Recently, promising clinical outcomes of cancer immunotherapy including administration of an anti PD-1 antibody targeting for T cell reactivation has gained particular attention worldwide. Adoptive cell therapy with tumor infiltrating lymphocytes and TCR/CAR (Chimeric Antigen Receptor) transgenic T cells are also under development. Although it has become clearer that the efficacy of adoptive cell therapy correlate with the quality of infusing T cells, antigen specific T cells in patients with chronic infection and cancer have been exhausted. We have succeeded to generate rejuvenated antigen specific T cells by reprogramming to pluripotency and differentiation. In this article, we introduce fundamentals of this technology and describe its potential for adoptive cell therapy in the future.

Salivary gland hypofunction, or xerostomia (dry mouth syndrome), induces various clinical problems, such as dental decay, bacterial infection, and swallowing dysfunction. Xerostomia caused by autoimmune disease and aging affects an increasing number of patients. The development of novel functional treatments for xerostomia is needed, as currently available therapies are only palliative in nature. Tissue stem cell transplantation and gene therapy are currently being investigated as potential approaches to the restoration of salivary gland function. The final goal of regenerative therapy is fully functional regenerative organ replacement for dysfunctional organs. Previously, we developed a technology to reconstitute the organ germ (Organ Germ Method) using epithelial and mesenchymal stem cells. We have recently reported the regeneration of fully functional organs, such as teeth, hair and lacrimal glands, can be achieved by the transplantation of bioengineered organ germs. In this review, we describe the regeneration of the salivary gland as part of a feasibility study of a next-generation regenerative therapy.

Mesenchymal stem cells (MSCs) possess multipotent capacity and exhibit immunoregulatory properties. In particular, MSCs can be easily isolated from various organs, can differentiate into various types of cells and generate regulatory T cells. Using human MSC derived from bone marrow and adipose tissue, we have clarified the following novel findings in vitro. 1) MSCs differentiated into osteoblasts or osteocytes under osteoblast-conditioned medium including the inflammatory stimuli such as IL-1. 2) The combination of IL-6 and soluble IL-6 receptor induced differentiation of MSCs to chondrocyte, whereas IL-17 inhibited their differentiation. 3) MSCs highly produced osteoprotegerin and inhibited osteoclastogenesis. Furthermore, we developed a local delivery system of MSCs by using nano-fiber scaffold. MSCs seeded on nano-fiber scaffold suppressed arthritis and bone destruction due to inhibition of systemic inflammatory reaction and immune response by suppressing T cell proliferation and reducing anti-type II collagen antibody production in vivo. Thus, our data may serve as a new strategy for MSC-based therapy in inflammatory diseases and an alternative delivery method for the treatment of destruction of bone and joints.

Potential for re-programming cells has become widely accepted as a tool for obtaining transplantation materials. There has been great interest in cell-based therapies, including retinal transplants, because there is a reduced risk of immune rejection. Stem cells have the capacity for self-renewal plus the capacity to generate several differentiated cells. They are derived from many sources including human adult-derived induced pluripotent stem (iPS) cells and have found early application in the context of ocular disease. In results, our established iPS-retinal pigment epithelial (RPE) cells are high-quality RPE cells. iPS cells-derived RPE cells clearly showed polygonal morphology (mostly hexagonal) and contained melanin. Moreover, RPE cells derived from iPS cells had many characteristics of mature RPE cells in vivo, but no characteristics of pluripotent stem cells. Recently, we transplanted RPE cell sheets to treat a patient with wet age-related macular degeneration (September, 2014). In addition, we are now conducting experiments to determine whether allogeneic T cells can recognize iPS-RPE cells from HLA-A, B, DRB1 locus homozygote donors. iPS bank might be useful as allografts in retinal disorders, if the recipient T cells cannot respond to allogeneic RPE cells because of match to some of main HLA antigens.

Cancer stem cells (CSCs) were first experimentally identified in leukemia in 1997, and following researches demonstrated the existence of CSCs in a variety of solid cancers. It has been reported that CSCs are responsible for tumorigenicity, chemoresistance, and metastasis as well as heterogeneity of cancers. Therefore, elimination of CSCs may be crucial to eradicate cancer. Recent reports indicate that CSCs from some solid cancers show phenotypic plasticity, and intrinsic or extrinsic signals can revert differentiated cancer cells back into CSCs. These traits of CSCs may cause problems in devising targeted therapy against CSCs in future. Here we discuss how we can achieve total elimination of cancer cells in light of recent progress in understanding of CSCs.

Gastrointestinal cancer such as gastric cancer and colon caner, occupies the majority of the cause of death due to the malignant tumor. Although some progress about the conquest of cancer is accepted through early diagnosis and the development of the endoscope treatment, the treatment, which directly targets the molecular mechanism of the carcinogenesis, is still poor. It therefore is necessary to analyze a process of carcinogenesis in detail. While the gene abnormality as the cause of cancer has been gradually clarified, the characteristic of the cancer stem cell has recently attracted as novel target for cancer therapy. In this report, I summarize about the present situation of a clarified cancer stem cell study and introduce a new trial to understand carcinogenesis mechanism by rebuilding the process of carcinogenesis until the acquisition of the cancer stem cell mainly on stomach and colon.

