It has been reported that hyaluronan tetrasaccharide (HA4) has an ability to accelerate the healing process of spinal cord injuries and to mitigate the symptoms of experimental autoimmune encephalitis (EAE) in animal models. However, the precise mechanisms of the effect of HA4 are unclear. This study examined the in vitro effects of HA4 on the differentiation of PC12 cells and neuronal progenitor cells from mouse embryo. The effect of HA4 on the proliferation and differentiation of rat oligodendrocyte precursor cells was examined. HA4 upregulated neurite-outgrowth of cultured PC12 cells in the presence of Nerve Growth Factor (NGF), which induces neuronal differentiation in PC12. HA4 also upregulated beta III-tubulin positive cells and increased the expression of neurofilament M, two of the marker proteins of nerve differentiation. The activation was observed in an HA4 dose-dependent manner, and the effect was observed at levels as low as femtogram/L. Furthermore, the enhancement of neurite-outgrowth was not observed in the presence of hyaluronan disaccharide, hexasaccharide, 12 mer or high molecular weight hyaluronan. Upregulation of neurite-outgrowth was observed only in the presence of NGF. While HA4 accelerated neurite-outgrowth of primary mouse neural progenitor cells, it did not enhance the proliferation or differentiation of oligodendrocyte precursor cells derived from rat primary neuronal progenitor cells in culture. These results suggest that the effects of HA4 alleviating some neuronal diseases may be ascribable, at least in part, to its capacity to enhance differentiation of neurons.

Adult stem cells exist in most mammalian tissues to maintain their homeostasis and help repair them. Reductions in adult stem cell function and/or number are clearly associated with aging, however, the causal correlations between such findings and the effects of aging are largely unknown. Some stem cell functional changes, such as the loss of lineage specificity and self-renewal capacity, senescence and transformation, arise in stem cells autonomously during the aging process. These autonomous changes of stem cell functions reflect the damaging effects of age on the genome, epigenome, and proteome. Other stem cell functional changes are influenced by the age-related changes in the local microenvironments (niches) or systemic environments. If stem cell-based therapy can be used not only for age-related degenerative diseases, but also normal functional declines associated with aging, consideration of the behavior of stem cells based on effects from the local microenvironments (niches) and systemic environments in older individuals will therefore be needed.

In bones, aging manifests itself as a shift towards production of myeloid cells in bone marrow, a condition associated with increased chronic inflammation by macrophages and decreased bone mass due to excess bone resorption by osteoclasts. An increase in the ratio of RANKL, a cytokine promoting osteoclast differentiation, to osteoprotegerin (OPG) , which acts as a decoy RANKL receptor, cannot explain the observed increase in osteoclast production, as serum OPG levels increase with age in humans, apparently to levels insufficient to counteract bone loss and prevent fracture. Age-related increases in osteoclastogenesis, decreases in lymphopoiesis, and inflammation including arthritis are likely best explained by a vicious cycle of myeloid skewing and inflammation occurring in bone marrow. These activities are due to aging of both hematopoietic stem cells themselves and the bone marrow microenvironment (niche cells) , which supports hematopoiesis. Impaired osteoblastogenesis and niche cell function are most likely pathologies emerging from increased oxidative stress, peroxisome proliferator-activated receptorγ (PPARγ) activity, and adipogenesis in the aging bone marrow. Currently, administration of either OPG or an anti-RANKL antibody has proved beneficial to prevent block bone dysfunction and osteoporosis in the elderly. However, anti-aging interventions targeting mesenchymal stem cell differentiation in the bone marrow may also help counteract inflammation and osteoclastic bone loss and enhance osteoblastic bone formation.

We aimed to clarify the clinical significance of circulating tumor cells (CTCs) in the tumor drainage vein blood of colorectal cancer(CRC) patients with Dukes' stage B and C. This study included 111 patients with Dukes' stage B and 86 patients with Dukes' stage C. We selected multiple genetic markers, including the cancer-associated marker (CEA), epithelial markers(CK19 and CK20), and cancer stem-like cell marker(CD133), and the mRNA levels of these genes were detected by quantitative real-time reverse transcription-polymerase chain reaction assays. In Kaplan-Meier survival curve analysis, overall survival(OS) and disease-free survival(DFS) of Dukes' stage B and C patients who were positive for CEA, CK19, CK20, and/or CD133 (CEA/CK/CD133) were significantly worse than that in patients who were negative for these markers. By Cox progression analysis, it was demonstrated that CEA/CK/CD133 mRNA in tumor drainage vein blood was an independent prognostic factor for OS and DFS in these patients. These results suggest that CEA/CK/CD133 mRNA detection in tumor drainage vein blood is a useful tool for the determination of high-risk CRC patients with Dukes' stage B and C who are in need of postoperative adjuvant therapy.

