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An important issue in cancer therapy is the presence of a population that is resistant to anticancer treatment. This resistance has been partly ascribed to the presence of quiescent stem cells in a cancer population. However, how the quiescent state is induced in a proliferating cancer population is totally obscure. We think that our study on the stem cell system of pigment cells will provide some insight into the molecular basis for cancer stem cells, because the quiescent melanocyte stem cell would be the ideal model for understanding the process generating quiescent stem cells. In this article, we review our latest understanding of the quiescent stem cells of the melanocyte lineage by referring to some related topics of cancer stem cells. [ABSTRACT FROM AUTHOR]

Transplantation of fetal dopaminergic (DA) neurons can produce symptomatic relief for patients with Parkinson's disease, but the technical and ethical difficulties have limited the application of this therapy. Neural precursor cells and embryonic stem cells (ESCs) are expected to be candidates of potential donor cells for transplantation. Human neural precursor cells obtained from the midbrain give rise to TH-positive neurons. The growth of the cells, however, is slow and the differentiation rate of DA neurons is still low for clinical application. Monkey ESCs give rise to midbrain DA neurons, and the transplanted ESC-derived neurospheres function as DA neurons, attenuating the neurological symptoms of the monkey Parkinson's disease model. These results suggest the possibility of using stem cells for the treatment of Parkinson's disease, but problems such as the low survival rate in vivo and tumor formation must be solved. [ABSTRACT FROM AUTHOR]

It has long been believed that the adult mammalian central nervous system does not regenerate after injury. However, recent advances in the field of stem cell biology, including the identification of Musashi-1-positive neural stem cells (NSCs) or NSC-like cells, has provided new insight for the development of novel therapeutic strategies aimed at inducing regeneration in the damaged central nervous system (CNS). The major strategies for inducing regeneration in the damaged CNS can be classified into two subgroups: (1) activation of endogenous neural stem cells and (2) cell transplantation therapies. In this paper, we would like to summarize our recent findings on the functions of the neural RNA-binding protein Musashi-1 expressed in neural stem cells in relation to insult-induced neurogenesis, and therapeutic interventions for spinal cord injury, especially focusing on the treatment of spinal cord injury in the acute phase with anti-IL-6 receptor blocking antibody. [ABSTRACT FROM AUTHOR]

Diseases of the brain and spinal cord are especially daunting challenges for cell-based strategies of repair, given the multiplicity of cell types within the adult central nervous system, and the precision with which they must interact in both space and time. Nonetheless, a number of diseases are especially appropriate for cell-based therapy, in particular those in which single phenotypes are lost. Foremost among these are the disorders of myelin, in which oligodendrocytes are the specific and often sole victims of the underlying disease process. These include not only the vascular, traumatic, and inflammatory demyelinations of adulthood, but also the congenital and childhood dysmyelinating syndromes of the pediatric leukodystrophies. These congenital disorders of myelin formation and maintenance may present especially compelling targets for cell-based neurological therapy. [ABSTRACT FROM AUTHOR]

Transplantation of male germ line stem cells from a fertile donor to the testis of an infertile recipient restores donor-derived spermatogenesis in the recipient testis and the resulting sperm pass the donor genotype to the offspring of the recipient. Germ cell transplantation has been an invaluable tool to elucidate the biology of male germ line stem cells and their niche in the testis, develop systems to isolate and culture spermatogonial stem cells, examine defects in spermatogenesis, correct male infertility and introduce genetic changes into the male germ line. Although most widely studied in rodents, germ cell transplantation has been applied to larger mammals, including primates. Recently, ectopic grafting of testis tissue from diverse donor species, including primates, into a mouse host has opened an additional possibility to study spermatogenesis and to produce fertile sperm from immature donors. Testis xenografts are ideally suitable to study toxicants or drugs with the potential to enhance or suppress male fertility without the necessity of performing experiments in the target species. Therefore, transplantation of germ cells or xenografting of testis tissue represent powerful approaches for the study, preservation, and manipulation of male fertility. [ABSTRACT FROM AUTHOR]

