Journal articles on the topic 'Neurospheres'
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1
Somredngan, Sirilak, Kasem Theerakittayakorn, Hong Thi Nguyen, Apichart Ngernsoungnern, Piyada Ngernsoungnern, Pishyaporn Sritangos, Mariena Ketudat-Cairns, et al. "The Efficiency of Neurospheres Derived from Human Wharton’s Jelly Mesenchymal Stem Cells for Spinal Cord Injury Regeneration in Rats." International Journal of Molecular Sciences 24, no. 4 (February 14, 2023): 3846. http://dx.doi.org/10.3390/ijms24043846.
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Spinal cord injury (SCI) causes inflammation and neuronal degeneration, resulting in functional movement loss. Since the availability of SCI treatments is still limited, stem cell therapy is an alternative clinical treatment for SCI and neurodegenerative disorders. Human umbilical cord Wharton’s jelly-derived mesenchymal stem cells (hWJ-MSCs) are an excellent option for cell therapy. This study aimed to induce hWJ-MSCs into neural stem/progenitor cells in sphere formation (neurospheres) by using neurogenesis-enhancing small molecules (P7C3 and Isx9) and transplant to recover an SCI in a rat model. Inducted neurospheres were characterized by immunocytochemistry (ICC) and gene expression analysis. The best condition group was selected for transplantation. The results showed that the neurospheres induced by 10 µM Isx9 for 7 days produced neural stem/progenitor cell markers such as Nestin and β-tubulin 3 through the Wnt3A signaling pathway regulation markers (β-catenin and NeuroD1 gene expression). The neurospheres from the 7-day Isx9 group were selected to be transplanted into 9-day-old SCI rats. Eight weeks after transplantation, rats transplanted with the neurospheres could move normally, as shown by behavioral tests. MSCs and neurosphere cells were detected in the injured spinal cord tissue and produced neurotransmitter activity. Neurosphere-transplanted rats showed the lowest cavity size of the SCI tissue resulting from the injury recovery mechanism. In conclusion, hWJ-MSCs could differentiate into neurospheres using 10 µM Isx9 media through the Wnt3A signaling pathway. The locomotion and tissue recovery of the SCI rats with neurosphere transplantation were better than those without transplantation.
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Blackwood, Christopher. "Quantitative approach to numbers and sizes: Generation of primary neurospheres from the dorsal lateral ganglionic eminence of late embryonic mice." F1000Research 8 (November 25, 2019): 1983. http://dx.doi.org/10.12688/f1000research.21208.1.
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Background: The neurosphere assay is a powerful tool to study neural stem cell biology. The objective of this protocol is to create a simple and rapid approach to generate neurospheres from the dorsal lateral ganglionic eminence of late embryonic (day 17) mice. This method predicts the average number of neurospheres and provides an approximation of its expected size after 7 days in vitro. Characterization of numbers and sizes will provide investigators with quantitative data to advise on the implementation of downstream applications, including immnocytochemistry, self-renewal and differentiation assays. Methods: Our method is based on a simple dissection technique, where tissue surrounding the dorsal lateral ventricle from a single mouse embryo is trimmed away to enrich for neural stem cell and progenitor populations. Following this dissection, tissue is mechanically dissociated by trituration. Cells are then cultured in media containing epidermal growth factor and other supplements to generate healthy primary neurospheres. Results: Using this approach, we found reproducible number of primary neurospheres after 7 days in vitro. Furthermore, we found this method yields different sizes of neurospheres. Lastly, using an anti-GFAP antibody, we confirm that these neurospheres can be used for immunocytochemistry studies. Conclusions: Future use of this protocol provides metrics on the generation of neurospheres that will be useful for further advances in the area of stem cell biology.
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Blackwood, Christopher. "Quantitative approach to numbers and sizes: Generation of primary neurospheres from the dorsal lateral ganglionic eminence of late embryonic mice." F1000Research 8 (March 11, 2020): 1983. http://dx.doi.org/10.12688/f1000research.21208.2.
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Background: The neurosphere assay is a powerful in vitro tool to investigate neural stem cells in the dorsal lateral ventricle (dLGE). In the dLGE, metrics of sizes and numbers of neurospheres generated using this assay has not been completely characterized. The objective of this protocol is to provide a stepwise method from a single isolation that predicts the average number of neurospheres generated and to estimate an approximation of its sizes after several days in vitro. The advantage of this protocol is that no expensive and specialized equipment is needed for tissue isolation. Estimates about the numbers and sizes of neurospheres will provide investigators with quantitative data to advise on how much starting dLGE tissue is required to generate the appropriate number of spheres for the implementation of downstream applications, including immunocytochemistry, self-renewal and differentiation assays. Methods: Our method is based on a simple dissection technique, where tissue surrounding the dorsal lateral ventricle from a single mouse embryo is trimmed away to enrich for neural stem cell and progenitor populations. Following this dissection, tissue is mechanically dissociated by trituration. Cells are then cultured in media containing epidermal growth factor and other supplements to generate healthy primary neurospheres. Results: Using this approach, we found reproducible number of primary neurospheres after 7 days in vitro (DIV). Furthermore, we observed that this method yields an average range of neurospheres sizes greater than 50 μm, but less than 100 μm after 7 DIV. Lastly, using an anti-GFAP antibody, we show that these neurospheres can be stained, confirming their use in future immunocytochemistry studies. Conclusions: Future use of this protocol provides metrics on the generation of primary neurospheres that will be useful for further advances in the area of stem cell biology.
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Tafreshi, Azita Parvaneh, Aude Sylvain, Guizhi Sun, Daniella Herszfeld, Keith Schulze, and Claude C. A. Bernard. "Lithium chloride improves the efficiency of induced pluripotent stem cell-derived neurospheres." Biological Chemistry 396, no. 8 (August 1, 2015): 923–28. http://dx.doi.org/10.1515/hsz-2014-0261.
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Abstract Induced pluripotent stem cell (iPSC)-derived neurospheres, which consist mainly of neural progenitors, are considered to be a good source of neural cells for transplantation in regenerative medicine. In this study, we have used lithium chloride, which is known to be a neuroprotective agent, in an iPSC-derived neurosphere model, and examined both the formation rate and size of the neurospheres as well as the proliferative and apoptotic status of their contents. Our results showed that lithium enhanced the formation and the sizes of the iPSC-derived neurospheres, increased the number of Ki67-positive proliferating cells, but reduced the number of the TUNEL-positive apoptotic cells. This increased number of Ki67 proliferating cells was secondary to the decreased apoptosis and not to the stimulation of cell cycle entry, as the expression of the proliferation marker cyclin D1 mRNA did not change after lithium treatment. Altogether, we suggest that lithium enhances the survival of neural progenitors and thus the quality of the iPSC-derived neurospheres, which may strengthen the prospect of using lithium-treated pluripotent cells and their derivatives in a clinical setting.
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Duval, Nathalie, Danielle Gomès, Viviane Calaora, Alessandra Calabrese, Paolo Meda, and Roberto Bruzzone. "Cell coupling and Cx43 expression in embryonic mouse neural progenitor cells." Journal of Cell Science 115, no. 16 (August 15, 2002): 3241–51. http://dx.doi.org/10.1242/jcs.115.16.3241.
