Comparison of human dental follicle cells (DFCs) and stem cells from human exfoliated deciduous teeth (SHED) after neural differentiation in vitro
Christian MorsczeckFlorian VöllnerM SaugspierCaroline BrandlTorsten E. ReichertOliver DriemelGottfried Schmalz
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Dental follicle
Neurosphere
Neural cell
Stem cell marker
Neural stem cells are defined as clonogenic cells with self-renewal capacity and the ability to generate all neural lineages (multipotentiality). Cells with these characteristics have been isolated from the embryonic and adult central nervous system. Under specific conditions, these cells can differentiate into neurons, glia, and non-neural cell types, or proliferate in long-term cultures as cell clusters termed "neurospheres". These cultures represent a useful model for neurodevelopmental studies and a potential cell source for cell replacement therapy. Because no specific markers are available to unequivocally identify neural stem cells, their functional characteristics (self-renewal and multipotentiality) provide the main features for their identification. Here, we review the experimental and ultrastructural studies aimed at identifying the morphological characteristics and the antigenic markers of neural stem cells for their in vitro and in vivo identification.
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Abstract Glioblastoma multiforme (GBM) is one of the most aggressive tumors characterised by bad prognosis and limited treatment response. Stem-cell like GBM subpopulations are believed to cause tumor initiation and treatment resistance. Aim of this study was to clarify whether the GBM stem-cell like subpopulations (GSCLS) present in GBM cell cultures are characterized by specific genomic alterations. Fourteen GBM primary cultures and 6 cell lines were analyzed for the presence of GSCLS compartments by investigating their ability to grow in serum-free neurobasal medium. Growth in neurospheres was compared to the expression of the stem cell markers CD133, nestin and SOX2. Nine out of 14 GBM primary cultures and 3 out of 7 GBM cell lines were able to grow as stable non-adherent neurospheres in NBE-medium for 3 up to 14 months. From those GBM samples, able to grow as neurospheres in NBE-medium, 33% expressed all 3 stem cell markers, 42% two markers and 25% one stem cell marker. The samples who grew as monolayers or died in NBE-medium, expressed only one (30%) or no stem cell marker (70%). Genomic changes in selected primary cultures (N=8) and a GBM cell line grown in parallel for 6 to 60 weeks in spheres and monolayer cultures were comparably analyzed by (array)CGH. In all cases analyzed, GSCLS grown as neurospheres contained widely the identical chromosomal gains/amplifications and losses as the respective parental cell cultures. Differences concerned random low-level gains/losses of large chromosomal regions or whole chromosome arms. However, on closer inspection by aCGH small but distinct amplifications/deletions could be found in the GSCLS grown in neurospheres mainly affecting single gene loci. Further changes at the SOX2 gene locus and SOX2 gene expression were analyzed in GBM cell lines and primary culture. An increase of SOX2 gene expression was observed in GSCLS grown as neurospheres compared to the respective monolayer cultures as determined by qRT PCR. Preliminary data show that the SOX2 expression is higher in GBM tumor samples compared to the respective cell lines indicating loss of stem cell features during in vitro cell culture. Consequently, the association between the expression of stem cell markers, like SOX2, and the survival time of GBM patients is currently established. In ongoing experiments genome-wide gene expression differences between GSCLS neurospheres and the respective monolayer cultures are established and compared with the aCGH data. In conclusion, we demonstrate that the majority of human GBM primary cell cultures/cell lines contain stem cell-like subpopulations. These sphere-forming cell clones harbor small distinct gene amplifications/deletions as compared to the respective monolayer cell cultures and the genes residing in the affected regions are currently characterized. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5142.
