CD44 and hyaluronan promote invasive growth of B35 neuroblastoma cells into the brain.

Hyaluronan and its receptor CD44 are known to contribute to the invasive growth of different tumors of the central nervous system. It is not known, however, if CD44 is sufficient to activate invasive growth into the brain tissue. This study examines how CD44 regulates the motility and invasive growth of B35 neuroblastoma cells into a hyaluronan-rich environment. A comprehensive experimental approach was used encompassing biochemical techniques, single molecule microscopy, correlative confocal and scanning electron microscopy, morphometry of cellular extensions, live-cell imaging and tracking, transplantation onto organotypic brain slices, two-photon imaging and invasion assays. We found that CD44-GFP fusion protein was localized in filopodia and in focal bleb-like protrusions where it provided binding sites for hyaluronan. Transient expression of CD44-GFP was sufficient to increase the length of filopodia, to enhance cell migration and to promote invasive growth into hyaluronan-rich brain tissue. Thus, CD44 controls molecular devices localized in filopodia and bleb-like specializations of the cell surface that enhance cell migration and invasive growth.

Adult germ line stem cells as a source of functional neurons and glia.

The derivation of autologous pluripotent cells has become a central goal in translational stem cell research. Although somatic cell nuclear transfer and transcription factor-based reprogramming enable the generation of pluripotent cells from adult tissue, both methodologies depend on complex epigenetic alterations. Recent data suggest that the adult germ line may represent an alternative and natural source of pluripotent stem cells. Multipotent adult germ line stem cells (maGSCs) with properties similar to those of embryonic stem cells have been derived from mouse spermatogonial stem cells. These cells exhibit extensive self-renewal, expression of pluripotency markers, and differentiation into derivatives of all three germ layers. Here we report the derivation of multipotent neural and glial precursors as well as adherently proliferating neural stem cells from maGSCs. Characterization of maGSC-derived neurons revealed segregation into GABAergic, glutamatergic, serotonergic, and tyrosine hydroxylase-positive phenotypes. On a functional level, maGSC-derived neurons generate spontaneously active functional networks, which use both glutamatergic and GABAergic synaptic transmission and engage in synchronized oscillatory activity. maGSC-derived oligodendrocytes undergo full maturation and ensheathe host axons in myelin-deficient tissue. Our data suggest that neural stem and precursor cells derived from maGSCs could provide a versatile and potentially autologous source of functional neurons and glia.

Neural cell adhesion molecule polysialylation enhances the sensitivity of embryonic stem cell-derived neural precursors to migration guidance cues.

The development of stem cell-based neural repair strategies requires detailed knowledge on the interaction of migrating donor cells with the host brain environment. Here we report that overexpression of polysialic acid (PSA), a carbohydrate polymer attached to the neural cell adhesion molecule (NCAM), in embryonic stem (ES) cell-derived glial precursors (ESGPs) strikingly modifies their migration behavior in response to guidance cues. ESGPs transduced with a retrovirus encoding the polysialyltransferase STX exhibit enhanced migration in monolayer cultures and an increased penetration of organotypic slice cultures. Chemotaxis assays show that overexpression of PSA results in an enhanced chemotactic migration toward gradients of a variety of chemoattractants, including fibroblast growth factor 2 (FGF2), platelet-derived growth factor, and brain-derived neurotrophic factor (BDNF), and that this effect is mediated via the phosphatidylinositol 3'-kinase (PI3K) pathway. Moreover, PSA-overexpressing ESGPs also exhibit an enhanced chemotactic response to tissue explants derived from different brain regions. The effect of polysialylation on directional migration is preserved in vivo. Upon transplantation into the adult striatum, PSA-overexpressing but not control cells display a targeted migration toward the subventricular zone. On the basis of these data, we propose that PSA plays a crucial role in modulating the ability of migrating precursor cells to respond to regional guidance cues within the brain tissue. Disclosure of potential conflicts of interest is found at the end of this article.

Generation and potential biomedical applications of embryonic stem cell-derived glial precursors.

Recent progress in embryonic and adult stem cell research has opened new perspectives for generating large numbers of different neural cell types in vitro and using them for nervous system repair. Several lines of arguments suggest that myelin diseases represent particularly attractive targets for cell-based therapies. First, in contrast to neuronal cell replacement, a single and uniform cell type, the oligodendrocyte progenitor, suffices for therapeutic remyelination in all areas of the CNS, with no need for complex circuit integration. Second, there is an increasing understanding of the mechanisms regulating the recruitment of stem and progenitor cells into CNS lesions. Third, stem cells represent excellent vehicles for cell-mediated gene transfer, enabling novel approaches, which combine classic cell replacement with the delivery of therapeutic factors. Among the various donor sources, embryonic stem (ES) cells stand out as a population featuring pluripotency, unlimited self-renewal and amenability to gene targeting. Here we discuss the advantages, challenges and perspectives of bringing this unique cell type closer to a clinical application for treating myelin diseases and other neurological disorders.

Retinoic acid induction of ES-cell-derived neurons: the radial glia connection.