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Embryonic neural progenitors isolated from the mouse striatal germinal zone grow in vitro as floating cell aggregates called neurospheres, which, upon adhesion, can be induced to differentiate into the three main cell types of the central nervous system (CNS), that is, astrocytes, neurons and oligodendrocytes. To study the possible role of connexins and junctional communication during differentiation of neural progenitors, we assessed cell-to-cell communication by microinjecting Lucifer Yellow into neurospheres at various times after adhesion. Cells located in neurospheres were strongly coupled, regardless of the differentiation time. Microinjections performed on the cell layers formed by differentiated cells migrating out of the neurosphere established that only astrocytes were coupled. These observations suggest the existence of at least three distinct communication compartments:coupled proliferating cells located in the sphere, uncoupled cells undergoing neuronal or oligodendrocytic differentiation and coupled differentiating astrocytes. A blockade of junctional communication by 18-β-glycyrrhetinic acid (βGA) reduced, in a concentration-dependent manner, the viability of undifferentiated neural progenitor cells. This effect appeared to be specific,inasmuch as it was reversible and that cell survival was not affected in the presence of the inactive analog glycyrrhyzic acid. Addition of βGA to adherent neurospheres also decreased cell density and altered the morphology of differentiated cells. Cx43 was strongly expressed in either undifferentiated or differentiated neurospheres, where it was found both within the sphere and in astrocytes, the two cell populations that were dye coupled. Western blot analysis further showed that Cx43 phosphorylation was strongly increased in adherent neurospheres, suggesting a post-translational regulation during differentiation. These results point to a major role of cell-to-cell communication and Cx43 during the differentiation of neural progenitor cells in vitro.
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Kühne, Britta Anna, Paula Vázquez-Aristizabal, Mercè Fuentes-Amell, Laura Pla, Carla Loreiro, Jesús Gómez-Catalán, Eduard Gratacós, Miriam Illa, and Marta Barenys. "Docosahexaenoic Acid and Melatonin Prevent Impaired Oligodendrogenesis Induced by Intrauterine Growth Restriction (IUGR)." Biomedicines 10, no. 5 (May 23, 2022): 1205. http://dx.doi.org/10.3390/biomedicines10051205.
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In this study, our aims were to characterize oligodendrogenesis alterations in fetuses with intrauterine growth restriction (IUGR) and to find therapeutic strategies to prevent/treat them using a novel rabbit in vitro neurosphere culture. IUGR was surgically induced in one uterine horn of pregnant rabbits, while the contralateral horn served as a control. Neural progenitor cells (NPCs) were obtained from pup’s whole brain and cultured as neurospheres mimicking the basic processes of brain development including migration and cell differentiation. Five substances, chosen based on evidence provided in the literature, were screened in vitro in neurospheres from untreated rabbits: Docosahexaenoic acid (DHA), melatonin (MEL), zinc, 3,3′,5-Triiodo-L-thyronine (T3), and lactoferrin (LF) or its metabolite sialic acid (SA). DHA, MEL and LF were further selected for in vivo administration and subsequent evaluation in the Neurosphere Assay. In the IUGR culture, we observed a significantly reduced percentage of oligodendrocytes (OLs) which correlated with clinical findings indicating white matter injury in IUGR infants. We identified DHA and MEL as the most effective therapies. In all cases, our in vitro rabbit neurosphere assay predicted the outcome of the in vivo administration of the therapies and confirmed the reliability of the model, making it a powerful and consistent tool to select new neuroprotective therapies.
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Hsu, Wesley, I.-Mei Siu, Gustavo Pradilla, Ziya L. Gokaslan, George I. Jallo, and Gary L. Gallia. "Animal model of intramedullary spinal cord glioma using human glioblastoma multiforme neurospheres." Journal of Neurosurgery: Spine 16, no. 3 (March 2012): 315–19. http://dx.doi.org/10.3171/2011.11.spine11492.
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Object Advances in the diagnosis and management of patients with spinal cord tumors have been limited because of the rarity of the disease and the limitations of current animal models for spinal cord glioma. The ideal spinal cord tumor model would possess a number of characteristics, including the use of human glioma cells that capture the growth pattern and local invasive nature of their human counterpart. In this study, the authors' goal was to develop a novel spinal cord tumor model using a human neurosphere cell line. Methods Eighteen female athymic rats were randomized into 3 experimental groups. Animals in the first group (6 rats) received a 3-ml intramedullary injection containing DMEM and were used as controls. Animals in the second group (6 rats) received a 3-ml intramedullary injection containing 100,000 glioblastoma multiforme (GBM) neurosphere cells in 3 ml DMEM. Animals in the third group (6 rats) received a 3-ml intramedullary injection containing 9L gliosarcoma cells in 3 ml DMEM. Functional testing of hindlimb strength was assessed using the Basso-Beattie-Bresnahan (BBB) scale. Once the functional BBB score of an animal was less than or equal to 5 (slight movement of 2 joints and extensive movement of the third), euthanasia was performed. Results Animals in the GBM neurosphere group had a mean survival of 33.3 ± 2.0 days, which was approximately twice as long as animals in the 9L gliosarcoma group (16.3 ± 2.3 days). There was a significant difference between survival of the GBM neurosphere and 9L gliosarcoma groups (p < 0.001). None of the control animals died (p < 0.001 for GBM neurosphere group vs controls and 9L vs controls). Histopathological examination of the rats injected with 9L gliosarcoma revealed that all animals developed highly cellular, well-circumscribed lesions causing compression of the surrounding tissue, with minimal invasion of the surrounding gray and white matter. Histopathological examination of animals injected with GBM neurospheres revealed that all animals developed infiltrative lesions with a high degree of white and gray matter invasion along with areas of necrosis. Conclusions The authors have established a novel animal model of spinal cord glioma using neurospheres derived from human GBM. When injected into the spinal cords of athymic nude rats, neurospheres gave rise to infiltrative, actively proliferating tumors that were histologically identical to spinal cord glioma in humans. On the basis of their results, the authors conclude that this is a reproducible animal model of high-grade spinal cord glioma based on a human GBM neurosphere line. This model represents an improvement over other models using nonhuman glioma cell lines. Novel therapeutic strategies can be readily evaluated using this model.
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Raja, Waseem K., Esther Neves, Christopher Burke, Xin Jiang, Ping Xu, Kenneth J. Rhodes, Vikram Khurana, Robert H. Scannevin, and Chee Yeun Chung. "Patient-derived three-dimensional cortical neurospheres to model Parkinson’s disease." PLOS ONE 17, no. 12 (December 1, 2022): e0277532. http://dx.doi.org/10.1371/journal.pone.0277532.
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There are currently no preventive or disease-modifying therapies for Parkinson’s Disease (PD). Failures in clinical trials necessitate a re-evaluation of existing pre-clinical models in order to adopt systems that better recapitulate underlying disease mechanisms and better predict clinical outcomes. In recent years, models utilizing patient-derived induced pluripotent stem cells (iPSC) have emerged as attractive models to recapitulate disease-relevant neuropathology in vitro without exogenous overexpression of disease-related pathologic proteins. Here, we utilized iPSC derived from patients with early-onset PD and dementia phenotypes that harbored either a point mutation (A53T) or multiplication at the α-synuclein/SNCA gene locus. We generated a three-dimensional (3D) cortical neurosphere culture model to better mimic the tissue microenvironment of the brain. We extensively characterized the differentiation process using quantitative PCR, Western immunoblotting and immunofluorescence staining. Differentiated and aged neurospheres revealed alterations in fatty acid profiles and elevated total and pathogenic phospho-α-synuclein levels in both A53T and the triplication lines compared to their isogenic control lines. Furthermore, treatment of the neurospheres with a small molecule inhibitor of stearoyl CoA desaturase (SCD) attenuated the protein accumulation and aberrant fatty acid profile phenotypes. Our findings suggest that the 3D cortical neurosphere model is a useful tool to interrogate targets for PD and amenable to test small molecule therapeutics.