Tumor therapies targeting tumor stem cells(TSCs) have been limited. One of the reasons is that TSC markers are often shared by normal stem cells (NSCs), and therapies targeting those marker-positive cells may cause severe injury to normal tissues. To solve the problem, we focused on doublecortin -like kinase 1 (Dclk1). In the normal intestines of Dclk1(creERT2/+); Rosa26(LacZ/+) mice, LacZ-labeled epithelial cells were scattered along villi after tamoxifen injection. In contrast, in Dclk1(creERT2/+); Rosa26(LacZ/+); Apc(Min/+) mice, intestinal tumors were occupied by LacZ-labeled tumor cells, and selective ablation of Dclk1-positive cells using iDTR system resulted in regression of intestinal tumors without apparent damage to the normal intestines. Thus, Dclk1 appeared to be a marker that discriminates TSCs from NSCs in the intestine.

Prognosis of pancreatic cancer remains dismal due to the resistance against conventional therapies. Metastasis and massive invasion toward surrounding organs hamper radical resection. Small part of entire cancer cells reveal resistance against chemotherapy or radiotherapy, increased tumorigenicity and migratory phenotype. These cells are called as cancer stem cells, as a counter part of normal stem cells. In pancreatic cancer, several cancer stem cell markers have been identified, which enabled detailed characterization of pancreatic cancer stem cells. Recent researches clarified that conventional chemotherapy itself could increase cancer cells with stem cell-phenotype, suggesting the necessity of cancer stem cell-targeting therapy. Based on these observations, pancreatic cancer stem cell-targeting therapies have been tested, which effectively eliminated cancer stem cell fraction and attenuated cancer progression in experimental models. Clinical efficacy of these therapies need to be evaluated, and cancer stem cell-targeting therapy will contribute to improve the prognosis of pancreatic cancer.

Glioblastoma multiforme (GBM) is the most frequent and aggressive brain tumor, and which harbors not only rapidly dividing cells but also small populations of slowly dividing and dormant cells with tumorigenesity-, self-renewal-, and multi-lineage differentiation capabilities. GBM stem cells (GSCs), which are resistant to conventional chemo -radiotherapy and may be a cause of local recurrence and dissemination. Additionally, heterogeneity of GSCs in the same tumor had been shown by the innovation of microarray and sequencing technology. However, outcome in patients with GBM remains unsatisfactory. Accumulation of the clinical evidence and basic research findings targeting for GSCs and their specific microenvironments (GSC niches) raise the possibility of new treatments to overcome GBM.

The existence of cancer stem cells in solid tumors was first reported in 2003 based on the analyses of human breast cancers. Analyses of clinical specimens are especially important for the advancement of human cancer stem cell research. For example, the analyses of breast cancer stem cells directly isolated from human breast cancer specimens identified the microRNAs that are involved in the regulation of human breast cancer stem cells. In addition, human breast cancer xenograft is an attractive model to analyze cancer stem cells, as well as cancer metastases that have profound effect on the long-term survival of breast cancer patients. We will present the current status of human breast cancer stem cell research that utilizes clinical specimens.

To achieve cure is difficult and progression after response to treatment is common for patients with several type of malignancies. One possible reason is existence of cancer stem cells, which have self-renewal activity and resistance to chemotherapy and radiotherapy. Several cellular markers and signal pathways are investigated to identify cancer stem cell. Targeting cancer stem cell is considered as promising to achieve cure in combination with other treatment. Although no cancer stem cell specific treatment is established at the present moment, several targeting agents are under development. We conducted a phase I study of specific inhibitor of xCT, a glutamate-cystine transporter, of CD44 splice variant (CD44v) expressed cancer stem like cell to confirm the mode of action. Clinical study of combination therapy with cisplatin is ongoing.

Multiple myeloma(MM) is characterized by the clonal expansion of malignant plasma cells. In xenograft models, CD19-CD38++ MM plasma cells engrafted and rapidly propagated MM, indicating that MM plasma cells, which are terminally differentiated cells, include MM-initiating cells. Bone marrow niche for MM-initiating cells are now being investigated extensively. MM patients harbor phenotypic CD19+ B cells expressing the immunoglobulin gene sequence and the idiotype unique to the individual myeloma clone. CD19+ clonotypic B cells include "pre-myeloma stem cells", and additional oncogenic hits are likely to be needed before developing myeloma disease from them. Prospective identification of CD19+ "pre-myeloma stem cells" is important for diagnosing pre-myeloma status and for developing the methods for the prevention of multiple myeloma.

