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Ingming Chiu - One of the best experts on this subject based on the ideXlab platform.
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Enzyme-Free Dissociation of Neurospheres by a Microfluidic Chip-Based Method
Methods of Molecular Biology, 2020Co-Authors: Hao-chen Chang, Ingming ChiuAbstract:Abstract Neurosphere assay is a common and robust method for identification of neural stem/progenitor cells, but obtaining large numbers of live single cells from dissociated Neurospheres is difficult using nonenzymatic methods. Here, we present an enzyme-free method for high-efficiency Neurosphere dissociation into single cells using microfluidic device technology. This method allows single cell dissociation of DC115 and KT98 cells with high cell viabilities (80-85 %), single-cell yield (91-95 %), and recovery (75-93 %).
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inhibition of Neurosphere formation in neural stem progenitor cells by acrylamide
Cell Transplantation, 2015Co-Authors: Jonghang Chen, Meishu Chen, Yingchin Ko, Ingming ChiuAbstract:Abstract Previous studies showed that transplantation of cultured neural stem/progenitor cells (NSPCs) could improve functional recovery for various neurological diseases. This study aims to develop a stem cell-based model for predictive toxicology of development in the neurological system after acrylamide exposure. Treatment of mouse (KT98/F1B-GFP) and human (U-1240 MG/F1B-GFP) NSPCs with 0.5 mM acrylamide resulted in the inhibition of Neurosphere formation (definition of self-renewal ability in NSPCs), but not inhibition of cell proliferation. Apoptosis and differentiation of KT98 (a precursor of KT98/F1B-GFP) and KT98/F1B-GFP are not observed in acrylamide-treated Neurospheres. Analysis of secondary Neurosphere formation and differentiation of neurons and glia illustrated that acrylamide-treated KT98 and KT98/F1B-GFP Neurospheres retain the NSPC properties, such as self-renewal and differentiation capacity. Correlation of acrylamide-inhibited Neurosphere formation with cell-cell adhesion was observed in mouse NSPCs by live cell image analysis and the presence of acrylamide. Protein expression levels of cell adhesion molecules [neural cell adhesion molecule (NCAM) and N-cadherin] and extracellular signal-regulated kinases (ERK) in acrylamide-treated KT98/F1B-GFP and U-1240 MG/F1B-GFP Neurospheres demonstrated that NCAM decreased and phospho-ERK (pERK) increased, whereas expression of N-cadherin remained unchanged. Analysis of AKT (protein kinase B, PKB)/β-catenin pathway showed decrease in phospho-AKT (p-AKT) and cyclin D1 expression in acrylamide-treated Neurospheres of KT98/F1B-GFP. Furthermore, PD98059, an ERK phosphorylation inhibitor, attenuated acrylamide-induced ERK phosphorylation, indicating that pERK contributed to the cell proliferation, but not in Neurosphere formation in mouse NSPCs. Coimmunoprecipitation results of KT98/F1B-GFP cell lysates showed that the complex of NCAM and fibroblast growth factor receptor 1 (FGFR1) is present in the Neurosphere, and the amount of this complex decreases after acrylamide treatment. Our results reveal that acrylamide inhibits Neurosphere formation through the disruption of the Neurosphere architecture in NSPCs. The downregulation of cell-cell adhesion resulted from decreasing the levels of NCAM as well as the formation of NCAM/FGFR complex.
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Inhibition of Neurosphere formation in neural stem/progenitor cells by acrylamide.
Cell Transplantation, 2013Co-Authors: Jonghang Chen, Meishu Chen, Yingchin Ko, Ingming ChiuAbstract:Abstract Previous studies showed that transplantation of cultured neural stem/progenitor cells (NSPCs) could improve functional recovery for various neurological diseases. This study aims to develop a stem cell-based model for predictive toxicology of development in the neurological system after acrylamide exposure. Treatment of mouse (KT98/F1B-GFP) and human (U-1240 MG/F1B-GFP) NSPCs with 0.5 mM acrylamide resulted in the inhibition of Neurosphere formation (definition of self-renewal ability in NSPCs), but not inhibition of cell proliferation. Apoptosis and differentiation of KT98 (a precursor of KT98/F1B-GFP) and KT98/F1B-GFP are not observed in acrylamide-treated Neurospheres. Analysis of secondary Neurosphere formation and differentiation of neurons and glia illustrated that acrylamide-treated KT98 and KT98/F1B-GFP Neurospheres retain the NSPC properties, such as self-renewal and differentiation capacity. Correlation of acrylamide-inhibited Neurosphere formation with cell-cell adhesion was observed in mouse NSPCs by live cell image analysis and the presence of acrylamide. Protein expression levels of cell adhesion molecules [neural cell adhesion molecule (NCAM) and N-cadherin] and extracellular signal-regulated kinases (ERK) in acrylamide-treated KT98/F1B-GFP and U-1240 MG/F1B-GFP Neurospheres demonstrated that NCAM decreased and phospho-ERK (pERK) increased, whereas expression of N-cadherin remained unchanged. Analysis of AKT (protein kinase B, PKB)/β-catenin pathway showed decrease in phospho-AKT (p-AKT) and cyclin D1 expression in acrylamide-treated Neurospheres of KT98/F1B-GFP. Furthermore, PD98059, an ERK phosphorylation inhibitor, attenuated acrylamide-induced ERK phosphorylation, indicating that pERK contributed to the cell proliferation, but not in Neurosphere formation in mouse NSPCs. Coimmunoprecipitation results of KT98/F1B-GFP cell lysates showed that the complex of NCAM and fibroblast growth factor receptor 1 (FGFR1) is present in the Neurosphere, and the amount of this complex decreases after acrylamide treatment. Our results reveal that acrylamide inhibits Neurosphere formation through the disruption of the Neurosphere architecture in NSPCs. The downregulation of cell-cell adhesion resulted from decreasing the levels of NCAM as well as the formation of NCAM/FGFR complex.
