| 1. |
- Andersson, Elin, et al.
(författare)
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Development of the mesencephalic dopaminergic neuron system is compromised in the absence of neurogenin 2.
- 2006
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Ingår i: Development: For advances in developmental biology and stem cells. - : The Company of Biologists. - 1477-9129. ; 133:3, s. 507-516
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Tidskriftsartikel (refereegranskat)abstract
- Neurogenin 2 (Ngn2) is a proneural gene involved in neuronal differentiation and subtype specification in various regions of the nervous system. In the ventral midbrain, Ngn2 is expressed in a spatiotemporal pattern that correlates with the generation of mesencephalic dopaminergic (mesDA) neurons. We show here that lack of Ngn2 impairs the development of mesDA neurons, such that less than half of the normal mesDA neuron number remain in Ngn2 mutant mice at postnatal stages. Analysis of Ngn2 mutant mice during mesDA neurogenesis show that medially located precursors are formed but are arrested in their differentiation at a stage when they have not yet acquired the characteristics of mesDA neuron precursors. Loss of Ngn2 function appears to specifically affect the generation of DA neurons, as the development of other types of neurons within the ventral midbrain is unaltered. Ngn2 is the first example of a gene expressed in progenitors in the ventricular zone of the mesDA neuron domain that is essential for proper mesDA neuron differentiation, and whose loss of function causes impaired mesDA neurogenesis without other major abnormalities in the ventral midbrain.
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| 2. |
- Andersson, Elin, et al.
(författare)
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Ngn2 and Nurr1 act in synergy to induce midbrain dopaminergic neurons from expanded neural stem and progenitor cells.
- 2007
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 313:6, s. 1172-1180
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Tidskriftsartikel (refereegranskat)abstract
- Parkinson's Disease (PD) is a debilitating motor function disorder due primarily to a loss of midbrain dopaminergic neurons and a subsequent reduction in dopaminergic innervation of the striatum. Several attempts have been made to generate dopaminergic neurons from progenitor cell populations in vitro for potential use in cell replacement therapy for PD. However, expanding cells from fetal brain with retained potential for dopaminergic differentiation has proven to be difficult. In this study, we sought to generate mesencephalic dopaminergic (mesDA) neurons from an expanded population of fetal mouse ventral midbrain (VM) progenitors through the use of retroviral gene delivery. We over-expressed Ngn2 and Nurr1, two genes present in the ventral midbrain and important for normal development of mesDA neurons, in multipassaged neurosphere-expanded midbrain progenitors. We show that over-expression of Ngn2 in these progenitors results in increased neuronal differentiation but does not promote mesDA formation. We also show that over-expression of Nurr1 alone is sufficient to generate tyrosine hydroxylase (TH) expressing cells with an immature morphology, however the cells do not express any additional markers of mesDA neurons. Overexpression of Nurr1 and Ngn2 in combination generates morphologically mature TH-expressing neurons that also express additional mesencephalic markers. (c) 2006 Elsevier Inc. All rights reserved.
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| 3. |
- Barraud, Perrine, et al.
(författare)
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In vitro characterization of a human neural progenitor cell coexpressing SSEA4 and CD133.
- 2007
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Ingår i: Journal of Neuroscience Research. - : Wiley. - 1097-4547 .- 0360-4012. ; 85, s. 250-259
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Tidskriftsartikel (refereegranskat)abstract
- The stage-specific embryonic antigen 4 (SSEA4) is commonly used as a cell surface marker to identify the pluripotent human embryonic stem (ES) cells. Immunohistochemistry on human embryonic central nervous system revealed that SSEA4 is detectable in the early neuroepithelium, and its expression decreases as development proceeds. Flow cytometry analysis of forebrain-derived cells demonstrated that the SSEA4-expressing cells are enriched in the neural stem/progenitor cell fraction (CD133+), but are rarely codetected with the neural stem cell (NSC) marker CD15. Using a sphere-forming assay, we showed that both subfractions CD133+/SSEA4+ and CD133+/CD15+ isolated from the embryonic forebrain are enriched in neurosphere-initiating cells. In addition CD133, SSEA4, and CD15 expression is sustained in the expanded neurosphere cells and also mark subfractions of neurosphere-initiating cells. Therefore, we propose that SSEA4 associated with CD133 can be used for both the positive selection and the enrichment of neural stem/progenitor cells from human embryonic forebrain.