Acute myelogenous leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. LSCs play a central role in the propagation of leukemia through their unique stem cell like properties, and LSCs share the many functional molecules with their normal counterpart hematopoietic stem cells (HSCs). For the establishment of LSCs-specific therapeutic approaches, it is quite important to understand the biological differences between LSCs and HSCs. Recent, studies have succeeded in clarifying these biological differences. In this review, I would like to introduce the biological significance of LSCs and discuss the molecular targeted therapy against LSCs.

The principle concept of cancer stem cells (CSCs) giving rise to the carcinogenesis, relapse or metastasis of malignancy is broadly recognized. On the other hand, circulating tumor cells (CTCs) also plays important roles in relapse or metastasis of malignancy, and there has been much focused on the association between CSCs and CTCs in cancer cases. The technical innovations for detection of CTCs enabled us to unveil the nature of CTCs. We now realize that CTCs isolated by cell surface antibodies, such as DCLK1, LGR5 indicated CSC properties, and CTCs with epitherial-mesenchymal transition(EMT) phenotype showed characteristics of CSCs.

Geminin regulates cellular proliferation and differentiation through the inhibition of DNA replication licensing and chromatin remodeling, respectively, to sustain the activity of hematopoietic stem cells (HSCs) and possibly that of leukemic stem cells (LSCs). Thus, Geminin is presumed to act as a cell fate determinant by turning on and off self-renewal and differentiation of the stem cells. We visualized Geminin expression by generating knock-in mice expressing Geminin fusion protein with enhanced yellow fluorescent protein. We further established a new method for manipulating the Geminin expression level and activity by generating cell -penetrating (CP) - Geminin. Here we argue for a new strategy for expanding HSCs ex vivo to provide a cellular source of HSCs for transplantation and further for eradicating LSCs, which are resistant to conventional chemotherapy.

Emerging evidences have revealed the role of tumor-associated immune cells and their derivatives in the regulation of tumor progression. In particular, recent studies have clarified that various tumor-associated immune regulatory systems, which include tumor-infiltrating myeloid cells and pro-inflammatory cytokines, for regulating the maintenance and activation of cancer stem cells. The networks formed by immune cells and cancer stem cells play a critical role in further amplifying infiltration of pro-tumor immune cells and inflammatory cascades, and promoting cancer stemness. Further clarification about the immune-mediated regulation of cancer stem cell activities should provide therapeutic implication against cancer stem cells and treatment-resistant variants of cancer cells.

CD44 was identified as a cell surface marker for cancer stem cells (CSCs) in various epithelial tumor. However, function-based evidence to support the role of CD44 in CSCs was not known. We found that expression of CD44, in particular variant forms of CD44 (CD44v), suppresses reactive oxygen species (ROS) levels by promoting the synthesis of reduced glutathione(GSH), a primary intracellular antioxidant. CD44v stabilizes xCT, a subunit of a glutamate-cystine transporter, and thereby promotes the uptake of cystine for GSH synthesis. Our findings reveal a novel function for CD44v in protection of CSCs from high levels of ROS in the tumor microenvironment.

Cancer stem cells are the cellular sources of the vast majority of mature cancer cells and are reportedly responsible for the recurrence of disease following anti-cancer therapy. Recent advantages in the cancer stem cell research field have indicated that TGF-β signaling pathway plays an essential role for the maintenance of cancer stem cells via regulating cancer stemness, quiescence, epithelial -mesenchymal transition (EMT), or therapeutic resistance in vivo. Therefore, the outcome of these investigations will hopefully be the development of novel agents that can specifically control both augmentation and suppression of TGF-β signaling pathway in cancer stem cells, and thereby provide a novel avenue for curative cancer patient therapy.

In addition to the properties of self-renewal and multipotency, cancer stem cells share the characteristics of their distinct cell cycle status with somatic stem cells. Cancer stem cells (CSCs) are maintained in a quiescent state with this characteristic conferring resistance to anticancer therapies that target dividing cells. Elucidation of the mechanisms of CSC quiescence might therefore be expected to provide further insight into CSC behaviors and lead to the elimination of cancer. This review summarizes several key regulators of the cell cycle in CSCs as well as attempts to define future challenges in this field, especially from the point of view of the application of our current understandings to the clinical medicine.

Proliferation of cancer cells requires activation of signals that response to nutrients. One of the major coordinators of nutrient signals is mammalian/mechanistic target of rapamycin (mTOR). mTOR complex 1 (mTORC1) controls multiple cellular processes, including protein synthesis, glucose metabolism, fatty acid and sterol synthesis, mitochondrial biogenesis, and autophagy. Although inhibitors of mTORC1 have been developed, effectiveness of the inhibitors for cancer eradication appears to be limited, because of presence of several feed-back signals. In addition, leukemia stem cells might survive against mTOR inactivation through common mechanisms by which normal hematopoietic stem cells are maintained in the niche. Advances in understanding of how mTOR signaling is involved in mechanisms of stem cells may lead to novel therapeutic approaches that can successfully eradicate cancer.