Olfactory bulb hypoplasia in Prokr2 null mice stems from defective neuronal progenitor migration and differentiation.

New neurons are added on a daily basis to the olfactory bulb (OB) of a mammal, and this phenomenon exists throughout its lifetime. These new cells are born in the subventricular zone and migrate to the OB via the rostral migratory stream (RMS). To examine the role of the prokineticin receptor 2 (Prokr2) in neurogenesis, we created a Prokr2 null mouse, and report a decrease in the volume of its OB and also a decrease in the number of bromodeoxyuridine (BrdU)-positive cells. There is disrupted architecture of the OB, with the glomerular layer containing terminal dUTP nick-end labeling (TUNEL) -positive nuclei and also a decrease in tyrosine hydroxylase-positive neurons in this layer. In addition, there are increased numbers of doublecortin-positive neuroblasts in the RMS and increased PSA-NCAM (polysialylated form of the neural cell adhesion molecule) -positive neuronal progenitors around the olfactory ventricle, indicating their detachment from homotypic chains is compromised. Finally, in support of this, Prokr2-deficient cells expanded in vitro as neurospheres are incapable of migrating towards a source of recombinant human prokineticin 2 (PROK2). Together, these findings suggest an important role for Prokr2 in OB neurogenesis

Secreted factors from olfactory mucosa cells expanded as free-floating spheres increase neurogenesis in olfactory bulb neurosphere cultures.

BACKGROUND: The olfactory epithelium is a neurogenic tissue comprising a population of olfactory receptor neurons that are renewed throughout adulthood by a population of stem and progenitor cells. Because of their relative accessibility compared to intra-cranially located neural stem/progenitor cells, olfactory epithelium stem and progenitor cells make attractive candidates for autologous cell-based therapy. However, olfactory stem and progenitor cells expand very slowly when grown as free-floating spheres (olfactory-spheres) under growth factor stimulation in a neurosphere assay. RESULTS: In order to address whether olfactory mucosa cells extrinsically regulate proliferation and/or differentiation of immature neural cells, we cultured neural progenitor cells derived from mouse neonatal olfactory bulb or subventricular zone (SVZ) in the presence of medium conditioned by olfactory mucosa-derived spheres (olfactory-spheres). Our data demonstrated that olfactory mucosa cells produced soluble factors that affect bulbar neural progenitor cell differentiation but not their proliferation when compared to control media. In addition, olfactory mucosa derived soluble factors increased neurogenesis, especially favouring the generation of non-GABAergic neurons. Olfactory mucosa conditioned medium also contained several factors with neurotrophic/neuroprotective properties. Olfactory-sphere conditioned medium did not affect proliferation or differentiation of SVZ-derived neural progenitors. CONCLUSION: These data suggest that the olfactory mucosa does not contain factors that are inhibitory to neural stem/progenitor cell proliferation but does contain factors that steer differentiation toward neuronal phenotypes. Moreover, they suggest that the poor expansion of olfactory-spheres may be in part due to intrinsic properties of the olfactory epithelial stem/progenitor cell population.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Secreted factors from olfactory mucosa cells expanded as free-floating spheres increase neurogenesis in olfactory bulb neurosphere cultures

<p>Abstract</p> <p>Background</p> <p>The olfactory epithelium is a neurogenic tissue comprising a population of olfactory receptor neurons that are renewed throughout adulthood by a population of stem and progenitor cells. Because of their relative accessibility compared to intra-cranially located neural stem/progenitor cells, olfactory epithelium stem and progenitor cells make attractive candidates for autologous cell-based therapy. However, olfactory stem and progenitor cells expand very slowly when grown as free-floating spheres (olfactory-spheres) under growth factor stimulation in a neurosphere assay.</p> <p>Results</p> <p>In order to address whether olfactory mucosa cells extrinsically regulate proliferation and/or differentiation of immature neural cells, we cultured neural progenitor cells derived from mouse neonatal olfactory bulb or subventricular zone (SVZ) in the presence of medium conditioned by olfactory mucosa-derived spheres (olfactory-spheres). Our data demonstrated that olfactory mucosa cells produced soluble factors that affect bulbar neural progenitor cell differentiation but not their proliferation when compared to control media. In addition, olfactory mucosa derived soluble factors increased neurogenesis, especially favouring the generation of non-GABAergic neurons. Olfactory mucosa conditioned medium also contained several factors with neurotrophic/neuroprotective properties. Olfactory-sphere conditioned medium did not affect proliferation or differentiation of SVZ-derived neural progenitors.</p> <p>Conclusion</p> <p>These data suggest that the olfactory mucosa does not contain factors that are inhibitory to neural stem/progenitor cell proliferation but does contain factors that steer differentiation toward neuronal phenotypes. Moreover, they suggest that the poor expansion of olfactory-spheres may be in part due to intrinsic properties of the olfactory epithelial stem/progenitor cell population.</p