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| 4. |
- Barraud, Perrine, et al.
(författare)
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Isolation and characterization of neural precursor cells from the Sox1-GFP reporter mouse.
- 2005
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Ingår i: European Journal of Neuroscience. - : Wiley. - 1460-9568 .- 0953-816X. ; 22:7, s. 1555-1569
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Tidskriftsartikel (refereegranskat)abstract
- We have made use of a reporter mouse line in which enhanced green fluorescence protein (GFP) is inserted into the Sox1 locus. We show that the GFP reporter is coexpressed with the Sox1 protein as well as with other known markers for neural stem and progenitor cells, and can be used to identify and isolate these cells by fluorescence-activated cell sorting (FACS) from the developing or adult brain and from neurosphere cultures. All neurosphere-forming cells with the capacity for multipotency and self-renewal reside in the Sox1–GFP-expressing population. Thus, the Sox1–GFP reporter system is highly useful for identification, isolation and characterization of neural stem and progenitor cells, as well as for the validation of alternative means for isolating neural stem and progenitor cells. Further, transplantation experiments show that Sox1–GFP cells isolated from the foetal brain give rise to neurons and glia in vivo, and that many of the neurons display phenotypic characteristics appropriate for the developing brain region from which the Sox1–GFP precursors were derived. On the other hand, Sox1–GFP cells isolated from the adult subventricular zone or expanded neurosphere cultures gave rise almost exclusively to glial cells following transplantation. Thus, not all Sox1–GFP cells possess the same capacity for neuronal differentiation in vivo.
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| 5. |
- Cacci, Emanuele, et al.
(författare)
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Generation of human cortical neurons from a new immortal fetal neural stem cell line.
- 2007
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 313:3, s. 588-601
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Tidskriftsartikel (refereegranskat)abstract
- Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia. and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology. (c) 2006 Elsevier Inc. All rights reserved.
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| 6. |
- Hebsgaard, Josephine, et al.
(författare)
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Dopamine neuron precursors within the developing human mesencephalon show radial glial characteristics.
- 2009
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Ingår i: GLIA. - : Wiley. - 1098-1136 .- 0894-1491. ; 57, s. 1648-1659
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Tidskriftsartikel (refereegranskat)abstract
- Specification and differentiation of neural precursors into dopaminergic neurons within the ventral mesencephalon has been subject to much attention due to the implication of dopaminergic neurons in Parkinson's disease and the perspective of generating sources of therapeutically active cells to be used for cell replacement therapy for the disease. However, despite intensive research efforts, little is known about the characteristics of the dopamine neuron progenitors in human. We show that the dopamine neuron determinant LMX1a is expressed in the diencephalic and mesencephalic dopaminergic neuron domains during human development. Within the mesencephalon, LMX1a is expressed in the dopaminergic neurons and their progenitors located in the ventricular zone of the floor plate region. Furthermore, the neural progenitors in the developing human ventral mesencephalon have a radial morphology and express the radial glial markers Vimentin and BLBP. These radial glia are mitotic and act as precursors for the dopaminergic neurons. Finally, we show that progenitors isolated from the human ventral mesencephalon maintain their radial glial characteristics and neurogenic capacity after expansion in vitro, making them a promising future source of cells to be used in cell replacement therapy for Parkinson's disease. (c) 2009 Wiley-Liss, Inc.
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| 7. |
- Hebsgaard, Josephine, et al.
(författare)
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Strengths and limitations of the neurosphere culture system.
- 2006
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Ingår i: Molecular Neurobiology. - 1559-1182. ; 34:3, s. 153-161
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Tidskriftsartikel (refereegranskat)abstract
- After the initial reports of free-floating cultures of neural stem cells termed neurospheres (1,2), a wide array of studies using this promising culture system emerged. In theory, this was a near-perfect system for large-scale production of neural cells for use in cell replacement therapies and to assay for and characterize neural stem cells. More than a decade later, after rigorous scrutiny and ample experimental testing of the neurosphere culture system, it has become apparent that the culture system suffers from several disadvantages, and its usefulness is limited for several applications. Nevertheless, the bulk of high-quality research produced over the last decade has also shown that under the right circumstances and for the appropriate purposes, neurospheres hold up to their initial promise. This article discusses the pros and cons of the neurosphere culture system regarding its three major applications: as an assay for neural stem cells, as a model system for neurogenesis and neural development, and for expansion of neural stem cells for transplantation purposes.