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Godoy, Paulo R. D. V., Ali Pour Khavari, Marzia Rizzo, Elza T. Sakamoto-Hojo, and Siamak Haghdoost. "Targeting NRF2, Regulator of Antioxidant System, to Sensitize Glioblastoma Neurosphere Cells to Radiation-Induced Oxidative Stress." Oxidative Medicine and Cellular Longevity 2020 (June 16, 2020): 1–17. http://dx.doi.org/10.1155/2020/2534643.
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The presence of glioma stem cells (GSCs), which are enriched in neurospheres, may be connected to the radioresistance of glioblastoma (GBM) due to their enhanced antioxidant defense and elevated DNA repair capacity. The aim was to evaluate the responses to different radiation qualities and to reduce radioresistance of U87MG cells, a GBM cell line. U87MG cells were cultured in a 3D model and irradiated with low (24 mGy/h) and high (0.39 Gy/min) dose rates of low LET gamma and high LET carbon ions (1-2 Gy/min). Thereafter, expression of proteins related to oxidative stress response, extracellular 8-oxo-dG, and neurospheres were determined. LD50 for carbon ions was significantly lower compared to LD50 of high and low dose rate gamma radiation. A significantly higher level of 8-oxo-dG was detected in the media of cells exposed to a low dose rate as compared to a high dose rate of gamma or carbon ions. A downregulation of oxidative stress proteins was also observed (NRF2, hMTH1, and SOD1). The NRF2 gene was knocked down by CRISPR/Cas9 in neurosphere cells, resulting in less self-renewal, more differentiated cells, and less proliferation capacity after irradiation with low and high dose rate gamma rays. Overall, U87MG glioma neurospheres presented differential responses to distinct radiation qualities and NRF2 plays an important role in cellular sensitivity to radiation.
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Zainal Abidin, Shahidee, Han-Chung Lee, Syahril Abdullah, Norshariza Nordin, Pike-See Cheah, and King-Hwa Ling. "The expression profile of miR-3099 during neural development of Ts1Cje mouse model of Down syndrome." Neuroscience Research Notes 4, no. 1 (March 15, 2021): 7–15. http://dx.doi.org/10.31117/neuroscirn.v4i1.62.
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MicroRNA-3099 (miR-3099) plays a crucial role in regulating neuronal differentiation and development of the central nervous system (CNS). The miR-3099 is a pro-neuronal miRNA that promotes neural stem/progenitor cell (NSPC) differentiation into neuronal lineage by suppressing astrogliogenesis. Down syndrome (DS) brain exhibited increased astrogliogenesis and reduced neuronal cell density. The involvement of miR-3099 in the neurodevelopment of DS has not been investigated and potentially responsible for the neurogenic-to-gliogenic shift phenomenon observed in DS brain. To investigate the role of miR-3099 during DS brain development, neural/progenitor cell proliferation and differentiation, we profiled miR-3099 expression level in the Ts1Cje, a mouse model for DS. We analysed the Ts1Cje whole brain at embryonic day (E) 10.5, E14.5 and P1.5, proliferating neurospheres and differentiating neurospheres at 3, 9 and 15 days in vitro (DIV). Expression of miR-3099 in both the developing mouse brain and the differentiating neurosphere was not significantly different between Ts1Cje and wild type controls. In contrast, the expression level of miR-3099 was significantly higher (p<0.05) in proliferating NSPC derived from the Ts1Cje compared to wild-type. Further molecular profiling of NPSC and glial cell markers indicated that the expression of Sox2 (p<0.01) and Gfap (p<0.05) were significantly downregulated in Ts1Cje neurospheres as compared to that of wild type, respectively. While there were no significant differences in Tuj1 and Nestin expression levels between the Ts1Cje and wild type neurospheres, their expression levels were ~3-fold upregulated and ~2.6 downregulated Ts1Cje group, respectively. The findings suggest that dysregulation of miR-3099 affects NSPC lineage commitment as indicated by altered postmitotic neuronal cell markers. Further molecular characterisation and gene expression profiling of other neuronal and glial markers will help refine the analysis of gene-gene interactions underlying the neuropathologies of DS.
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Peixoto, Joana, Sudha Janaki-Raman, Lisa Schlicker, Werner Schmitz, Susanne Walz, Alina M. Winkelkotte, Christel Herold-Mende, Paula Soares, Almut Schulze, and Jorge Lima. "Integrated Metabolomics and Transcriptomics Analysis of Monolayer and Neurospheres from Established Glioblastoma Cell Lines." Cancers 13, no. 6 (March 16, 2021): 1327. http://dx.doi.org/10.3390/cancers13061327.
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Altered metabolic processes contribute to carcinogenesis by modulating proliferation, survival and differentiation. Tumours are composed of different cell populations, with cancer stem-like cells being one of the most prominent examples. This specific pool of cells is thought to be responsible for cancer growth and recurrence and plays a particularly relevant role in glioblastoma (GBM), the most lethal form of primary brain tumours. Here, we have analysed the transcriptome and metabolome of an established GBM cell line (U87) and a patient-derived GBM stem-like cell line (NCH644) exposed to neurosphere or monolayer culture conditions. By integrating transcriptome and metabolome data, we identified key metabolic pathways and gene signatures that are associated with stem-like and differentiated states in GBM cells, and demonstrated that neurospheres and monolayer cells differ substantially in their metabolism and gene regulation. Furthermore, arginine biosynthesis was identified as the most significantly regulated pathway in neurospheres, although individual nodes of this pathway were distinctly regulated in the two cellular systems. Neurosphere conditions, as opposed to monolayer conditions, cause a transcriptomic and metabolic rewiring that may be crucial for the regulation of stem-like features, where arginine biosynthesis may be a key metabolic pathway. Additionally, TCGA data from GBM patients showed significant regulation of specific components of the arginine biosynthesis pathway, providing further evidence for the importance of this metabolic pathway in GBM.
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Yokoi, Takuya, Takuya Uemura, Kiyohito Takamatsu, Kosuke Shintani, Ema Onode, Shunpei Hama, Yusuke Miyashima, Mitsuhiro Okada, and Hiroaki Nakamura. "Fate and contribution of induced pluripotent stem cell-derived neurospheres transplanted with nerve conduits to promote peripheral nerve regeneration in mice." Bio-Medical Materials and Engineering 32, no. 3 (May 18, 2021): 171–81. http://dx.doi.org/10.3233/bme-201182.
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BACKGROUND: We previously demonstrated that a bioabsorbable nerve conduit coated with mouse induced pluripotent stem cell (iPSC)-derived neurospheres accelerated peripheral nerve regeneration in mice. OBJECTIVE: We examined the fate and utility of iPSC-derived neurospheres transplanted with nerve conduits for the treatment of sciatic nerve gaps in mice. METHODS: Complete 5-mm defects were created in sciatic nerves and reconstructed using nerve conduits that were either uncoated or coated with mouse iPSC-derived neurospheres. The survival of the neurospheres on the nerve conduits was tracked using an in vivo imaging. The localization of the transplanted cells and regenerating axons was examined histologically. The gene expression levels in the nerve conduits were evaluated. RESULTS: The neurospheres survived for at least 14 days, peaking at 4--7 days after implantation. The grafted neurospheres remained as Schwann-like cells within the nerve conduits and migrated into the regenerated axons. The expression levels of ATF3, BDNF, and GDNF in the nerve conduit coated with neurospheres were upregulated. CONCLUSIONS: Mouse iPSC-derived neurospheres transplanted with nerve conduits for the treatment of sciatic nerve defects in mice migrated into regenerating axons, survived as Schwann-like cells, and promoted axonal growth with an elevation in the expression of nerve regeneration-associated trophic factors.