Laminin enhances the growth of human neural stem cells in defined culture media

<p>Abstract</p> <p>Background</p> <p>Human neural stem cells (hNSC) have the potential to provide novel cell-based therapies for neurodegenerative conditions such as multiple sclerosis and Parkinson's disease. In order to realise this goal, protocols need to be developed that allow for large quantities of hNSC to be cultured efficiently. As such, it is important to identify factors which enhance the growth of hNSC. <it>In vivo</it>, stem cells reside in distinct microenvironments or niches that are responsible for the maintenance of stem cell populations. A common feature of niches is the presence of the extracellular matrix molecule, laminin. Therefore, this study investigated the effect of exogenous laminin on hNSC growth.</p> <p>Results</p> <p>To measure hNSC growth, we established culture conditions using B27-supplemented medium that enable neurospheres to grow from human neural cells plated at clonal densities. Limiting dilution assays confirmed that neurospheres were derived from single cells at these densities. Laminin was found to increase hNSC numbers as measured by this neurosphere formation. The effect of laminin was to augment the proliferation/survival of the hNSC, rather than promoting the undifferentiated state. In agreement, apoptosis was reduced in dissociated neurospheres by laminin in an integrin β1-dependent manner.</p> <p>Conclusion</p> <p>The addition of laminin to the culture medium enhances the growth of hNSC, and may therefore aid their large-scale production.</p

Astrocytes in a dish:Using pluripotent stem cells to model neurodegenerative and neurodevelopmental disorders

Neuroscience and Neurobiology have historically been neuron biased, yet up to 40% of the cells in the brain are astrocytes. These cells are heterogeneous and regionally diverse but universally essential for brain homeostasis. Astrocytes regulate synaptic transmission as part of the tripartite synapse, provide metabolic and neurotrophic support, recycle neurotransmitters, modulate blood flow and brain blood barrier permeability and are implicated in the mechanisms of neurodegeneration. Using pluripotent stem cells (PSC), it is now possible to study regionalised human astrocytes in a dish and to model their contribution to neurodevelopmental and neurodegenerative disorders. The evidence challenging the traditional neuron-centric view of degeneration within the CNS is reviewed here, with focus on recent findings and disease phenotypes from human PSC-derived astrocytes. In addition we compare current protocols for the generation of regionalised astrocytes and how these can be further refined by our growing knowledge of neurodevelopment. We conclude by proposing a functional and phenotypical characterisation of PSC-derived astrocytic cultures that is critical for reproducible and robust disease modelling

Efficient and Scalable Generation of Human Ventral Midbrain Astrocytes from Human-Induced Pluripotent Stem Cells

In Parkinson's disease, progressive dysfunction and degeneration of dopamine neurons in the ventral midbrain cause life-changing symptoms. Neuronal degeneration has diverse causes in Parkinson's, including non-cell autonomous mechanisms mediated by astrocytes. Throughout the CNS, astrocytes are essential for neuronal survival and function, as they maintain metabolic homeostasis in the neural environment. Astrocytes interact with the immune cells of the CNS, microglia, to modulate neuroinflammation, which is observed from the earliest stages of Parkinson's, and has a direct impact on the progression of its pathology. In diseases with a chronic neuroinflammatory element, including Parkinson's, astrocytes acquire a neurotoxic phenotype, and thus enhance neurodegeneration. Consequently, astrocytes are a potential therapeutic target to slow or halt disease, but this will require a deeper understanding of their properties and roles in Parkinson's. Accurate models of human ventral midbrain astrocytes for in vitro study are therefore urgently required. We have developed a protocol to generate high purity cultures of ventral midbrain-specific astrocytes (vmAstros) from hiPSCs that can be used for Parkinson's research. vmAstros can be routinely produced from multiple hiPSC lines, and express specific astrocytic and ventral midbrain markers. This protocol is scalable, and thus suitable for high-throughput applications, including for drug screening. Crucially, the hiPSC derived-vmAstros demonstrate immunomodulatory characteristics typical of their in vivo counterparts, enabling mechanistic studies of neuroinflammatory signaling in Parkinson's

Connexin 36 Expression Regulates Neuronal Differentiation from Neural Progenitor Cells

