RESUMEN
BACKGROUND: Neurogenesis, the production of neural cell-types from neural stem cells (NSCs), occurs during development as well as within select regions of the adult brain. NSCs in the adult subependymal zone (SEZ) exist in a well-categorized niche microenvironment established by surrounding cells and their molecular products. The components of this niche maintain the NSCs and their definitive properties, including the ability to self-renew and multipotency (neuronal and glial differentiation). RESULTS: We describe a model in vitro NSC niche, derived from embryonic stem cells, that produces many of the cells and products of the developing subventricular zone (SVZ) and adult SEZ NSC niche. We demonstrate a possible role for apoptosis and for components of the extracellular matrix in the maintenance of the NSC population within our niche cultures. We characterize expression of genes relevant to NSC self-renewal and the process of neurogenesis and compare these findings to gene expression produced by an established neural-induction protocol employing retinoic acid. CONCLUSIONS: The in vitro NSC niche shows an identity that is distinct from the neurally induced embryonic cells that were used to derive it. Molecular and cellular components found in our in vitro NSC niche include NSCs, neural progeny, and ECM components and their receptors. Establishment of the in vitro NSC niche occurs in conjunction with apoptosis. Applications of this culture system range from studies of signaling events fundamental to niche formation and maintenance as well as development of unique NSC transplant platforms to treat disease or injury.
Asunto(s)
Neurogénesis , Células Madre/ultraestructura , Animales , Apoptosis , Encéfalo/embriología , Encéfalo/ultraestructura , Células Madre Embrionarias/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Ratones , Modelos NeurológicosRESUMEN
The prognosis of patients diagnosed with malignant gliomas including glioblastoma multiforme (GBM) is poor and there is an urgent need to develop and translate novel therapies into the clinic. Neural stem cells display remarkable tropism toward GBMs and thus may provide a platform to deliver oncolytic agents to improve survival. First we provide a brief review of clinical trials that have used intra-tumoral herpes simplex virus thymidine kinase (HSV/tk) gene therapy to treat brain tumors. Then, we review recent evidence that neural stem cells can be used to deliver HSV/tk to GBMs in animal models. While previous clinical trials used viruses or non-migratory vector-producing cells to deliver HSV/tk, the latter approaches were not effective in humans, primarily because of satellite tumor cells that escaped surgical resection and survived due to low efficiency delivery of HSV/tk. To enhance delivery of HSV/tk to kill gliomas cells, recent animal studies have focused on the ability of neural stem cells, transduced with HSV/tk, to migrate efficiently and selectively to regions occupied by GBM cells. This approach holds the promise of targeting GBM cells that have infiltrated the brain well beyond the original site of the tumor epicenter.
Asunto(s)
Terapia Genética/métodos , Glioblastoma/terapia , Neuronas/metabolismo , Simplexvirus/genética , Trasplante de Células Madre , Timidina Quinasa/metabolismo , Transducción Genética , Proteínas Virales/metabolismo , Animales , Antineoplásicos , Movimiento Celular/genética , Ensayos Clínicos como Asunto , Glioblastoma/genética , Glioblastoma/patología , Humanos , Metástasis de la Neoplasia , Neoplasias Experimentales/genética , Neoplasias Experimentales/terapia , Neuronas/patología , Viroterapia Oncolítica , Simplexvirus/enzimología , Timidina Quinasa/genética , Proteínas Virales/genéticaRESUMEN
Recent studies show that adult neural tissues can harbor stem cells within unique niches. In the mammalian central nervous system, neural stem cell (NSC) niches have been identified in the dentate gyrus and the subventricular zone (SVZ). Stem cells in the well-characterized SVZ exist in a microenvironment established by surrounding cells and tissue components, including transit-amplifying cells, neuroblasts, ependymal cells, blood vessels, and a basal lamina. Within this microenvironment, stem cell properties, including proliferation and differentiation, are maintained. Current NSC culture techniques often include the addition of molecular components found within the in vivo niche, such as mitogenic growth factors. Some protocols use bio-scaffolds to mimic the physical growth environment of living tissue. We describe a novel NSC culture system, derived from embryonic stem (ES) cells, that displays elements of an NSC niche in the absence of exogenously applied mitogens or complex physical scaffolding. Mouse ES cells were neuralized with retinoic acid and plated on an entactin-collagen-laminin-coated glass surface at high density (250,000 cells/cm(2)). Six to eight days after plating, complex multicellular structures consisting of heterogeneous cell types developed spontaneously. NSC and progenitor cell proliferation and differentiation continued within these structures. The identity of cellular and molecular components within the cultures was documented using RT-PCR, immunocytochemistry, and neurosphere-forming assays. We show that ES cells can be induced to form structures that exhibit key properties of a developing NSC niche. We believe this system can serve as a useful model for studies of neurogenesis and stem cell maintenance in the NSC niche as well as for applications in stem cell transplantation.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Sistema Nervioso/citología , Animales , División Celular , Cartilla de ADN , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Proteínas de Homeodominio/genética , Inmunohistoquímica , Ratones , Moléculas de Adhesión de Célula Nerviosa/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genéticaRESUMEN
Recent studies suggest that adult stem cells can cross germ layer boundaries. For example, bone marrow-derived stem cells appear to differentiate into neurons and glial cells, as well as other types of cells. How can stem cells from bone marrow, pancreas, skin, or fat become neurons and glia; in other words, what molecular and cellular events direct mesodermal cells to a neural fate? Transdifferentiation, dediffereniation, and fusion of donor adult stem cells with fully differentiated host cells have been proposed to explain the plasticity of adult stem cells. Here we review the origin of select adult stem cell populations and propose a unifying hypothesis to explain adult stem cell plasticity. In addition, we outline specific experiments to test our hypothesis. We propose that peripheral, tissue-derived, or adult stem cells are all progeny of the neural crest.