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Barton, Jack, Katherine Pacey, Neha Jain, Tessa Kasia, Darren Edwards, Christine Thevanesan, Karin Straathof, Giuseppe Barone, and John Anderson. "Establishment and phenotyping of neurosphere cultures from primary neuroblastoma samples." F1000Research 8 (June 10, 2019): 823. http://dx.doi.org/10.12688/f1000research.18209.1.
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Background: Primary cell culture using serum free media supplemented with growth factors has been used in a number of cancers to propagate primary cells with stem like properties, which form as spherical cellular aggregates. Methods: We systematically evaluated the capacity of freshly disaggregated neuroblastoma tumors to become established as neurospheres in stem cell media using a uniform protocol. 67 primary neuroblastoma samples from patients treated at a single institution were prospectively evaluated for their ability to become established in culture. Samples, either solid tissue or cells from surgical transit fluid both post chemotherapy and chemotherapy naïve, were evaluated from diagnostic needle biopsies or surgical resections. Results: Overall 37 neurosphere cultures were successfully established from 67 samples. In 11 out of 14 cases investigated by flow cytometry, uniform staining for neuroblastoma markers CD56 and GD2 was demonstrated in CD45 negative non-hemopoietic cells, confirming neuroblastoma origin. Conclusion: We present a simple and reproducible approach for producing primary neurospheres from neuroblastoma samples, which provides a reliable resource for future work including genetic analysis, stem cell research and models for therapeutics.
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Miki, Shunichiro, Tomoyuki Koga, Kasey R. Skinner, Robert F. Gruener, Daisuke Kawauchi, R. Stephanie Huang, C. Ryan Miller, and Frank Furnari. "TB-1 ADDITIONAL GENETIC ALTERATIONS DIFFERENTIALLY ALTER THE TRANSCRIPTOMIC LANDSCAPE OF H3 K27M-MUTANT DIFFUSE MIDLINE GLIOMA." Neuro-Oncology Advances 4, Supplement_3 (December 1, 2022): iii6. http://dx.doi.org/10.1093/noajnl/vdac167.020.
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Abstract Histone H3 K27M mutation is a hallmark mutation for H3 K27M-mutant diffuse midline glioma (DMG), but targeting this mutation has yet to achieve a significant survival benefit in clinical trials. Recent analyses revealed alterations in several genes, such as NF1 and PDGFRA, are observed in substantial subpopulations of H3 K27M-mutant DMG patients in addition to H3 mutation and recurrent TP53 mutations, indicating patient-to-patient tumor heterogeneity and the potential necessity of tailored target therapy for the treatment of this disease. Here, using our human induced pluripotent stem cells (iPSC)-derived glioma avatar platform, we designed DMG models by introducing TP53R248Q with or without heterozygous H3 K27M mutation in combination with further genetic modifications of either NF1 knockout or PDGFRAD842V overexpression to recapitulate DMG subpopulations. Mice with TP53R248Q; H3F3AK27M (QM) tumors survived significantly longer than those harboring QM;NF1-/- (QMN) tumors and QM; PDGFRAD842Voe (QMP) tumors. RNA-sequencing of those induced DMG (iDMG) neurospheres revealed altered patterns of upregulation of MAPK pathway genes both in QMN and QMP-iDMG neurospheres compared to their H3 wildtype counterparts with the same combinations of genetic alterations, suggesting that those additional mutations modifies the oncogenic signaling associated with H3 K27M mutation. Further, differential expression analysis comparing QMN and QMP-iDMG neurospheres revealed 405 differentially expressed genes. Gene set enrichment analysis showed upregulation of transcriptional programs related to mesenchymal signature in QMN-iDMG neurospheres and proneural signature in QMP-iDMG neurosphere as expected. These data show that NF1 deletion and PDGFRAD842V overexpression significantly alter gene expression in H3 K27M-mutant iDMG tumors, potentially opening up a new therapeutic avenue in these devastating tumors with patient-to-patient heterogeneity. Further work using these models will shed light on the development of tailored therapy based on detailed genetic information on each patient sample, such as combining targeted kinase inhibition with HDAC inhibitors that have shown promise in the clinic.
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Wang, Lei, Zheng Gang Zhang, Rui Lan Zhang, and Michael Chopp. "Activation of the PI3-K/Akt Pathway Mediates cGMP Enhanced-Neurogenesis in the Adult Progenitor Cells Derived from the Subventricular Zone." Journal of Cerebral Blood Flow & Metabolism 25, no. 9 (March 30, 2005): 1150–58. http://dx.doi.org/10.1038/sj.jcbfm.9600112.
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The intracellular mechanisms that regulate neurogenesis remain unclear. Using neurospheres isolated from the subventricular zone (SVZ) of the adult rat, we investigated the effect of cyclic guanosine monophosphate (cGMP) and its signaling pathway on the induction of neurogenesis. Neurospheres expressed phosphodiesterase 5 (PDE5) and treatment of neurospheres with Sildenafil, a specific inhibitor of PDE5, significantly increased cGMP levels and neurogenesis. In addition, incubation of neurospheres with Sildenafil significantly phosphorylated Akt, which was associated with an increase of phosphorylation of glycogen synthase kinase 3 (GSK-3), a downstream target of Akt. Coincubation of neurospheres with Sildenafil and LY 294002, a pharmacological inhibitor of PI3-K/Akt, abolished Sildenafil-induced phosphorylated Akt and GSK-3. Furthermore, LY 294002 blocked Sildenafil-increased SVZ cell proliferation. These data suggest that Sildenafil-enhanced neurogenesis likely occurs through activation of the PI3-K/Akt/GSK-3 pathway.
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Ibrahim, Ahmed N., Daisuke Yamashita, Joshua C. Anderson, Moaaz Abdelrashid, Amr Alwakeal, Dagoberto Estevez-Ordonez, Svetlana Komarova, et al. "Intratumoral spatial heterogeneity of BTK kinomic activity dictates distinct therapeutic response within a single glioblastoma tumor." Journal of Neurosurgery 133, no. 6 (December 2020): 1683–94. http://dx.doi.org/10.3171/2019.7.jns191376.