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Single-cell enzyme-free dissociation of Neurospheres using a microfluidic chip.
Analytical Chemistry, 2013Co-Authors: Hao-chen Chang, Ingming ChiuAbstract:Obtaining single dissociated cells from Neurospheres is difficult using nonenzymatic methods. In this paper we report the development of a microfluidic-chip-based approach that utilizes flow and microstructures to dissociate Neurospheres. We show that this microfluidic-chip-based Neurosphere-dissociation method can generate high yields of single cells from dissociated Neurospheres of mouse KT98 and DC115 cell models (passage number, 3–8; diameter range, 40–250 μm): 90% and 95%, respectively. The microfluidic-chip-dissociated cells had high viabilities (80–85%) and the ability to regrow into Neurospheres, demonstrating the applicability of this device to Neurosphere assay applications. In addition, the dissociated cells retained their normal differentiation potentials, as shown by their capabilities to differentiate into three neural lineages (neurons, astroglia, and oligodendrocytes) when cultured in differentiation culture conditions. Since this microfluidic-chip-based method does not require the use of ...
Albrecht M Muller - One of the best experts on this subject based on the ideXlab platform.
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genetic instability and diminished differentiation capacity in long term cultured mouse Neurosphere cells
Mechanisms of Ageing and Development, 2010Co-Authors: Vladimir Vukicevic, Anna Jauch, Timo C Dinger, Linda Gebauer, Veronika Hornich, Stefan R Bornstein, Monika Ehrhartbornstein, Albrecht M MullerAbstract:Summary The potential use of neural stem cells in basic research, drug testing and for development of therapeutic strategies requires large scale in vitro amplification, increasing the probability of genetic instability and transformation. Little is known, however, about potential correlations between long-term culture of neural stem and progenitor cells (NSPCs), changed differentiation and self-renewal capacities, and the occurrence of chromosomal instability. This study investigates the effect of extended culture time on self-renewal, differentiation capacity, cell cycle phase distribution, telomere length, telomerase activity and chromosomal stability on fetal brain-derived cells that form floating sphere colonies (Neurospheres). We observed that increased sphere-forming capacity indicative of increased proliferation was accompanied by a decreased ability to differentiate into neural lineages. The high mobility group A (Hmga2) gene positively regulates self-renewal via repression of p16Ink4a and p19ARF gene expression. This study discerned an upregulation of Hmga2 gene and protein expression and decreased p16Ink4a and p19ARF gene expression, suggesting that Hmga2 might promote the proliferation of Neurosphere cells in long-term culture. Further, our analyses revealed a significant decrease in telomere length after 4 weeks of culturing that is paralleled by a moderate upregulation of telomerase activity. Importantly, regular gain of chromosome 1 with random structural chromosomal aberrations was observed within 16 weeks of Neurosphere cell culture. Genetic instability and diminished differentiation capacity seem to be a consequence of long-term culture of Neurosphere cells. These data indicate the necessity to analyze self-renewal, differentiation capacity, telomere length, tumor suppressor genes and chromosomal stability in Neurosphere cultures prior to their usage in basic research, drug testing or the development of therapeutic strategies.
