antibody and Schirmer's test were positive. Histological examination of the salivary glands revealed infiltration of inflammatory cells, leading to the diagnosis of SS. There was an inverse relation between the platelet count and serum levels of PA-IgG. When the platelet count was low, the number of bone marrow megakaryocytes and the colony counts of CFU-Meg decreased. The serum thrombopoietin level increased at the nadir of the platelet count and decreased as platelets increased. After prednisolone therapy, her platelet count increased along with the improvement of sicca symptoms. These findings suggest that platelet fluctuation is due to the periodic increase of platelet destruction, as well as periodic failure of platelet production.

Gemtuzumab Ozogamicin (GO) targets leukemia cells expressing CD33 by means of a monoclonal antibody conjugated to a cytotoxic agent, calicheamicin. GO has been approved in Japan as monotherapy for the treatment of patients with relapsed/refractory acute myeloid leukemia (AML)since 2005. GO administered as a single agent has resulted in overall response rates of about 30% in previously relapsed adult AML. Preliminary data indicate a potential role for GO also as a component of induction or consolidation regimen. Although caution is advised when administering GO within 115 days of a stem cell transplantation (SCT) procedure because of veno-occlusive disease, recent clinical studies overseas suggest that GO can be integrated into reduced-intensity conditioning therapy before allogeneic SCT in patients with relapsed AML. In order to reduce toxicity and improve efficacy, its optimal dose and schedule should be defined by large clinical trials.