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OBJECTIVEDespite significant recent efforts applied toward the development of efficacious therapies for glioblastoma (GBM) through exploration of GBM’s genome and transcriptome, curative therapeutic strategies remain highly elusive. As such, novel and effective therapeutics are urgently required. In this study, the authors sought to explore the kinomic landscape of GBM from a previously underutilized approach (i.e., spatial heterogeneity), followed by validation of Bruton’s tyrosine kinase (BTK) targeting according to this stepwise kinomic-based novel approach.METHODSTwelve GBM tumor samples were obtained and characterized histopathologically from 2 patients with GBM. PamStation peptide-array analysis of these tissues was performed to measure the kinomic activity of each sample. The Ivy GBM database was then utilized to determine the intratumoral spatial localization of BTK activity by investigating the expression of BTK-related transcription factors (TFs) within tumors. Genetic inhibition of BTK family members through lentiviral short hairpin RNA (shRNA) knockdown was performed to determine their function in the core-like and edge-like GBM neurosphere models. Finally, the small-molecule inhibitor of BTK, ONO/GS-4059, which is currently under clinical investigation in nonbrain cancers, was applied for pharmacological inhibition of regionally specified newly established GBM edge and core neurosphere models.RESULTSKinomic investigation identified two major subclusters of GBM tissues from both patients exhibiting distinct profiles of kinase activity. Comparatively, in these spatially defined subgroups, BTK was the centric kinase differentially expressed. According to the Ivy GBM database, BTK-related TFs were highly expressed in the tumor core, but not in edge counterparts. Short hairpin RNA–mediated gene silencing of BTK in previously established edge- and core-like GBM neurospheres demonstrated increased apoptotic activity with predominance of the sub-G1 phase of core-like neurospheres compared to edge-like neurospheres. Lastly, pharmacological inhibition of BTK by ONO/GS-4059 resulted in growth inhibition of regionally derived GBM core cells and, to a lesser extent, their edge counterparts.CONCLUSIONSThis study identifies significant heterogeneity in kinase activity both within and across distinct GBM tumors. The study findings indicate that BTK activity is elevated in the classically therapy-resistant GBM tumor core. Given these findings, targeting GBM’s resistant core through BTK may potentially provide therapeutic benefit for patients with GBM.
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HUNG, CHIH-HUANG, and TAI-HORNG YOUNG. "NGF IMPROVES NEURITE FASCICULATION OF EMBRYONIC RAT CEREBRAL CORTICAL NEURAL STEM CELLS ON EVAL SUBSTRATES UNDER SERUM FREE CONDITIONS." Biomedical Engineering: Applications, Basis and Communications 18, no. 04 (August 25, 2006): 207–13. http://dx.doi.org/10.4015/s1016237206000336.
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The behaviors of multipotential neural stem cells are regulated by several factors, including culture substrates, soluble factors, and cell-cell interactions. The purpose of this study was to explore the effects of cell-cell and cell-substrate interactions on developmental potential of neural stem cells from embryonic rat cerebral cortex at neurosphere level in the presence of nerve growth factors (NGF). The results suggested that, in combination with NGF, EVAL substrates could induce neurite fasciculation of differentiated forming-neurosphere cells under serum free conditions. Quantitative analysis of process growth reveals that, under serum free conditions, when NGF was present the process breadth was significantly larger than that of neurospheres cultured without NGF. In contrast, when serum were added in the medium, regardless of the addition of NGF or not, the neurospheres were induced into typically an extensive cellular substratum of protoplasmic cells upon which process-bearing cells spread. It indicated that, when serum was present, the differentiated GFAP-positive astrocytes layer could serve as a supported architectural for growth and survival of process-bearing cells. At this time, neurites were failed to fasciculate. Here, we proposed that the mediation of cell-biomaterial interaction by cell-cell interaction should be taken into account for regulation of neurites fasciculation. Moreover, the pattern of neural stem cell growth on EVAL subtrates with or without NGF in the absence or presence of serum in this studies suggested that neurons are more adherent to glail cells than to other neurons but are more adherent to other neurons than to EVAL substrates.
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Liard, Olivier, Stéphanie Segura, Emmanuel Sagui, André Nau, Aurélie Pascual, Melissa Cambon, Jean-Luc Darlix, Thierry Fusai, and Emmanuel Moyse. "Adult-Brain-Derived Neural Stem Cells Grafting into a Vein Bridge Increases Postlesional Recovery and Regeneration in a Peripheral Nerve of Adult Pig." Stem Cells International 2012 (2012): 1–11. http://dx.doi.org/10.1155/2012/128732.
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We attempted transplantation of adult neural stem cells (ANSCs) inside an autologous venous graft following surgical transsection ofnervis cruraliswith 30 mm long gap in adult pig. The transplanted cell suspension was a primary culture of neurospheres from adult pig subventricular zone (SVZ) which had been labeledin vitrowith BrdU or lentivirally transferred fluorescent protein. Lesion-induced loss of leg extension on the thigh became definitive in controls but was reversed by 45–90 days after neurosphere-filled vein grafting. Electromyography showed stimulodetection recovery in neurosphere-transplanted pigs but not in controls. Postmortem immunohistochemistry revealed neurosphere-derived cells that survived inside the venous graft from 10 to 240 post-lesion days and all displayed a neuronal phenotype. Newly formed neurons were distributed inside the venous graft along the severed nerve longitudinal axis. Moreover, ANSC transplantation increased CNPase expression, indicating activation of intrinsic Schwann cells. Thus ANSC transplantation inside an autologous venous graft provides an efficient repair strategy.
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Uspenskaya, Yu A., N. A. Malinovskaya, A. V. Morgun, E. D. Osipova, and A. B. Salmina. "Mouse hippocampal neurospheres negatively regulate cerebral angiogenesis." Fundamental and Clinical Medicine 5, no. 3 (September 30, 2020): 18–23. http://dx.doi.org/10.23946/2500-0764-2020-5-3-18-23.
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Aim. To evaluate the features of cerebral angiogenesis in the presence of neurospheres, free-floating clusters of neural stem and progenitor cells. Materials and Methods. Endothelial cells were optionally co-cultured with neurospheres were isolated from the hippocampus of 10-14-days-old C57Bl6 mice. Angiogenesis was evaluated in vitro by means of the angiogenesis assay kit (Abcam, ab204726) according to the manufacturer's protocol. Results. The number of vascular loops was decreased in the presence of neurospheres. Other angiogenic parameters (average loop area and perimeter, total vessel length and number of contacts per mm2) had no statistically significant differences after 18 hours of culture. Conclusion. The proliferative activity of neurospheres inhibits in vitro cerebral angiogenesis.
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Radojevic, Vesna, and Josef P. Kapfhammer. "Directed Fiber Outgrowth from Transplanted Embryonic Cortex-Derived Neurospheres in the Adult Mouse Brain." Neural Plasticity 2009 (2009): 1–7. http://dx.doi.org/10.1155/2009/852492.
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Neural transplantation has emerged as an attractive strategy for the replacement of neurons that have been lost in the central nervous system. Multipotent neural progenitor cells are potentially useful as donor cells to repopulate the degenerated regions. One important aspect of a transplantation strategy is whether transplanted cells are capable of fiber outgrowth with the aim of rebuilding axonal connections within the host brain. To address this issue, we expanded neuronal progenitor from the cortex of embryonic day 15 ubiquitously green fluorescent protein-expressing transgenic mice as neurospheres in vitro and grafted them into the entorhinal cortex of 8-week-old mice immediately after a perforant pathway lesion. After transplantation into a host brain with a lesion of the entorhino-hippocampal projection, the neurosphere-derived cells extended long fiber projections directed towards the dentate gyrus. Our results indicate that transplantation of neurosphere-derived cells might be a promising strategy to replace lost or damaged axonal projections.
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Stricker, Priscila Elias Ferreira, Daiany de Souza Dobuchak, Ana Carolina Irioda, Bassam Felipe Mogharbel, Celia Regina Cavichiolo Franco, José Roberto de Souza Almeida Leite, Alyne Rodrigues de Araújo, et al. "Human Mesenchymal Stem Cells Seeded on the Natural Membrane to Neurospheres for Cholinergic-like Neurons." Membranes 11, no. 8 (August 7, 2021): 598. http://dx.doi.org/10.3390/membranes11080598.