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Cell fate determination
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Neural stem cells are a potential therapeutic source for cellular transplantation therapy in neurological diseases. The present paper was aimed to investigate whether neural stem cells could be obtained from the spinal cords of low temperature preserved abortuses. Fourteen weeks old abortuses were stored in a refrigerator at 4 degrees C without any additional treatments for 2, 6 and 12 h before use. The spinal cords were anatomized out and divided into cervical cords, thoracic cords and lumbar/sacral cords. Then the spinal cord segments were used for cell culture separately. Neural stem cells were isolated from the segments and cultured in bFGF, EGF and N2 supplement containing free-serum DMEM/F12 (1:1) medium. In order to examine the differentiation potential, the stem cells were induced to differentiate with 5% fetal bovine serum on poly-l-lysine substrate. Clonal culture was carried out to demonstrate that the isolated cells met the standard of stem cells. Indirect fluorescent immunocytochemistry was used to examine the expressions of neural stem cell marker (nestin), neuron marker (MAP2), astrocyte marker (GFAP) and cholinergic marker (ChAT). The stem cells in different cultures were compared. One-way analysis of variance and Kruskal-Wallis test were used for the statistical comparison. As a result, neural stem cells were obtained from all the spinal cord segments with different postmortem intervals. Both the cells on the surface and inside the neurospheres showed nestin immunoreactivity. Therefore, nearly all the cells that composed the neurospheres were nestin-positive undifferentiated cells. When the spheres were induced to differentiate, they could yield GFAP-positive astrocytes and MAP2-positive neurons including ChAT-positive cholinergic neurons. Primary neurospheres could be dissociated mechanically, expand in subcultures and maintain the differentiation potential. In clonal cultures, single cells from a single primary sphere could give rise to new neurospheres, which had the same differentiation potential as the primary spheres. The lumbar/sacral cord cultures gave rise to the most abundant primary neurospheres. When the preservation time of the fetus was prolonged to 12 h, the number of primary neurospheres decreased sharply. The clonal formation and phenotype capacity were similar in all cultures. In conclusion, spinal neural stem cells can be isolated from low temperature preserved abortuses and represent an alternative source for experimental and potential therapeutic purposes.
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Objective To investigate the feasibility of isolation of brain tumor stem cell (BTSC) in gliomas by labeling with CD133. Methods Nine human gliomas specimens,two glioma cell lines (C6 and U87),were involved in this study. The methods of isolation included primary culture and CD133+ cells culture after magnetic cell sorting by CD133 cell isolation kit. Cells cultures were performed under the condition of neuron specific enolase (NSE). The BTSC was identified by immunocytochemical staining of neurospheres by detected the types of molecular markers of nestin,NSE,glial fibrillary acidic protein (GFAP),and by differentiation tests as well. Results Classical neurospheres formed in vitro by normal NSC were detected in two glioma specimens cultures established from pediatric malignant gliomas. They were medulloblastoma and high-grade glioma. The neurospheres derived from high-grade glioma could have long-term cultures,while from medulloblastoma could not. Conversely,clone formation was never observed in cultures from adult gliomas. The neurospheres were also detected in C6 and U87 cell lines either by primary culture or by CD133+ cells culture. The neurospheres could proliferate and increase in their diameters. The labeling test showed nestin positive,but NSE and GFAP negative.The induced cells ceuld defferentiate into the cells which had the same from as the original cells and expressed NSE and GFAP.Conclusion The results show that gliomas contain neural stem-like cells,which are BTSC. The BTSC can be exclusively isolated with the cell fraction expressing the neural stem cell surface marker CD133.
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The large variety of neural and non-neural derivatives that arise from the neural crest raises the fundamental question of how cell fates become specified during its ontogeny. In other words, what is the relative influence of intrinsic cell commitment compared to cell–cell interactions in promoting differentiation of distinct cell types.
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Abstract Cadherins constitute a superfamily of cell adhesion molecules involved in cell‐cell interaction, histogenesis and cellular transformation. They have been implicated in the development of various lineages, including derivatives of the neural crest. Neural crest cells (NCC) emerge from the dorsal part of the neural tube after an epithelio‐mesenchymal transition (EMT) and migrate through the embryo. After homing and differentiation, NCC give rise to many cell types, such as neurons, Schwann cells and melanocytes. During these steps, the pattern of expression of the various cadherins studied is very dynamic. Cadherins also display plasticity of expression during the transformation of neural crest cell derivatives. Here, we review the pattern of expression and the role of the main cadherins involved in the development and transformation of neural crest cell derivatives. © 2001 Wiley‐Liss, Inc.
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Objective:To explore two methods of culturing mouse embryo neural stem cells in vitro and identification of their differentiation.Methods:The neural stem cells were derived from the cortex tissue of mouse embryo at gestation day of 14~16 with two methods: culturing of neurosphere in floating and culturing of monolayer adherent cells.To observe the development of the neural stem cell,the expression of the stem cell marker Nestin and the neural marker MAP-2 and the glial marker GFAP was examined with immunofluorescence.Results:The cultured neural stem cells proliferated.The maintenance of stem cell property was preserved with both methods as revealed by Nestin immunofluorescence.These stem cells are able to differentiate into glial(GFAP positive) and neuronal lineage(MAP-2 positive).Conclusion: With the help of growth factors and serum-free technique,the mouse neural stem cells cultivated by methods of neural stem cell spheres and adherent monolayer cells,have potential of proliferation and differentiation.
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