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| 8. |
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| 9. |
- Nelander Wahlestedt, Jenny, et al.
(författare)
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Organization of the human embryonic ventral mesencephalon.
- 2009
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Ingår i: Gene Expression Patterns. - : Elsevier BV. - 1872-7298 .- 1567-133X. ; 9, s. 555-561
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Tidskriftsartikel (refereegranskat)abstract
- The neurons in the ventral mesencephalon (VM) are organized into several nuclei consisting of distinct neuronal populations. These include the dopaminergic (DA) neurons of the substania nigra and ventral tegmental area, the oculomotor (OM) neurons that innervate the muscles controlling eye movement, and the reticular neurons of the red nucleus (RN) involved in motor control and coordination reviewed in Puelles (2007). The factors and genes that control the differentiation of the various neuronal populations in the VM have been extensively studied in the mouse and other model organisms but little is known about the progenitors and their protein expression in the developing human brain. In this study we analyze if key regulators identified in rodents are also expressed in the human VM during embryonic development. We report that BLBP and LMX1A mark the floor plate and that FOXA2 is expressed in both the floor plate and basal plate of the human VM. The proneural transcription factors NGN2 and MASH1 are expressed in the ventricular zone of the human VM within and lateral to the floor plate. The post-mitotic DA neurons express TH as well as NURR1 and PITX3. ISL1 and BRN3A can be used to detect the cells of OM and RN, respectively. We show that many key developmental control factors are expressed in a temporal and spatial manner in the human VM essentially corresponding to what has been observed in the mouse. This data therefore suggest similar roles for these factors also in human VM development and dopamine neurogenesis.
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| 10. |
- Parmar, Malin, et al.
(författare)
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Early specification of dopaminergic phenotype during ES cell differentiation
- 2007
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Ingår i: BMC Developmental Biology. - 1471-213X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Understanding how lineage choices are made during embryonic stem (ES) cell differentiation is critical for harnessing strategies for controlled production of therapeutic somatic cell types for cell transplantation and pharmaceutical drug screens. The in vitro generation of dopaminergic neurons, the type of cells lost in Parkinson's disease patients' brains, requires the inductive molecules sonic hedgehog and FGF8, or an unknown stromal cell derived inducing activity (SDIA). However, the exact identity of the responding cells and the timing of inductive activity that specify a dopaminergic fate in neural stem/progenitors still remain elusive. RESULTS: Using ES cells carrying a neuroepithelial cell specific vital reporter (Sox1-GFP) and FACS purification of Sox1-GFP neural progenitors, we have investigated the temporal aspect of SDIA mediated dopaminergic neuron specification during ES cell differentiation. Our results establish that SDIA induces a dopaminergic neuron fate in nascent neural stem or progenitor cells at, or prior to, Sox1 expression and does not appear to have further instructive role or neurotrophic activity during late neuronal differentiation of neural precursors. Furthermore, we show that dopaminergic neurons could be produced efficiently in a monolayer differentiation paradigm independent of SDIA activity or exogenous signalling molecules. In this case, the competence for dopaminergic neuron differentiation is also established at the level of Sox1 expression. CONCLUSION: Dopaminergic neurons are specified early during mouse ES cell differentiation. The subtype specification seems to be tightly linked with the acquisition of a pan neuroectoderm fate.
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| 11. |
- Rogelius, Nina, et al.
(författare)
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Reprogramming of neonatal SVZ progenitors by Islet-1 and Neurogenin-2.
