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1.
2.
Sci Rep ; 6: 23579, 2016 Mar 31.
Article in English | MEDLINE | ID: mdl-27030542

ABSTRACT

Many neurological injuries are likely too extensive for the limited repair capacity of endogenous neural stem cells (NSCs). An alternative is to isolate NSCs from a donor, and expand them in vitro as transplantation material. Numerous groups have already transplanted neural stem and precursor cells. A caveat to this approach is the undefined phenotypic distribution of the donor cells, which has three principle drawbacks: (1) Stem-like cells retain the capacity to proliferate in vivo. (2) There is little control over the cells' terminal differentiation, e.g., a graft intended to replace neurons might choose a predominantly glial fate. (3) There is limited ability of researchers to alter the combination of cell types in pursuit of a precise treatment. We demonstrate a procedure for differentiating human neural precursor cells (hNPCs) in vitro, followed by isolation of the neuronal progeny. We transplanted undifferentiated hNPCs or a defined concentration of hNPC-derived neurons into mice, then compared these two groups with regard to their survival, proliferation and phenotypic fate. We present evidence suggesting that in vitro-differentiated-and-purified neurons survive as well in vivo as their undifferentiated progenitors, and undergo less proliferation and less astrocytic differentiation. We also describe techniques for optimizing low-temperature cell preservation and portability.


Subject(s)
Cryopreservation/methods , Neural Stem Cells/cytology , Neurogenesis/physiology , Neurons/cytology , Neurons/transplantation , Stem Cell Transplantation , Animals , Cell Differentiation , Cell Proliferation , Cell Survival , Cell Tracking/methods , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Immunomagnetic Separation/methods , Injections, Intraventricular , Mice , Mice, Inbred NOD , Mice, SCID , Neural Stem Cells/physiology , Neuroglia/cytology , Neuroglia/physiology , Neurons/physiology , Phenotype , Stereotaxic Techniques , Transplantation, Heterologous
3.
Cell Rep ; 11(7): 1031-42, 2015 May 19.
Article in English | MEDLINE | ID: mdl-25959821

ABSTRACT

The coordination of complex tumor processes requires cells to rapidly modify their phenotype and is achieved by direct cell-cell communication through gap junction channels composed of connexins. Previous reports have suggested that gap junctions are tumor suppressive based on connexin 43 (Cx43), but this does not take into account differences in connexin-mediated ion selectivity and intercellular communication rate that drive gap junction diversity. We find that glioblastoma cancer stem cells (CSCs) possess functional gap junctions that can be targeted using clinically relevant compounds to reduce self-renewal and tumor growth. Our analysis reveals that CSCs express Cx46, while Cx43 is predominantly expressed in non-CSCs. During differentiation, Cx46 is reduced, while Cx43 is increased, and targeting Cx46 compromises CSC maintenance. The difference between Cx46 and Cx43 is reflected in elevated cell-cell communication and reduced resting membrane potential in CSCs. Our data demonstrate a pro-tumorigenic role for gap junctions that is dependent on connexin expression.


Subject(s)
Brain Neoplasms/pathology , Connexin 43/metabolism , Connexins/metabolism , Glioblastoma/pathology , Neoplastic Stem Cells/pathology , Animals , Cell Communication/physiology , Fluorescent Antibody Technique , Gap Junctions/metabolism , Glioblastoma/metabolism , Heterografts , Humans , Immunoblotting , Membrane Potentials/physiology , Neoplastic Stem Cells/metabolism , Patch-Clamp Techniques , Polymerase Chain Reaction
4.
PLoS One ; 10(3): e0120281, 2015.
Article in English | MEDLINE | ID: mdl-25763840

ABSTRACT

This study investigates the electrophysiological properties and functional integration of different phenotypes of transplanted human neural precursor cells (hNPCs) in immunodeficient NSG mice. Postnatal day 2 mice received unilateral injections of 100,000 GFP+ hNPCs into the right parietal cortex. Eight weeks after transplantation, 1.21% of transplanted hNPCs survived. In these hNPCs, parvalbumin (PV)-, calretinin (CR)-, somatostatin (SS)-positive inhibitory interneurons and excitatory pyramidal neurons were confirmed electrophysiologically and histologically. All GFP+ hNPCs were immunoreactive with anti-human specific nuclear protein. The proportions of PV-, CR-, and SS-positive cells among GFP+ cells were 35.5%, 15.7%, and 17.1%, respectively; around 15% of GFP+ cells were identified as pyramidal neurons. Those electrophysiologically and histological identified GFP+ hNPCs were shown to fire action potentials with the appropriate firing patterns for different classes of neurons and to display spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs). The amplitude, frequency and kinetic properties of sEPSCs and sIPSCs in different types of hNPCs were comparable to host cells of the same type. In conclusion, GFP+ hNPCs produce neurons that are competent to integrate functionally into host neocortical neuronal networks. This provides promising data on the potential for hNPCs to serve as therapeutic agents in neurological diseases with abnormal neuronal circuitry such as epilepsy.


Subject(s)
Neocortex/cytology , Neural Stem Cells/cytology , Neural Stem Cells/transplantation , Animals , Cell Survival , Fetus/cytology , Heterografts , Humans , Mice , Neocortex/metabolism , Neural Stem Cells/chemistry , Neural Stem Cells/metabolism , Neurons/chemistry , Neurons/cytology , Neurons/metabolism , Telencephalon/cytology
5.
J Vis Exp ; (62)2012 Apr 29.
Article in English | MEDLINE | ID: mdl-22565048

ABSTRACT

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies(1). To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia(2-8). The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma(2,7,8). The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds(9). The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division(10). CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity(2). This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry(2). The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.


Subject(s)
Brain Neoplasms/pathology , Flow Cytometry/methods , Fluoresceins/chemistry , Fluorescent Dyes/chemistry , Glioblastoma/pathology , Staining and Labeling/methods , Succinimides/chemistry , Cell Growth Processes/physiology , Humans , Tumor Cells, Cultured
6.
J Vis Exp ; (62)2012 Apr 22.
Article in English | MEDLINE | ID: mdl-22546813

ABSTRACT

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.


Subject(s)
Cytological Techniques/methods , Flow Cytometry/methods , Neural Stem Cells/cytology , Neurons/cytology , Animals , Cell Differentiation/physiology , Mice
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