Germ cells provide an enduring link between generations and therefore must possess the fundamental ability of reprogramming their genome to generate a totipotent state. We wish to understand the molecular basis of the unique properties of the mammalian germ line. Recently we identified Blimp1, a potent transcriptional repressor of a histone methyltransferase subfamily, as a critical determinant of the germ cell lineage in mice. Surprisingly, Blimp1 expression marks the origin of the germ line in proximal epiblast cells in pregastrulation embryos, substantially earlier than previously thought. Furthermore, we showed that established primordial germ cells undergo extensive erasure of genome-wide histone H3 lysine 9 dimethylation (H3K9me2) and DNA methylation, two major repressive epigenetic modifications, and instead acquire high levels of H3-K27 trimethylation (H3K27me3) in their migration period. We suggest that germline specification is a genetic system for the orderly reprogramming of the cells' epigenome toward a totipotent state, with reacquisition of totipotency-associated transcription factors and continued Blimp1 expression preventing their reversion to an explicit pluripotent state or somatic differentiation. [ABSTRACT FROM AUTHOR]

Embryonic stem cells (ESCs), derivatives of cells of early mammalian embryos, have proven to be one of the most powerful tools in developmental and stem cell biology. When injected into embryos, ESCs can contribute to tissues derived from all three germ layers and to the germline. Prior studies have successfully shown that ESCs can recapitulate features of embryonic development by spontaneously forming somatic lineages in culture. Amazingly, recently it has been shown that mouse ESCs can also give rise to primordial germ cells (PGCs) in culture that are capable of undergoing meiosis and forming both male and female gametes. While the full potential of these ES-derived germ cells and gametes remains to be demonstrated, these discoveries provide a new approach for studying reproductive biology and medicine. [ABSTRACT FROM AUTHOR]

Nuclear transfer can be used to generate embryonic stem cell (ntESC) lines from a patient's own somatic cells. We have shown that ntESCs can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. Several reports have already demonstrated that ntESCs can be used in regenerative medicine in order to rescue immunodeficient or infertile phenotypes. However, it is unclear whether ntES cells are identical to fertilized embryonic stem cells (ESCs). This review seeks to describe the phenotype and possible abnormalities of ntESC lines. [ABSTRACT FROM AUTHOR]

Stem cells have been targeted to many organ systems specifically to replace scarred organs and to rejuvenate diseased organs. Even though our understanding of the versatility of stem cells is slowly unraveling, tracking these cells as they enter the body has become a very important field of study. In this chapter, we review various modalities for imaging stem cells and assess the advantages and shortcomings of each technique. [ABSTRACT FROM AUTHOR]

There are few systems which enable adult tissue stem cells to be studied. However, the gastrointestinal tract with its high degree of polarity, well-defined cell migratory pathways, and dynamic cell replacement is a model tissue providing unique opportunities for stem cell study. Lineage tracking indicates that all cell replacement originates at well-defined stem cell positions, with an associated slower cell cycle. Radiobiological studies suggest a hierarchical stem cell compartment (actual and potential stem cells). Actual stem cells have an intolerance of genotoxic damage and die via apoptosis. Stem cells also selectively sort the old and new DNA strands at division, retaining the replication error free strands in the stem cell daughter. High genotoxic sensitivity and selective sorting of old and new DNA strands, provides extremely effective protective mechanisms against both replication and random errors. This provides a new explanation for the low cancer risk in the small intestine. [ABSTRACT FROM AUTHOR]

Stem cell commitment and differentiation entails the successive loss of self-renewal and developmental potential, and results in the final restriction to a terminally differentiated mature cell type. Hematopoiesis, the development of blood cells from hematopoietic stem cells in bone marrow, is particularly well studied, and at different branching points within the hematopoietic system multiple developmental intermediates have been identified. Here we describe a Flt3+CD11b+ multipotent progenitor that can be amplified in vitro by a specific cytokine combination to high cell numbers, and following adoptive transfer into syngeneic mice, it generates dendritic cells but also additional mature cell types. By employing gene expression profiling with DNA microarrays and knockout mouse models, we demonstrate that the helix-loop-helix (HLH) transcription factor Id2 (inhibitor of DNA binding/differentiation 2) acts as a molecular switch in development of Langerhans cells (LCs), the cutaneous contingent of dendritic cells (DCs), and of specific DC subsets and B cells. [ABSTRACT FROM AUTHOR]

The possibility of turning one somatic cell type into another may in the long run have beneficial applications in regenerative medicine. Somatic cell nuclear transfer (therapeutic cloning) may offer this possibility; however, ethical guidelines prevent application of this technology in many in countries. As a result, alternative approaches are being developed for altering cell fate. This communication discusses recent non-nuclear transfer-based in vitro approaches for reprogramming cells and enhancing their potential for differentiation toward various lineages. [ABSTRACT FROM AUTHOR]