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This study aimed to differentiate human mesenchymal stem cells (hMSCs) from the human umbilical cord in cholinergic-like neurons using a natural membrane. The isolation of hMSCs from Wharton’s jelly (WJ) was carried out using “explant” and mononuclear cells by the density gradient from umbilical blood and characterized by flow cytometry. hMSCs were seeded in a natural functional biopolymer membrane to produce neurospheres. RT-PCR was performed on hMSCs and neurospheres derived from the umbilical cord. Neural precursor cells were subjected to a standard cholinergic-like neuron differentiation protocol. Dissociated neurospheres, neural precursor cells, and cholinergic-like neurons were characterized by immunocytochemistry. hMSCs were CD73+, CD90+, CD105+, CD34- and CD45- and demonstrated the trilineage differentiation. Neurospheres and their isolated cells were nestin-positive and expressed NESTIN, MAP2, ßIII-TUBULIN, GFAP genes. Neural precursor cells that were differentiated in cholinergic-like neurons expressed ßIII-TUBULIN protein and choline acetyltransferase enzyme. hMSCs seeded on the natural membrane can differentiate into neurospheres, obtaining neural precursor cells without growth factors or gene transfection before cholinergic phenotype differentiation.
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Hung, Chih-Huang, Yi-Chen Li, and Tai-Horng Young. "INDUCTION OF NEURONAL DIFFERENTIATION OF EMBRYONIC RAT CORTICAL NEUROSPHERES BY NERVE GROWTH FACTOR AND FETAL BOVINE SERUM ON THE NONADHERENT AND ADHERENT SUBSTRATES." Biomedical Engineering: Applications, Basis and Communications 25, no. 01 (February 2013): 1250053. http://dx.doi.org/10.4015/s1016237212500536.
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Regulating neural stem cells will greatly aid in developing regenerative medicine for the repair purpose of central nervous system (CNS). In this study, we explored the effect of nerve growth factor (NGF) on the differentiation of neural stem cells from embryonic rat cerebral cortex on nonadherent and adherent substrates at neurosphere level. The results showed that NGF could not promote adhesion and differentiation of neural stem cells when neurospheres were cultured on nonadherent tissue culture polystyrene (TCPS) substrates under serum-free condition. However, when 10% fetal bovine serum (FBS) was added to the culture system, FBS could induce the attachment of neurospheres onto an originally nonadherent substrate and almost all of the neurosphere-forming cells would migrate away from the spheres with the protoplasmic astrocyte morphology. Furthermore, when NGF was added into the FBS-containing medium, a significant number of differentiated neurons developed and distributed on the astrocyte layer. For comparison, adherent poly-D-lysine (PDL) substrates were also used. It was found that NGF could enhance differentiation of neural stem cells into neurons on PDL under serum-free condition but neurons and astrocytes could not migrate far away from the spheres. Similar to the TCPS results were also observed on PDL that differentiated neurons migrated out from the spheres and developed on the astrocyte layer when the medium contained NGF and FBS. Since the development of neurons needs glia cells to form a cellular substratum and to support neuronal migration to appropriate location, these results provide evidences that FBS-induced glia cells, serving as an architectural support layer, are essential for the growth and migration of NGF-induced neurons.
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Herrera, Alejandro, Sara Morcuende, Rocío Talaverón, Beatriz Benítez-Temiño, Angel M. Pastor, and Esperanza R. Matarredona. "Purinergic Receptor Blockade with Suramin Increases Survival of Postnatal Neural Progenitor Cells In Vitro." International Journal of Molecular Sciences 22, no. 2 (January 12, 2021): 713. http://dx.doi.org/10.3390/ijms22020713.
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Neural progenitor cells (NPCs) are self-renewing and multipotent cells that persist in the postnatal and adult brain in the subventricular zone and the hippocampus. NPCs can be expanded in vitro to be used in cell therapy. However, expansion is limited, since the survival and proliferation of adult NPCs decrease with serial passages. Many signaling pathways control NPC survival and renewal. Among these, purinergic receptor activation exerts differential effects on the biology of adult NPCs depending on the cellular context. In this study, we sought to analyze the effect of a general blockade of purinergic receptors with suramin on the proliferation and survival of NPCs isolated from the subventricular zone of postnatal rats, which are cultured as neurospheres. Treatment of neurospheres with suramin induced a significant increase in neurosphere diameter and in NPC number attributed to a decrease in apoptosis. Proliferation and multipotency were not affected. Suramin also induced an increase in the gap junction protein connexin43 and in vascular endothelial growth factor, which might be involved in the anti-apoptotic effect. Our results offer a valuable tool for increasing NPC survival before implantation in the lesioned brain and open the possibility of using this drug as adjunctive therapy to NPC transplantation.
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Kobolak, Julianna, Annamaria Teglasi, Tamas Bellak, Zofia Janstova, Kinga Molnar, Melinda Zana, Istvan Bock, Lajos Laszlo, and Andras Dinnyes. "Human Induced Pluripotent Stem Cell-Derived 3D-Neurospheres Are Suitable for Neurotoxicity Screening." Cells 9, no. 5 (May 1, 2020): 1122. http://dx.doi.org/10.3390/cells9051122.
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We present a hiPSC-based 3D in vitro system suitable to test neurotoxicity (NT). Human iPSCs-derived 3D neurospheres grown in 96-well plate format were characterized timewise for 6-weeks. Changes in complexity and homogeneity were followed by immunocytochemistry and transmission electron microscopy. Transcriptional activity of major developmental, structural, and cell-type-specific markers was investigated at weekly intervals to present the differentiation of neurons, astrocytes, and oligodendrocytes. Neurospheres were exposed to different well-known toxicants with or without neurotoxic effect (e.g., paraquat, acrylamide, or ibuprofen) and examined at various stages of the differentiation with an ATP-based cell viability assay optimized for 3D-tissues. Concentration responses were investigated after acute (72 h) exposure. Moreover, the compound-specific effect of rotenone was investigated by a panel of ER-stress assay, TUNEL assay, immunocytochemistry, electron microscopy, and in 3D-spheroid based neurite outgrowth assay. The acute exposure to different classes of toxicants revealed distinct susceptibility profiles in a differentiation stage-dependent manner, indicating that hiPSC-based 3D in vitro neurosphere models could be used effectively to evaluate NT, and can be developed further to detect developmental neurotoxicity (DNT) and thus replace or complement the use of animal models in various basic research and pharmaceutical applications.
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Bayram, Keziban Korkmaz, Juliette Fitremann, Arslan Bayram, Zeynep Yılmaz, Ecmel Mehmetbeyoğlu, Yusuf Özkul, and Minoo Rassoulzadegan. "Gene Expression of Mouse Hippocampal Stem Cells Grown in a Galactose-Derived Molecular Gel Compared to In Vivo and Neurospheres." Processes 9, no. 4 (April 18, 2021): 716. http://dx.doi.org/10.3390/pr9040716.