Cancer stem cells are relatively resistant to chemotherapy, and cause relapse of cancer. Thus, various strategies to eliminate cancer stem cells have recently been exploited. One of them is immunotherapy. To develop the immunotherapy targeting cancer stem cells, tumor antigens expressed in cancer stem cells have been identified, and their use in the immunotherapy is expected. However, cancer stem cells may have an immunosuppressive ability. Therefore, blockade of the immunosuppressive mechanisms of cancer stem cells may also be required for development of effective immunotherapies against cancer stem cells.

Wilms' tumor gene WT1 encodes a transcription factor and functions as an oncogene. WT1 gene product WT1 protein is a promising par-tumor-associated antigen. WT1 peptide-based immunotherapy has been performing for more than six hundred patients with leukemias and various types of solid tumors. This immunotherapy is safe and has clinical benefit especially for leukemia, glioblastoma multiforme, advanced pancreatic cancer, and ovarian cancer. As a new strategy for cancer treatment, it should be recommended to initiate immunotherapy that had a potential of eradication of cancer stem cells before surgery, chemo- and radio-therapy.

Stem cells are pluripotent and self renewing, and possess an ability to differentiate into the various cell types of a particular tissue. In cancer tissues, existence of cells showing biological similarities with stem cells, named cancer stem cells (CSC), are known to regulate the growth of the tissue and determine its prognosis. Stem cells and CSCs usually exist as minor populations of cells in a tissue. Detection and analysis of these cells are usually laborious even using fluorescence activated cell sorting (FACS) with the conventional protocols. Considering these drawbacks, we developed a novel analytical method named mRNA quantification after FACS (FACS-mQ). In FACS-mQ, cells are labeled with a fluorescent dye in a manner that minimizes RNA degradation, then cells sorted by FACS are examined by analyzing their gene expression profile. We established protocols to obtain single cells from clinical samples for flow cytometry analysis. Further, we performed FACS-mQ analysis using fluorescence-labeled antibodies, cRNA probes and locked nucleic acid (LNA) probes. Evident decrease of intracellular RNAs did not observed in FACS-mQ using immunocytochemistry. Approximately 60% of intracellular RNA was preserved after in situ hybridization using cRNA probes. These RNAs from a small number of sorted cells were suitable for quantitative analysis to establish gene expression profiles. FACS-mQ does not require laborious and time-consuming procedures; thus, it is expected to facilitate research on stem cells or cancer stem cells.

Stroke is a major neurologic disorder. Induced pluripotent stem (iPS) cells can be produced from basically any part of patients, with high reproduction ability and pluripotency to differentiate into various types of cells, suggesting that iPS cells can provide a hopeful therapy for cell transplantation. However, transplantation of iPS cells into ischemic brain has not been reported. In this study, we showed that the iPS cells fate in a mouse model of transient middle cerebral artery occlusion (MCAO). Undifferentiated iPS cells (5×10(5)) were transplanted into ipsilateral striatum and cortex at 24 h after 30 mins of transient MCAO. Behavioral and histologic analyses were performed at 28 day after the cell transplantation. To our surprise, the transplanted iPS cells expanded and formed much larger tumors in mice postischemic brain than in sham-operated brain. The clinical recovery of the MCAO+iPS group was delayed as compared with the MCAO+PBS (phosphate-buffered saline) group. iPS cells formed tridermal teratoma, but could supply a great number of Dcx-positive neuroblasts and a few mature neurons in the ischemic lesion. iPS cells have a promising potential to provide neural cells after ischemic brain injury, if tumorigenesis is properly controlled.