Asunto(s)
Linaje de la Célula/fisiología , Cresta Neural/citología , Células Madre/citología , Adulto , Animales , Biomarcadores/análisis , Diferenciación Celular/fisiología , Ectodermo/citología , Endodermo/citología , Humanos , Mesodermo/citología , Modelos Biológicos , Células Madre/químicaRESUMEN
Embryonic stem (ES) cells differentiate into all cell types of the body during development, including those of the central nervous system (CNS). After transplantation, stem cells have the potential to replace host cells lost due to injury or disease or to supply host tissues with therapeutic factors and thus provide a functional benefit. In the current study, we assessed whether mouse neuralized ES cells can incorporate into retinal tissue and prevent retinal degeneration in mnd mice. These mice have an inherited lysosomal storage disease characterized by retinal and CNS degeneration. Sixteen weeks after intravitreal transplantation into adult mice, donor cells had incorporated into most layers of the retina, where they resembled retinal neurons in terms of morphology, location in the retina, and expression of cell type-specific marker proteins. Presence of these donor cells was correlated with a reduction in the sizes and numbers of lysosomal storage bodies in host retinal cells. The presence of transplanted donor cells was also accompanied by enhanced survival of host retinal neurons, particularly photoreceptors. These results demonstrate that neuralized ES cells protect host neurons from degeneration and appear to replace at least some types of lost neurons.
Asunto(s)
Embrión de Mamíferos/citología , Neuronas/fisiología , Células Fotorreceptoras/fisiología , Retina/fisiología , Degeneración Retiniana/prevención & control , Trasplante de Células Madre , Células Madre/fisiología , Animales , Biomarcadores/análisis , Diferenciación Celular , Células Cultivadas , Estudios de Factibilidad , Lisosomas , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes Neurológicos , Retina/metabolismo , Sinapsis/metabolismoRESUMEN
Embryonic stem (ES) cells can differentiate into many specialized cell types, including those of the nervous system. We evaluated the differentiation of enhanced green fluorescent protein (EGFP)-expressing B5 mouse ES cells in vitro and in vivo after transplantation into the eyes of mice with hereditary retinal degeneration. After neural induction with retinoic acid, the majority of cells in embryoid bodies expressed markers for neural progenitors as well as for immature and mature neurons and glial cells. When induced ES cells were plated in vitro, further differentiation was observed and the majority of cells expressed beta-III Tubulin, a marker for immature neurons. In addition, many plated cells expressed markers for mature neurons or glial cells. Four days after intravitreal transplantation into the eyes of rd1 mice (a model of rapid retinal degeneration), donor cells appeared attached to the vitreal surface of the retina. After 6 weeks in vivo, most transplanted cells remained adherent to the inner retinal surface, and some donor cells had integrated into the retina. Transplanted cells exhibited some properties typical of neurons, including extensive process outgrowth with numerous varicosities and expression of neuronal and synaptic markers. Therefore, after induction B5 ES cells can acquire the morphologies of neural cells and display markers for neuronal and glial cells in vitro and in vivo. Furthermore, when placed in the proper microenvironment ES-derived neural precursors can associate closely with or migrate into nervous tissue where differentiation appears to be determined by cues provided by the local environment, in this case the degenerating neural retina.
Asunto(s)
Neuronas/patología , Retina/patología , Degeneración Retiniana/patología , Degeneración Retiniana/terapia , Trasplante de Células Madre , Animales , Biomarcadores/análisis , Diferenciación Celular , Células Cultivadas , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso/análisis , Nestina , Neuroglía/química , Neuroglía/patología , Neuronas/química , Retina/química , Tubulina (Proteína)/análisisRESUMEN
Propylthiouracil (PTU) is an anti-thyroid drug that reportedly can impair olfactory function in humans and mice. In the mouse, PTU treatment disrupts survival and differentiation of olfactory precursor neurons. While the mechanism responsible for this effect is not understood, it is suspected that these changes are consequent to localized toxicity due to PTU metabolism. In vitro and in vivo studies in other biological systems demonstrate that PTU can significantly alter glutathione S-transferase (GST) enzyme expression and activity. The localization of GST biotransformation enzymes in basal cells, sustentacular cells and Bowman's glands of the olfactory mucosa suggests that these cells play a significant role in olfactory physiology. This study investigated the effects of PTU treatment, T(4) replacement therapy and thyroidectomy on GST expression, GST and glutathione peroxidase (GSH-PX) activity in mouse olfactory tissue. One month treatment with PTU revealed a significant decrease in expression of GST alpha (37%) as identified by Western blot analysis. In contrast, no change in GST mu expression was observed after 1 month of treatment. Concomitant treatment with T(4) caused a significant induction of GST alpha, and mu isozymes. GST enzyme activity significantly decreased after 1 month of PTU treatment (53%) and remained suppressed, despite the presence of exogenous T(4). GSH-PX activity significantly decreased after 1 month of PTU treatment (30%) and remained at control levels with T(4) supplementation. Thyroidectomy caused a 25% reduction in olfactory GST alpha expression. Total GST and GSH-PX activity were not altered in these animals. Supplementation with T(4) in thyroidectomized animals prevented the suppression of GST alpha expression. These results suggest that the combined action of localized PTU toxicity and altered levels of circulating thyroid hormone contribute to PTU-mediated abnormalities in the olfactory system.