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Background: N-heptyl-D-galactonamide (GalC7) is a small synthetic carbohydrate derivative that forms a biocompatible supramolecular hydrogel. In this study, the objective was to analyze more in-depth how neural cells differentiate in contact with GalC7. Method: Direct (ex vivo) cells of the fresh hippocampus and culture (In vitro) of the primary cells were investigated. In vitro, investigation performed under three conditions: on culture in neurospheres for 19 days, on culture in GalC7 gel for 7 days, and on culture in both neurospheres and GalC7 gel. Total RNA was isolated with TRIzol from each group, Sox8, Sox9, Sox10, Dcx, and Neurod1 expression levels were measured by qPCR. Result: Sox8 and Sox10, oligodendrocyte markers, and Sox9, an astrocyte marker, were expressed at a much higher level after 7 days of culture in GalC7 hydrogel compared to all other conditions. Dcx, a marker of neurogenesis, and Neurod1, a marker of neuronal differentiation, were expressed at better levels in the GalC7 gel culture compared to the neurosphere. Conclusions: These results show that the GalC7 hydrogel brings different and interesting conditions for inducing the differentiation and maturation of neural progenitor cells compared with polymer-based scaffolds or cell-only conditions. The differences observed open new perspectives in tissue engineering, induction, and transcript analysis.
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Seifert, Carolin, Ellen Balz, Susann Herzog, Anna Korolev, Sebastian Gaßmann, Heiko Paland, Matthias A. Fink, et al. "PIM1 Inhibition Affects Glioblastoma Stem Cell Behavior and Kills Glioblastoma Stem-like Cells." International Journal of Molecular Sciences 22, no. 20 (October 15, 2021): 11126. http://dx.doi.org/10.3390/ijms222011126.
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Despite comprehensive therapy and extensive research, glioblastoma (GBM) still represents the most aggressive brain tumor in adults. Glioma stem cells (GSCs) are thought to play a major role in tumor progression and resistance of GBM cells to radiochemotherapy. The PIM1 kinase has become a focus in cancer research. We have previously demonstrated that PIM1 is involved in survival of GBM cells and in GBM growth in a mouse model. However, little is known about the importance of PIM1 in cancer stem cells. Here, we report on the role of PIM1 in GBM stem cell behavior and killing. PIM1 inhibition negatively regulates the protein expression of the stem cell markers CD133 and Nestin in GBM cells (LN-18, U-87 MG). In contrast, CD44 and the astrocytic differentiation marker GFAP were up-regulated. Furthermore, PIM1 expression was increased in neurospheres as a model of GBM stem-like cells. Treatment of neurospheres with PIM1 inhibitors (TCS PIM1-1, Quercetagetin, and LY294002) diminished the cell viability associated with reduced DNA synthesis rate, increased caspase 3 activity, decreased PCNA protein expression, and reduced neurosphere formation. Our results indicate that PIM1 affects the glioblastoma stem cell behavior, and its inhibition kills glioblastoma stem-like cells, pointing to PIM1 targeting as a potential anti-glioblastoma therapy.
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Yang, Erfang, Na Liu, Yingxin Tang, Yang Hu, Ping Zhang, Chao Pan, Shasha Dong, Youping Zhang, and Zhouping Tang. "Generation of Neurospheres from Human Adipose-Derived Stem Cells." BioMed Research International 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/743714.
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Transplantation of neural stem cells (NSCs) to treat neurodegenerative disease shows promise; however, the clinical application of NSCs is limited by the invasive procurement and ethical concerns. Adipose-derived stem cells (ADSCs) are a source of multipotent stem cells that can self-renew and differentiate into various kinds of cells; this study intends to generate neurospheres from human ADSCs by culturing ADSCs on uncoated culture flasks in serum-free neurobasal medium supplemented with B27, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF); the ADSCs-derived neurospheres were terminally differentiated after growth factor withdrawal. Expression of Nestin, NeuN, MAP2, and GFAP in ADSCs and terminally differentiated neurospheres was shown by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting, and immunocytochemistry; cell proliferation in neurospheres was evaluated by cell cycle analyses, immunostaining, and flow cytometry. These data strongly support the conclusion that human ADSCs can successfully differentiate into neurospheres efficiently on uncoated culture flasks, which present similar molecular marker pattern and proliferative ability with NSCs derived from embryonic and adult brain tissues. Therefore, human ADSCs may be an ideal alternative source of stem cells for the treatment of neurodegenerative diseases.
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Zhou, Sheng, Soghra Fatima, and Brian P. Sorrentino. "Abcg2 Expression Marks Neural Stem Cells in the Murine Cerebellum." Blood 108, no. 11 (November 16, 2006): 1674. http://dx.doi.org/10.1182/blood.v108.11.1674.1674.
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Abstract Neural stem cells have been identified in both the cerebellum and forebrain of fetal and adult mice. These cells can form neurospheres in culture and differentiate into both glia and neurons either in vitro or in vivo. In embryonic day 14 forebrain, neural stem cells are found to exist exclusively in a subpopulation with the Side Population (SP) phenotype, and express Abcg2, a member of the ABC transporter family that is responsible for the SP phenotype in hematopoietic stem cells (HSCs). The expression of Abcg2 in stem cells in the cerebellum has not been characterized. We have generated an Abcg2/GFP knock-in mouse model in which expression of GFP is under control of the endogenous Abcg2 locus and used this model to demonstrate that Abcg2 expression can be used for HSC enrichment. Here we report the use of this mouse model to explore the relationship between Abcg2 expression and neural stem cell function in neonatal cerebellum. Single cells were prepared from cerebellum of 4–9 day old mice by digesting with papain. We then stained the cells with anti-CD45 and anti-Ter119 antibody to exclude the resident hematopoietic cells in subsequent flow cytometry analysis and cell sorting. We found that a small but consistent subpopulation of cells, comprising 0.7±0.12% of total CD45−Ter119- single cell preparations, expressed the Abcg2/GFP allele. To determine whether these GFP+ cells were enriched for neural stem cells, we sorted the CD45−Ter119- cells into GFP+ and GFP− subpopulations and analyzed for their neurosphere forming activity in the presence of epidermal growth factor and basic fibroblast growth factor. We found that the GFP+ subpopulation formed 21 fold more neurospheres compared with the GFP− subpopulation. These neurosphere forming cells can self-renew as evidenced by their capacity to form secondary neurospheres when replated. These results demonstrate that similar to what is seen with HSCs and with embryonic forebrain cells, Abcg2 is expressed in the neural stem cells in neonatal cerebellum, and Abcg2/GFP expression in this mouse model could also be used as a marker to prospectively purify neural stem cells from cerebellum. Ongoing studies are focused on defining the in vivo multilineage differentiation potential of the Abcg2/GFP+ cells and determining whether Abcg2 expression could be used as a marker for purification of medulloblastoma stem cells.
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Eisenmann, Kathryn M., Kenneth Lloyd, Krista M. Pettee, Kathryn M. Becker, Jason Schroeder, Kevin Reinard, James R. Tonra, Ramon Mohanlal, and Lan Huang. "Abstract 5498: Plinabulin, a novel tubulin targeting agent, collapses the proinvasion tumor microtube network in primary and recurrent patient derived glioblastoma cell lines to inhibit neurosphere invasion and survival." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5498. http://dx.doi.org/10.1158/1538-7445.am2022-5498.