Background: Gap junction communication has been shown in glial and neuronal cells and it is thought they mediate interand intra-cellular communication. Connexin 36 (Cx36) is expressed extensively in the developing brain, with levels peaking at P14 after which its levels fall and its expression becomes entirely neuronal. These and other data have led to the hypothesis that Cx36 may direct neuronal coupling and neurogenesis during development. Methodology/Principal Findings: To investigate Cx36 function we used a neurosphere model of neuronal cell development and developed lentiviral Cx36 knockdown and overexpression strategies. Cx36 knockdown was confirmed by western blotting, immunocytochemistry and functionally by fluorescence recovery after photobleaching (FRAP). We found that knockdown of Cx36 in neurosphere neuronal precursors significantly reduced neuronal coupling and the number of differentiated neurons. Correspondingly, the lentiviral mediated overexpression of Cx36 significantly increased the number of neurons derived from the transduced neurospheres. The number of oligodendrocytes was also significantly increased following transduction with Cx36 indicating they may support neuronal differentiation. Conclusions/Significance: Our data suggests that astrocytic and neuronal differentiation during development are governed by mechanisms that include the differential expression of Cx36

Induced Pluripotent Stem Cell Neuronal Models for the Study of Autophagy Pathways in Human Neurodegenerative Disease

Human induced pluripotent stem cells (hiPSCs) are invaluable tools for research into the causes of diverse human diseases, and have enormous potential in the emerging field of regenerative medicine. Our ability to reprogramme patient cells to become hiPSCs, and to subsequently direct their differentiation towards those classes of neurons that are vulnerable to stress, is revealing how genetic mutations cause changes at the molecular level that drive the complex pathogeneses of human neurodegenerative diseases. Autophagy dysregulation is considered to be a major contributor in neural decline during the onset and progression of many human neurodegenerative diseases, meaning that a better understanding of the control of non-selective and selective autophagy pathways (including mitophagy) in disease-affected classes of neurons is needed. To achieve this, it is essential that the methodologies commonly used to study autophagy regulation under basal and stressed conditions in standard cell-line models are accurately applied when using hiPSC-derived neuronal cultures. Here, we discuss the roles and control of autophagy in human stem cells, and how autophagy contributes to neural differentiation in vitro. We also describe how autophagy-monitoring tools can be applied to hiPSC-derived neurons for the study of human neurodegenerative disease in vitro

Identification of unique reciprocal and non reciprocal cross packaging relationships between HIV-1, HIV-2 and SIV reveals an efficient SIV/HIV-2 lentiviral vector system with highly favourable features for in vivo testing and clinical usage.

BACKGROUND: Lentiviral vectors have shown immense promise as vehicles for gene delivery to non-dividing cells particularly to cells of the central nervous system (CNS). Improvements in the biosafety of viral vectors are paramount as lentiviral vectors move into human clinical trials. This study investigates the packaging relationship between gene transfer (vector) and Gag-Pol expression constructs of HIV-1, HIV-2 and SIV. Cross-packaged vectors expressing GFP were assessed for RNA packaging, viral vector titre and their ability to transduce rat primary glial cell cultures and human neural stem cells. RESULTS: HIV-1 Gag-Pol demonstrated the ability to cross package both HIV-2 and SIV gene transfer vectors. However both HIV-2 and SIV Gag-Pol showed a reduced ability to package HIV-1 vector RNA with no significant gene transfer to target cells. An unexpected packaging relationship was found to exist between HIV-2 and SIV with SIV Gag-Pol able to package HIV-2 vector RNA and transduce dividing SV2T cells and CNS cell cultures with an efficiency equivalent to the homologous HIV-1 vector however HIV-2 was unable to deliver SIV based vectors. CONCLUSION: This new non-reciprocal cross packaging relationship between SIV and HIV-2 provides a novel way of significantly increasing bio-safety with a reduced sequence homology between the HIV-2 gene transfer vector and the SIV Gag-Pol construct thus ensuring that vector RNA packaging is unidirectional.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Induction of A9 dopaminergic neurons from neural stem cells improves motor function in an animal model of Parkinson's disease

Neural stem cells (NSCs) are widely endorsed as a cell source for replacement strategies in neurodegenerative disease. However, their usefulness is currently limited by the inability to induce specific neurotransmitter phenotypes in these cells. In order to direct dopaminergic neuronal fate, we overexpressed Pitx3 in NSCs that were then exposed to E11 developing ventral mesencephalon (VM) in explant culture. This resulted in a significant potentiation of dopaminergic differentiation of the cells. When transplanted into the 6-hydroxydopamine lesioned Parkinsonian rats, these cografts of VM and Pitx3 overexpressing NSCs resulted in a significant restitution of motor function. In addition, there were greater numbers of Girk2 positive A9 neurons in the periphery of the transplants that were NSC derived. This demonstrates that given the correct signals, NSCs can be induced to become dopaminergic neurons that can differentiate into the correct nigrastriatal phenotype required for the treatment of Parkinson's diseas