Embryonic stem cells (ESCs), embryonic germ cells (EGCs), and embryonic carcinoma cells (ECCs) are three types of pluripotent cells derived from mammalian embryos. The three cell types are capable not only of self-renewal, but also of having the potential to give rise to cells of all tissue types in the fetal and adult body. In several reports, ESCs, ECCs, and EGCs have been described to reprogram somatic cells in vitro. After reprogramming caused by fusion, somatic cells exhibit various features of pluripotent cells: expression of pluripotency markers (e.g., Oct4, nanog, and Rex-1), absence of tissue-specific gene expression, reactivation of inactive X chromosome of female somatic cells, demethylation, as well as histone modification. An activity in pluripotent stem cells appears to be capable of inducing the global changes inherent in the reprogramming of somatic cells. Investigations involving pluripotent stem cells will yield substantial insight into various fundamental biological processes, such as cellular differentiation and de-differentiation. Most importantly for the public, however, is that such studies might lead into cell-based therapies and as such have the potential to change regenerative medicine. [ABSTRACT FROM AUTHOR]

The functional difference between the maternal and paternal genome, which is characterized by epigenetic modifications during gametogenesis, that is genomic imprinting, prevents mammalian embryos from parthenogenesis. Genomic imprinting leads to nonequivalent expression of imprinted genes from the maternal and paternal alleles. However, our research showed that alteration of maternal imprinting by oocyte reconstruction using nongrowing oocytes together with deletion of the H19 gene, provides appropriate expression of maternally imprinted genes. Here we discuss that further alteration of paternally imprinted gene expressions at chromosomes 7 and 12 allows the ng/fg parthenogenetic embryos to develop to term, suggesting that the paternal contribution is obligatory for the descendant. [ABSTRACT FROM AUTHOR]

At present, the limitation of Phenotype-based genetic screening in embryonic stem cells (ESCs) is the diploid nature of the genome. Since it is known that cells deficient in the Bloom's syndrome gene (Blm) show an increased rate of homologous recombination, we have developed a new system to conditionally regulate the Blm allele for introduction of bi-allelic mutations across the genome. Transient deficiency of Blm induces homologous recombination not only between sister chromatids but also between homologous chromosomes, resulting in a high rate of loss of heterozygosity (LOH). Introduction of genome-wide mutations in ESCs can be achieved by retroviral vector. In combination, using genome-wide mutagenesis and transient loss of Blm expression, we have generated ES libraries with bi-allelic mutations. These results show that this new system is very efficient for identifying gene functions in ESCs. [ABSTRACT FROM AUTHOR]

Niche has become the most important issue in stem cell biology, but it is still a hypothetical notion that cannot be defined in a better way than the microenvironment surrounding stem cells. Using a melanocyte stem cell system as a model, we have analyzed the cellular and molecular requirements for differentiation of quiescent stem cells. Our results demonstrate the multiple subsets within the stem cell compartment and thus suggests the complexity of niche. [ABSTRACT FROM AUTHOR]

Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much optimism for their contribution to human medicine. [ABSTRACT FROM AUTHOR]

The isolation of endothelial progenitor cells (EPCs) derived from bone marrow (BM) was one epoch-making event for the recognition of neovessel formation in adults occurring as physiological and pathological responses. The finding that EPCs home to sites of neovascularization and differentiate into endothelial cells (ECs) in situ is consistent with vasculogenesis, a critical paradigm that has been well described for embryonic neovascularization, but proposed recently in adults in which a reservoir of stem or progenitor cells contribute to vascular organogenesis. EPCs have also been considered as therapeutic agents to supply the potent origin of neovascularization under pathological conditions. This chapter highlights an update of EPC biology as well as its potential use for therapeutic regeneration. [ABSTRACT FROM AUTHOR]

We have recently identified a stromal cell-derived inducing activity (SDIA), which induces differentiation of neural cells from mouse embryonic stem (ES) cells. Particularly, midbrain TH+dopaminergic neurons are generated efficiently in this system. These dopaminergic neurons are transplantable and survive well in the 6-OHDA-treated mouse striatum. SDIA induces co-cultured ES cells to differentiate into rostral central nervous system (CNS) tissues containing both ventral and dorsal cells. While early exposure of SDIA-treated ES cells to BMP4 suppresses neural differentiation and promotes epidermogenesis, late BMP4 exposure after the 4th day of co-culture causes differentiation of neural crest cells and dorsal-most CNS cells, with autonomic system and sensory lineages induced preferentially by high and low BMP4 concentrations, respectively. In contrast, Sonic Hedgehog (Shh) suppresses differentiation of neural crest lineages and promotes that of ventral CNS tissues such as motor neurons and HNF3ß+ floor plate cells with axonal guidance activities. Thus, SDIA-treated ES cells generate naïve precursors that have the competence of differentiating into the "full" dorsal-ventral range of neuroectodermal derivatives in response to patterning signals. I also discuss the role of SDIA and the mode of rostral-caudal specification of neuralized ES cells. In addition, I would like to discuss them in the light of control of in vitro neural production for the use in regenerative medicine for parkinsonism and retinal degeneration. [ABSTRACT FROM AUTHOR]