- 2008
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Ingår i: Molecular and Cellular Neuroscience. - : Elsevier BV. - 1044-7431. ; 38:3, s. 453-459
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Tidskriftsartikel (refereegranskat)abstract
- The subventricular zone (SVZ) lining the lateral walls of the lateral ventricles is one of the major neurogenic areas in the postnatal brain. Precursor cells in the SVZ migrate via the rostral migratory stream to the olfactory bulb where they differentiate into neurons. Cell replacement strategies utilizing the recruitment of these endogenous progenitors and their progeny to different areas of the brain hold great promise for the future, but much research is needed in order to understand the sequence of molecular signals necessary to induce proliferation, migration and site-specific differentiation of these cells. In this study we show that the SVZ cells can be redirected from their normal migration route and directed towards other brain regions when they are infected with retroviruses encoding the developmentally important transcription factors Islet-1 and Neurogenin-2. After co-transduction with these transcription factors, transduced cells could be detected in several areas of the brain. When located in the striatum, the reprogrammed cells displayed neuroblast-like morphology. Once removed from the striatal parenchyma and allowed to further differentiation in vitro they developed into beta-III-tubulin positive neurons.
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| 12. |
- Rogelius, Nina, et al.
(författare)
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Retrovirally delivered Islet-1 increases recruitment of Ng2 expressing cells from the postnatal SVZ into the striatum.
- 2006
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Ingår i: Experimental Neurology. - : Elsevier BV. - 0014-4886. ; 201:Jun 23, s. 388-398
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Tidskriftsartikel (refereegranskat)abstract
- Neural stem and progenitor cells hold the promise to be used in cell-based therapies to treat both acute and degenerative neurological diseases. To date, most research has been focused on the use of in vitro propagated stem cells used as a source of cells in cell replacement therapies. However, mobilization of endogenous neural stem cells to generate a specific differentiated cell type offers an attractive alternative. In this study, we investigate the possibility to direct the formation of specific cells from the endogenous stem and progenitor cells residing in the subventricular region of the postnatal brain. With the aim to induce postnatal generation of striatal neurons, we ectopically expressed Islet-1, a LIM homeodomain transcription factor expressed by striatal progenitors during development, in cells of the subventricular zone (SVZ) of neonatal and adult rats. Ectopic expression of Islet-1 in the neonatal, but not adult, SVZ resulted in the appearance of a population of cells in the striatum. These cells were primarily located in the ventrolateral area of the striatum where they differentiate into Ng2 expressing cells. However, no neurogenesis was observed in the striatum, nor was ectopic striatal differentiation observed in any other area of the brain after retroviral expression of Islet-1 in the SVZ. Thus, although ectopic expression of Islet-1 is sufficient to direct the migration of cells into the striatum in neonatal animals, it does not specify a striatal projection neuron phenotype in cells generated from the SVZ after birth. (c) 2006 Elsevier Inc. All rights reserved.
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| 13. |
- Thompson, Lachlan, et al.
(författare)
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Neurogenin2 identifies a transplantable dopamine neuron precursor in the developing ventral mesencephalon.
- 2006
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Ingår i: Experimental Neurology. - : Elsevier BV. - 0014-4886. ; 198:1, s. 183-198
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Tidskriftsartikel (refereegranskat)abstract
- In neural transplantation studies, there is an interest in identifying and isolating mesencephalic dopamine (mesDA) neuron precursors that have the capacity to differentiate into fully mature mesDA neurons after transplantation. We report here that in the developing ventral mesencephalon (VM) the proneural gene Neurogenin2 (Ngn2) is expressed exclusively in the part of the ventricular zone that gives rise to the migrating mesDA neuroblasts, but not in the differentiated mesDA neurons. From other studies, we know that Ngn2 is involved in the generation of mesDA neurons and that the development of mesDA neurons is severely compromised in Ngn2-null mutant mice. We show here that cells isolated by FACS from the developing VM of Ngn2-GFP knock-in mice are capable of generating mesDA neurons, both in vitro and after transplantation to the striatum of neonatal rats. All mesDA neuron precursors, but not the serotonergic or GABAergie neuron precursors, are contained in the Ngn2-GFP-expressing population. Moreover, all glial cells were generated from cells contained in the GFP-negative cell fraction. The results show that surviving mesDA neurons in VM grafts are derived from early postmitotic, probably Nurr1-expressing precursors before they have acquired their fully differentiated neuronal phenotype. The Ngn2-GFP reporter construct used here thus provides a tool for the identification of inesDA neuron precursors in the VM and selective isolation of transplantable mesDA neuron precursors for transplantation. (c) 2005 Elsevier Inc. All rights reserved.
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