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in vivo haematopoietic activity is induced in Neurosphere cells by chromatin modifying agents
The EMBO Journal, 2005Co-Authors: Carolin Schmittwolf, Nicole Kirchhof, Anna Jauch, Michael Durr, Friedrich Harder, Martin Zenke, Albrecht M MullerAbstract:Modifications of DNA and chromatin are fundamental for the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. To test whether the developmental potential of fetal brain-derived cells that form floating sphere colonies (Neurospheres) can be modified by destabilizing their epigenotype, Neurosphere cells were treated with chemical compounds that alter the acetylation and methylation patterns of chromatin and DNA. Intravenous infusion of bulk or clonally derived Neurosphere cells treated with a combination of trichostatin A (TSA) plus 5-aza-2′-deoxycytidine (AzaC) (TSA/AzaC Neurosphere cells) yielded long-term, multilineage and transplantable Neurosphere-derived haematopoietic repopulation. Untreated Neurosphere cells exhibited no haematopoietic repopulation activity. The Neurosphere-derived haematopoietic cells showed a diploid karyotype, indicating that they are unlikely to be products of cell fusion events, a conclusion strengthened by multicolour fluorescence in situ hybridization. Our results indicate that altering the epigenotype of Neurosphere cells followed by transplantation enables the generation of Neurosphere-derived haematopoietic cells.
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In vivo haematopoietic activity is induced in Neurosphere cells by chromatin‐modifying agents
The EMBO Journal, 2005Co-Authors: Carolin Schmittwolf, Nicole Kirchhof, Anna Jauch, Michael Durr, Friedrich Harder, Martin Zenke, Albrecht M MullerAbstract:Modifications of DNA and chromatin are fundamental for the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. To test whether the developmental potential of fetal brain-derived cells that form floating sphere colonies (Neurospheres) can be modified by destabilizing their epigenotype, Neurosphere cells were treated with chemical compounds that alter the acetylation and methylation patterns of chromatin and DNA. Intravenous infusion of bulk or clonally derived Neurosphere cells treated with a combination of trichostatin A (TSA) plus 5-aza-2′-deoxycytidine (AzaC) (TSA/AzaC Neurosphere cells) yielded long-term, multilineage and transplantable Neurosphere-derived haematopoietic repopulation. Untreated Neurosphere cells exhibited no haematopoietic repopulation activity. The Neurosphere-derived haematopoietic cells showed a diploid karyotype, indicating that they are unlikely to be products of cell fusion events, a conclusion strengthened by multicolour fluorescence in situ hybridization. Our results indicate that altering the epigenotype of Neurosphere cells followed by transplantation enables the generation of Neurosphere-derived haematopoietic cells.
Seok Chung - One of the best experts on this subject based on the ideXlab platform.
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endothelial Neurosphere crosstalk in microwell arrays regulates self renewal and differentiation of human neural stem cells
Journal of Industrial and Engineering Chemistry, 2019Co-Authors: Kisuk Yang, Gyeong Eon Chang, Eunji Cheong, Ji Hun Yang, Seok ChungAbstract:Abstract Control of neural stem cell (NSC) self-renewal and differentiationis of great importance to improve its therapeutic efficacy in the treatment of neurodegenerative diseases. Neurosphere culture for NSC expansion under undifferentiation condition determines the self-renewal capacity and differentiation propensity of NSCs. In this study, we examined the effects of controlled crosstalk between endothelial cells (ECs) and NSC Neurospheres on self-renewal, differentiation, and functions of NSCs. Cultures of human fetal NSCs (hfNSCs) or human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (NPCs) in a microwell array with 500-μm well diameter facilitated cell–cell interaction and self-renewal ability, leading to increased neuronal differentiation and improved electrophysiological functions. Incorporation of ECs into size-controlled hfNSC Neurospheres further promoted cell–cell interaction and self-renewal capacity. The decrease in EC density in hfNSC Neurospheres effectively promoted cell–cell interaction and self-renewal. Under spontaneous differentiation condition, EC-containing hfNSC Neurospheres differentiated into astrocytes rather than neuronal lineages. Therefore, we suggest the engineering of NSCs at Neurosphere stage using microwell culture to control Neurosphere size and that EC co-culture for vascularization may regulate behaviors, phenotypes, and functions of NSCs, leading to modulation of their therapeutic and regenerative potentials.
Sufan Wu - One of the best experts on this subject based on the ideXlab platform.
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bone marrow stromal cells enhance differentiation of cocultured Neurosphere cells and promote regeneration of injured spinal cord
Journal of Neuroscience Research, 2003Co-Authors: Sufan Wu, Yoshihisa Suzuki, Yoko Ejiri, Toru Noda, Masaaki Kitada, Kazuya Kataoka, Masayoshi Ohta, Hirotomi ChouAbstract:Transplantation of bone marrow stromal cells (MSCs) has been regarded as a potential approach for promoting nerve regeneration. In the present study, we investigated the influence of MSCs on spinal cord Neurosphere cells in vitro and on the regeneration of injured spinal cord in vivo by grafting. MSCs from adult rats were cocultured with fetal spinal cord-derived Neurosphere cells by either cell mixing or making monolayered-feeder cultures. In the mixed cell cultures, neuroshpere cells were stimulated to develop extensive processes. In the monolayered-feeder cultures, numerous processes from Neurosphere cells appeared to be attracted to MSCs. In an in vivo experiment, grafted MSCs promoted the regeneration of injured spinal cord by enhancing tissue repair of the lesion, leaving apparently smaller cavities than in controls. Although the number of grafted MSCs gradually decreased, some treated animals showed remarkable functional recovery. These results suggest that MSCs might have profound effects on the differentiation of Neurosphere cells and be able to promote regeneration of the spinal cord by means of grafting. © 2003 Wiley-Liss, Inc.