Neuronal induction by retinoic acid (RA) is commonly used in embryonic stem (ES) cell differentiation. Two recent papers show that this paradigm induces a population of neurogenic precursors with properties of radial glia. Upon differentiation, RA-treated cells give rise to a defined and developmentally restricted neuronal lineage. This role of RA in cell fate specification provides new perspectives for studying the radial glia-neuron transition and for generating homogenous populations of neurons from ES cells.

Generation of purified oligodendrocyte progenitors from embryonic stem cells.

Demyelination is a key component in the pathogenesis of many neurological disorders. Transplantation of myelinating cells may offer a therapeutic approach to restore neurological function in these diseases. Recent findings suggest that pluripotent embryonic stem (ES) cells can serve as an unlimited donor source for neural transplantation. The clinical application of ES cells for myelin repair will depend critically on the ability to enrich oligodendroglial progenitors in high purity. Combining controlled differentiation in the presence of growth factors and genetic lineage selection, we devised a cell culture protocol yielding highly purified oligodendrocyte progenitors. Murine ES cell clones stably transfected with a construct encoding the beta-galactosidase-neomycine phosphotransferase fusion protein (beta(geo)) under control of the 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter were differentiated into bipotential glial precursors. Subsequent induction of a CNP-positive stage and selection in neomycine resulted in a homogenous cell population with a pre-oligodendrocyte phenotype. The selected cells continued to proliferate in the presence of FGF-2 and PDGF and, upon growth factor withdrawal, differentiated into mature galactocerebroside (GalC)-positive oligodendrocytes. Transplantation studies in myelin-deficient (md) rats indicate that ES cell-derived oligodendrocyte progenitors generated with this method may serve as an attractive donor source for myelin repair.

ES cell-derived glial precursors contribute to remyelination in acutely demyelinated spinal cord lesions.

Pluripotent embryonic stem (ES) cells have emerged as a powerful tool for disease modeling and neural regeneration. Transplantation studies in rodents indicate that ES cell-derived glial precursors (ESGPs) efficiently restore myelin in dysmyelinating mutants and chemically induced foci of myelin loss. Here we explore the myelination potential of ESGPs in an antibody/complement-induced demyelination model. Microinjection of an antibody to myelin oligodendrocyte glycoprotein (MOG) and complement was employed to generate circumscribed areas of demyelination in the adult rat spinal cord. ESGPs transplanted into 2-day-old lesions were found to survive and differentiate into both oligodendrocytes and astrocytes. The engrafted cells remained largely confined to the lesion site and showed no evidence of tumor formation up until 4 weeks after transplantation. Within areas of pronounced microglial activation and macrophage extravasation, engrafted ES cell-derived oligodendrocytes contacted and enwrapped host axons and alongside endogenous glia, contributed to the formation of new myelin sheaths. These findings demonstrate that ESGPs transplanted into acutely demyelinated lesions can contribute to myelin repair.

Induction of pluripotency in adult unipotent germline stem cells.

Mouse and human stem cells with features similar to those of embryonic stem cells have been derived from testicular cells. Although pluripotent stem cells have been obtained from defined germline stem cells (GSCs) of mouse neonatal testis, only multipotent stem cells have been obtained so far from defined cells of mouse adult testis. In this study we describe a robust and reproducible protocol for obtaining germline-derived pluripotent stem (gPS) cells from adult unipotent GSCs. Pluripotency of gPS cells was confirmed by in vitro and in vivo differentiation, including germ cell contribution and transmission. As determined by clonal analyses, gPS cells indeed originate from unipotent GSCs. We propose that the conversion process requires a GSC culture microenvironment that depends on the initial number of plated GSCs and the length of culture time.

Tripotential differentiation of adherently expandable neural stem (NS) cells.

BACKGROUND: A recent study has shown that pure neural stem cells can be derived from embryonic stem (ES) cells and primary brain tissue. In the presence of fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF), this population can be continuously expanded in adherent conditions. In analogy to continuously self-renewing ES cells, these cells were termed 'NS' cells (Conti et al., PLoS Biol 3: e283, 2005). While NS cells have been shown to readily generate neurons and astrocytes, their differentiation into oligodendrocytes has remained enigmatic, raising concerns as to whether they truly represent tripotential neural stem cells. METHODOLOGY/PRINCIPAL FINDINGS: Here we provide evidence that NS cells are indeed tripotent. Upon proliferation with FGF2, platelet-derived growth factor (PDGF) and forskolin, followed by differentiation in the presence of thyroid hormone (T3) and ascorbic acid NS cells efficiently generate oligodendrocytes ( approximately 20%) alongside astrocytes ( approximately 40%) and neurons ( approximately 10%). Mature oligodendroglial differentiation was confirmed by transplantation data showing that NS cell-derived oligodendrocytes ensheath host axons in the brain of myelin-deficient rats. CONCLUSIONS/SIGNIFICANCE: In addition to delineating NS cells as a potential donor source for myelin repair, our data strongly support the view that these adherently expandable cells represent bona fide tripotential neural stem cells.