The Polycomb group (PcG) gene Bmi-1 has recently been implicated in the maintenance of hematopoietic stem cells (HSCs). However, the role of each component of PcG complex in HSCs and the impact of forced expression of PcG genes on stem cell self-renewal remain to be elucidated. To address these issues, we performed both loss-of-function and gain-of-function analysis on various PcG proteins. Expression analysis revealed that not only Bmi-1 but also other PcG genes are predominantly expressed in HSCs. Loss-of-function analyses, however, demonstrated that absence of Bmi-1 is preferentially linked with a profound defect in HSC self-renewal, indicating a central role for Bmi-1, but not the other components, in the maintenance of HSC self-renewal. Overexpression analysis of PcG genes also confirmed an important role of Bmi-1 in HSC self-renewal. Our findings indicate that the expression level of Bmi-1 is the critical determinant for the self-renewal capacity of HSCs. These findings uncover novel aspects of stem cell regulation exerted through epigenetic modifications by the PcG proteins. [ABSTRACT FROM AUTHOR]

In cases of corneal epithelial stem cell deficiency where ocular surface reconstruction is required, corneal epithelial replacement using a tissue engineering technique shows great potential. Autologous cultivated corneal epithelial stem cell sheets are the safest and most reliable forms of sheet we can use for such treatment; however, they are not useful for treating bilaterally affected ocular surface disorders. In order to treat such cases, we must choose either an allogeneic cultivated corneal epithelial sheet or an autologous cultivated oral mucosal epithelial sheet. If we use the former, the threat of immunological reaction must be dealt with. Therefore, it is imperative that we have a basic understanding of the immunological aspects of ocular surface reconstruction using allogeneic tissues. When using an autologous cultivated oral mucosal epithelial sheet, a basic understanding of ocular surface epithelial biology is required as the sheet is not exactly the same as corneal epithelium. [ABSTRACT FROM AUTHOR]

Recent studies are beginning to reveal that our basic concepts of epidermal stem cell biology may be based on somewhat tenuous ground. For example, it is often assumed that colony-forming cells represent epidermal stem cells, although this has not proved to be the case in hematopoietic cell lineages. In addition, although most stem cells are not cycling, label-retaining cells are used as a primary measure of epidermal stem cells. Moreover, the locations of stem cell niches in epidermis are still being debated. Finally, while putative stem cell markers abound, the most effective isolation procedure for stem cells has not been determined, and the relative efficiency of various methods of stem cell isolation remains unknown. With a functional assay for epidermal stem cells (analogous to the in-vivo competitive assay used for hematopoiesis), we appear to be in a better position to more clearly define the molecular signature of the true long-term repopulating cell/stem cell of the epidermis. Nonetheless, significant progress has been made in regenerative therapy of the epidermis for ulcer and burn treatment, and for corrective gene therapy for inherited skin diseases [ABSTRACT FROM AUTHOR]

We have established several embryonic stem (ES) cell lines of the cynomolgus monkey. They maintain the normal karyotype and pluripotency in culture for long periods. We obtained government approval and grants to produce human ES cell lines from frozen surplus embryos in April 2002. We have established and characterized three human ES cell lines (KhES-1, KhES-2, KhES-3). We started distribution of human ES cells to other research groups in March 2004. It would be important to produce genetically modified monkey and human ES cells for various purposes. After improvement of the transfection and selection methods, we have produced monkey ES cells with integrated transgenes at efficient and reliable rates. We are also investigating reprogramming of somatic cells into pluripotent stem cells by cell fusion with ES cells. Such reprogramming could be used to produce pluripotent stem cells for each patient without therapeutic cloning, which would raise ethical concerns. [ABSTRACT FROM AUTHOR]

There are many notions in stem cell biology that lack proof. The stem cell niche is the most typical example. While it is a convenient terminology for designating anything that supports stem cells, the cellular basis of the niche is poorly understood for many stem cell systems. In this chapter, we describe how useful the melanocyte system would be for investigating the molecular and cellular basis of the niche. [ABSTRACT FROM AUTHOR]