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migration integration and differentiation of hippocampus derived Neurosphere cells after transplantation into injured rat spinal cord
Neuroscience Letters, 2001Co-Authors: Sufan Wu, Yoshihisa Suzuki, Masaaki Kitada, Kazuya Kataoka, Miyako Kitaura, Jun Takahashi, Yoshihiko NishimuraAbstract:Hippocampus-derived Neurospheres were prepared from transgenic rat fetuses expressing green fluorescent protein (GFP), and transplanted into an alginate-filled lesion of young rat spinal cord. One, two and four weeks after transplantation, a large number of grafted cells survived, many of which expressed immunoreactivity for glial fibrillary acidic protein, and a few expressed immunoreactivity for β-tubulin III. The grafted cells closely attached to the host tissue including astrocytes at the border of the lesion. It was notable that numerous GFP-positive cells had migrated within host spinal cord tissue up to 2 mm away from the implanted site 4 weeks postoperation. These results demonstrate that rat fetal hippocampus-derived Neurosphere cells could survive, differentiate, extensively migrate, and integrate well into the host spinal cord tissue.
Gunther K H Zupanc - One of the best experts on this subject based on the ideXlab platform.
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Stochastic cellular automata model of tumorous Neurosphere growth: Roles of developmental maturity and cell death
Journal of Theoretical Biology, 2019Co-Authors: Gunther K H Zupanc, Frederick B. Zupanc, Rifat SipahiAbstract:Abstract The Neurosphere assay is a powerful in vitro system for studying stem/progenitor-cell-driven tissue growth. By employing a stochastic cellular automata model, we simulated the development of tumorous Neurospheres in response to transformation of a randomly selected progenitor cell into a brain tumor stem cell. Simulated tumorous Neurospheres were distinguished from normal Neurospheres by their size, which exceeded that of normal Neurospheres typically manifold. A decisive factor that determined whether brain tumor stem cells gave rise to tumorous Neurospheres was their ability to escape encapsulation by neighboring cells, which suppressed mitotic activity through contact inhibition. In our simulations, the likelihood of tumorigenesis was strongly negatively correlated with the developmental maturity of the Neurospheres in which the transformation of a progenitor cell into a brain tumor stem cell was induced. This likelihood was furthermore modulated by the probability of the progeny of dividing cells to undergo cell death. In developmentally immature Neurospheres, the number of normal Neurospheres, relative to the number of tumorous Neurospheres, increased with increasing cell death probability. Markedly, in developmentally mature Neurospheres the opposite effect was observed. This dichotomous effect of cell death on simulated tumor progression provides theoretical support for the seemingly paradoxical finding made by other authors in experimental studies that anti-cancer therapies based on induction of apoptosis may both promote and suppress tumor growth.
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stochastic cellular automata model of Neurosphere growth roles of proliferative potential contact inhibition cell death and phagocytosis
Journal of Theoretical Biology, 2018Co-Authors: Rifat Sipahi, Gunther K H ZupancAbstract:Abstract Neural stem and progenitor cells isolated from the central nervous system form, under specific culture conditions, clonal cell clusters known as Neurospheres. The Neurosphere assay has proven to be a powerful in vitro system to study the behavior of such cells and the development of their progeny. However, the theory of Neurosphere growth has remained poorly understood. To overcome this limitation, we have, in the present paper, developed a cellular automata model, with which we examined the effects of proliferative potential, contact inhibition, cell death, and clearance of dead cells on growth rate, final size, and composition of Neurospheres. Simulations based on this model indicated that the proliferative potential of the founder cell and its progenitors has a major influence on Neurosphere size. On the other hand, contact inhibition of proliferation limits the final size, and reduces the growth rate, of Neurospheres. The effect of this inhibition is particularly dramatic when a stem cell becomes encapsulated by differentiated or other non-proliferating cells, thereby suppressing any further mitotic division – despite the existing proliferative potential of the stem cell. Conversely, clearance of dead cells through phagocytosis is predicted to accelerate growth by reducing contact inhibition. A surprising prediction derived from our model is that cell death, while resulting in a decrease in growth rate and final size of Neurospheres, increases the degree of differentiation of Neurosphere cells. It is likely that the cellular automata model developed as part of the present investigation is applicable to the study of tissue growth in a wide range of systems.