[Neural stem and progenitor cells of human embryos and fetuses as a basis of biomedical new technologies].
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Isolation and cultivation of stem and progenitor cells of human embryos and fetuses at the age of 7-12 weeks of gestation have been described. The embryonic cells of human brain formed neurospheres with heterogenous composition. Cell differentiation took place not only in the presence of serum or as a result of attachment of neurosphere to a sublayer, but also in floating neurospheres in the presence of mitogens. In most neurospheres, the nestin-immunopositive cells were located near the surface while the cells stained for beta-tubulin III and glial fibrillar acid protein, as compact groups inside the neurospheres.Keywords:
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Isolation and cultivation of stem and progenitor cells of human embryos and fetuses at the age of 7-12 weeks of gestation have been described. The embryonic cells of human brain formed neurospheres with heterogenous composition. Cell differentiation took place not only in the presence of serum or as a result of attachment of neurosphere to a sublayer, but also in floating neurospheres in the presence of mitogens. In most neurospheres, the nestin-immunopositive cells were located near the surface while the cells stained for beta-tubulin III and glial fibrillar acid protein, as compact groups inside the neurospheres.
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Embryonic stem cells can be applicable for neural cell transplantation. Here we differentiated mouse embryonic stem cells into neuroepithelial stem cells with tertiary structures in floating cell aggregates. We first cultured ES cells with 0.5μM retinoic acid to induce neural differentiation. RT-PCR and immunostainning studies showed most of the RA treated cells at day 8 differentiated into Nestin expressing neural stem cells without tertiary structures. About 80% of the treated cells become panNCAM+ neural cells by FACS analysis. The findings indicated the RA treatment successfully induced neural stem cells from ES cells. The RA treated cells at day 8 were recovered and were cultured in FCS depleted medium consisted of DMEM/F 12, N 2 supplement and fibronectin on nontreated dishes for additional 6-8 days. The cell aggregates contained a cluster of pseudostratified Nestin+ neuroepithelial stem cells surrounded by the Masson staining+ basement membrane, whereas mature neural cells located outside of the structures. Thus, we have generated neuroepithelial stem cells from ES cells. These findings may get access to induce purposive and transplantable neurons for several diseases and get a cue to resolve inaccessible problems of neurogenesis.
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Abstract Advancement in our understanding of the biology of adult stem cells and their therapeutic potential relies heavily on meaningful functional assays that can identify and measure stem cell activity in vivo and in vitro. In the mammalian nervous system, neural stem cells (NSCs) are often studied using a culture system referred to as the neurosphere assay. We previously challenged a central tenet of this assay, that all neurospheres are derived from a NSC, and provided evidence that it overestimates NSC frequency, rendering it inappropriate for quantitation of NSC frequency in relation to NSC regulation. Here we report the development and validation of the neural colony-forming cell assay (NCFCA), which discriminates stem from progenitor cells on the basis of their proliferative potential. We anticipate that the NCFCA will provide additional clarity in discerning the regulation of NSCs, thereby facilitating further advances in the promising application of NSCs for therapeutic use. Disclosure of potential conflicts of interest is found at the end of this article.
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Objective To investigate the culture,proliferation and differentiation of human neural progenitor cells derived from embryonic brains of different age.Methods The free-floating cells were cultured as 6-9 weeks embryonic whole brain group,14-17 weeks embryonic whole brain group,14-17 weeks embryonic striatum group.The expression of Nestin,self-renewal and multipotential properties of the cell clusters were identified.The growth and proliferation of the cell aggregations of each group were observed.After the neurospheres from each group were induced,the percentage of neuron or astrocyte was investigated by immunocytochemical staining.Results The cell aggregations of each group were positive of Nestin and Brdu immunochemical stainings,and could differentiate into MAP2 or GFAP positive cells.One week in vitro,there were the most neurospheres in 14-17 weeks embryonic striatum group,and a few in 6-9 weeks embryonic whole brain group,and fewer in 14-17 weeks embryonic whole brain group.Single cell from 14-17 weeks embryonic striatum group could form cmone.After differentiation of neural progenitor cells from each group,the percentage of MAP2 or GFAP positive cells had no significant difference among the three groups.Conclusion Neural progenitor cells can be derived from embryonic brains of different age.With gestational age increasing,the neural progenitor cells become more difficult to be isolated from the whole brains.Different regions should be obtained as primary culture according to the different embryo age.The percentage of differentiated neuron or astrocyte is coincident in neural progenitor cells from embryonic brains of different age.
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Neural stem cells play major roles in brain development. The precise regulation of neural stem cell survival and proliferation is important for determining numbers of terminally differentiated neurons and glia. In this study, we demonstrate that embryonic neural stem cells positively regulate their own survival by secreting diffusible factors. Single cells isolated from embryonic day 14 (E14) rat cortices were plated at clonal densities to avoid direct cell-cell contact. Clones positive for nestin, a neural stem cell marker, were formed from single cells in the presence of basic fibroblast factor (bFGF). The numbers of viable cells and clones were markedly increased by supplementing conditioned medium (stem cell conditioned medium, SCM) prepared from dense cultures of embryonic cortical stem cells. TUNEL and LDH assays confirmed SCM-mediated survival of embryonic cortical stem cells.
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The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies ≥2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies < 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are < 2mm and the ones that are ≥2mm in reference to the number of cells that were initially plated.
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Multipotent Stem Cell
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Abstract Background Pannexin 1 forms ion and metabolite permeable hexameric channels and is abundantly expressed in the brain. After discovering pannexin 1 expression in postnatal neural stem and progenitor cells we sought to elucidate its functional role in neuronal development. Results We detected pannexin 1 in neural stem and progenitor cells in vitro and in vivo . We manipulated pannexin 1 expression and activity in Neuro2a neuroblastoma cells and primary postnatal neurosphere cultures to demonstrate that pannexin 1 regulates neural stem and progenitor cell proliferation likely through the release of adenosine triphosphate (ATP). Conclusions Permeable to ATP, a potent autocrine/paracine signaling metabolite, pannexin 1 channels are ideally suited to influence the behavior of neural stem and progenitor cells. Here we demonstrate they play a robust role in the regulation of neural stem and progenitor cell proliferation. Endogenous postnatal neural stem and progenitor cells are crucial for normal brain health, and their numbers decline with age. Furthermore, these special cells are highly responsive to neurological injury and disease, and are gaining attention as putative targets for brain repair. Therefore, understanding the fundamental role of pannexin 1 channels in neural stem and progenitor cells is of critical importance for brain health and disease.
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