Human artificial chromosomes (HACs) are stable episomal gene vectors that can carry large gene inserts. We have reported complete correction of a genetic deficiency following the transfer of a HAC carrying the genomic dystrophin sequence (DYS-HAC) into induced pluripotent stem (iPS) cells derived from either a Duchenne muscular dystrophy (DMD) model mouse or a human DMD patient. The engineered iPS cells could differentiate in immunodeficient nude mice, and human dystrophin expression was detected in muscle-like tissues. Furthermore, chimeric mice generated from the engineered cells showed tissue-specific expression of dystrophin. Recently, Giulio's group has isolated and characterized a population of blood vessel-associated stem cells, called mesoangioblasts, that can differentiate into multiple mesoderm cell types, including skeletal muscle. The DYS-HAC was transferred to mesoangioblasts from the DMD-model mouse. Thus, when delivered in the arterial circulation, mesoangioblasts crossed the blood vessel wall and participated in skeletal muscle regeneration, ameliorating signs of muscular dystrophy in the DMD model mice. Most recently, the iPS cells from a DMD patient corrected with the DYS-HAC, were successfully differentiated to mesoangioblasts. Therefore, autologous transfer of genetically corrected iPS cells and muscle progenitor cells will be desirable therapeutic cells because immune suppression would not be required.

Disease modeling of ALS using patient-specific iPS cells provides several insight into ALS pathogensis(1)～3)). Solutions, including rapid differentiation from iPS cells to motor neurons(4)), to technical hurdle of iPS cell technology(5)) will advance the field of disease modeling of ALS.

Alzheimer's disease (AD) is the most common form of age-related dementia, characterized by progressive memory loss and cognitive disturbance. According to the amyloid cascade hypothesis, a prevailing theory of AD pathology, accumulation of toxic Aβ42, in the brain is the initiator of AD pathogenesis, subsequently leading to the formation of neurofibrillary tangles, and consequently neuronal loss. Mutations of presenilin 1 (PS1) and presenilin 2 (PS2), which are catalytic components of γ-secretase, are causative factors for autosomal dominant early-onset familial AD (FAD). Induced pluripotent stem cell (iPSC) technology provides a new method for elucidating the molecular basis of human diseases, including neurodegenerative diseases. Here we generate iPSCs from fibroblasts of FAD patients with mutations in PS1 (A246E) and PS2 (N141I), and characterize the differentiation of these cells into neurons. We find that FAD-iPSC-derived differentiated neurons have increased toxic Aβ42 secretion, recapitulating the molecular pathogenesis of mutant presenilins. Furthermore, secretion of Aβ42 from these neurons sharply responds to γ secretase inhibitors and modulators, indicating the potential for identification and validation of candidate drugs. Our findings demonstrate that the FAD-iPSC-derived neuron is a valid model of AD and provides an innovative strategy for the study of late-onset neurodegenerative diseases.

Currently, there is no effective treatment for the neuronal loss caused by neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or ischemic stroke. However, recent studies have shown that endogenous neural progenitor cells continuously generate new neurons in the subventricular zone (SVZ) of the adult mammalian brain. Some of these new neurons migrate to the injured site and differentiate into mature neurons. Such new neurons may be able to replace degenerated neurons and improve or repair neurological deficits. To establish a neuroregenerative therapy using this endogenous system, endogenous regulatory mechanisms must be understood. Here, we review current knowledge on the generation of new neurons in the adult brain and discuss their potential for use in replacing neurons lost to neurodegenerative diseases, including ALS, and to ischemic stroke.

The rapid and efficient induction of neural stem cells (NSCs) from pluripotent stem cells is required for the research of patient-specific iPS cells and regenerative medicine to induce their own neural cells. Here, we induced NSCs from human pluripotent stem cells within 2 weeks and these clonal NSCs were expanded efficiently by their self-renewal ability. Further, we directly induced NSCs from both mouse and human fibroblasts using four reprogramming factors (Oct4, Sox2, Klf4, and cMyc) without the clonal isolation of induced pluripotent stem cells (iPSCs). Since these NSCs rapidly developed into mature gliogenic neural stem cells, we were able to purify these rapidly differentiating NSCs without contamination of differentiation-resistant pluripotent cells. These methods will facilitate high throughput screening of phonotypes appeared in neural cells induced from the somatic cells derived from sporadic neurodegenerative diseases.