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Abstract Glioblastoma multiforme (GBM) is the most common adult primary brain malignancy. Aggressive, diffuse invasion is regulated by a dynamic cytoskeleton and significantly contributes to poor survival. Intraparenchymal tumor cell dissemination prevents complete surgical resection and compromises essential brain functions. Invasive cells are inherently less sensitive to traditional cytotoxic therapies. Recent investigation into targeted therapies has failed to address invasion/cytoskeleton based GBM therapy resistance. Furthermore, established GBM cell line modeling in 2 or 3 dimensions (3D) does not capture the genetic diversity or clinical relevance of patient derived primary (1') GBM cell lines, which may uncover/predict differential responses in tumors to novel therapies. Cytoskeleton remodeling and cell invasion in patient-derived primary GBM cells generates unique structures called tumor microtubes (TMs) not seen in established, cultured GBM cell lines. TMs are ultralong, pro-invasive, tubulin-enriched protrusions that promote chemo- and radioresistance in GBM patient derived primary cells. Using our extensive GBM cell line biobank derived from 1' and/or therapy resistant recurrent (2') tumors, we investigated the effects of a novel drug targeting the microtubule cytoskeleton. Plinabulin is a tubulin targeting agent that crosses the blood brain barrier and demonstrated anticancer efficacy in Phase III trial patients with non-small cell lung carcinoma. Plinabulin also has antiproliferative properties in 2D GBM cells. Here, we evaluate the efficacy of plinabulin treatment upon 3D neurosphere viability and invasion in 5 GBM 1' and 2' recurrent patient derived cell lines. Within 24h of matrigel embedding, 3D GBM patient neurospheres generate robust pro-invasive TMs. Plinabulin treatment (10-30 nM) at embedding followed by drug washout after 24h eliminated existing TMs and inhibited single cell invasion (>80% relative to controls) in both 1' and 2' GBM patient neurospheres. Cell survival was significantly reduced in plinabulin treated neurospheres (>30 nM). Plinabulin efficacy was evaluated in a clinically relevant therapeutic ITR model: GBM patient neurospheres invaded (I) for 48h to establish TM networks; were treated (T) with 1-50 nM plinabulin for 24h prior to drug washout; and recovered (R) for 96h. In 1', 2', or matched 1' and 2' cell line pairs from the same patient, 10-40 nM plinabulin sustained invasion inhibition (>90%) through 96h post washout and completely blocked TM extension relative to controls, consistent with established clinical concentrations of plinabulin. This study reveals plinabulin as a potent GBM therapeutic targeting pro-invasion TM networks and survival in 1' and therapy resistant 2' GBM patient cell lines. Citation Format: Kathryn M. Eisenmann, Kenneth Lloyd, Krista M. Pettee, Kathryn M. Becker, Jason Schroeder, Kevin Reinard, James R. Tonra, Ramon Mohanlal, Lan Huang. Plinabulin, a novel tubulin targeting agent, collapses the proinvasion tumor microtube network in primary and recurrent patient derived glioblastoma cell lines to inhibit neurosphere invasion and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5498.
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Vanderluit, Jacqueline L., Kerry L. Ferguson, Vassiliki Nikoletopoulou, Maura Parker, Vladimir Ruzhynsky, Tania Alexson, Stephen M. McNamara, David S. Park, Michael Rudnicki, and Ruth S. Slack. "p107 regulates neural precursor cells in the mammalian brain." Journal of Cell Biology 166, no. 6 (September 7, 2004): 853–63. http://dx.doi.org/10.1083/jcb.200403156.
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Here we show a novel function for Retinoblastoma family member, p107 in controlling stem cell expansion in the mammalian brain. Adult p107-null mice had elevated numbers of proliferating progenitor cells in their lateral ventricles. In vitro neurosphere assays revealed striking increases in the number of neurosphere forming cells from p107−/− brains that exhibited enhanced capacity for self-renewal. An expanded stem cell population in p107-deficient mice was shown in vivo by (a) increased numbers of slowly cycling cells in the lateral ventricles; and (b) accelerated rates of neural precursor repopulation after progenitor ablation. Notch1 was up-regulated in p107−/− neurospheres in vitro and brains in vivo. Chromatin immunoprecipitation and p107 overexpression suggest that p107 may modulate the Notch1 pathway. These results demonstrate a novel function for p107 that is distinct from Rb, which is to negatively regulate the number of neural stem cells in the developing and adult brain.
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Semenova, V., M. Lisyany, V. Rosumenko, D. Egorova, and L. Staino. "The biological properties of cancer stem cells in malignant gliomas in tissue culture: a review." Cell and Organ Transplantology 3, no. 2 (May 31, 2018): 163–68. http://dx.doi.org/10.22494/cot.v3i2.12.
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This review presents recent data on the biological properties of cancer stem cells in brain gliomas. The possibilities of using of experimental methods of cell culture and immunohistochemical identification of tumor stem cells as a part of neurospheres (tumorospheres) are shown. Methodological features of neurospheres culture are described.Literature data about the dependence of neurospheres formation activity in the culture on the degree of anaplasia of original glioma tissues are presented considering the disease outcomes in neuro-oncological patients. Possibilities of the practical use of cultured tumorospheres in the study of selective impact of antiblastic drugs on tumor stem cells in gliomas are described.
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Chiang, Yung H., Vincenzo Silani, and Feng C. Zhou. "Morphological Differentiation of Astroglial Progenitor Cells from Egf-Responsive Neurospheres in Response to Fetal Calf Serum, Basic Fibroblast Growth Factor, and Retinol." Cell Transplantation 5, no. 2 (March 1996): 179–89. http://dx.doi.org/10.1177/096368979600500208.
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Procurement of multipotential neuroglial stem cells is possible with the addition of epidermal growth factor (EGF). Stem cells will differentiate into neurons and glia upon the removal of EGF from the culture medium. We have previously characterized the neuronal differentiation of stem cells derived from long-term cultured nonpassage neurospheres. In the current study, we (1) characterize the morphological differentiation of the astroglial progenitor cell from 3-mo-old neurospheres, (2) examine whether the astroglial progenitor cells from neurospheres of different brain areas exhibit different differentiation responses to the same exogenous signals, and (3) test the effects of basic fibroblast growth factor (bFGF) and retinol on differentiation. Cerebral cortex, striatum, and mesencephalon cells were obtained from Embryonic Day 14 (E-14) rat fetuses and were dissociated for the procurement of neurospheres in chemically defined medium supplemented with EGF. After 3 mo in culture, the neurospheres, derived from each of the three brain areas, were subcultured into three groups on chamber slides: (1) basal medium, (2) the basal medium plus 20 ng/mL bFGF, and (3) the basal medium plus 10 μM retinol. Phenotypic expression of astroglial cells was examined after 14 days subculture. Our findings indicate that the 3-mo-old cultured nonpassage neurospheres contained numerous multipotential stem cells that stained positive with nestin, and that environmental factors played an important role in influencing the differentiation of astroglial progenitor cells. As detected by glial fibrillary acid protein (GFAP), astroglial progenitor cells turned into protoplasmic astrocytes in the FCS-containing basal medium, fibrous astrocytes in the presence of bFGF, and spindle-shaped astrocytes in the presence of retinol. There were no noticeable differences in differentiation among astroglial progenitor cells of the various brain region-derived neurospheres in any of the three medium conditions. Peculiar varicosity-and growth cone-like structures on the long slender GFAP-positive processes suggest that neuroblasts and glioblast may share common morphologies, features, or common progenitor cells during initial differentiation in vitro.
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Sheikh, Abdullah Md, Shozo Yano, Shatera Tabassum, Koji Omura, Asuka Araki, Shingo Mitaki, Yoshie Ito, Shuai Huang, and Atsushi Nagai. "Alteration of Neural Stem Cell Functions in Ataxia and Male Sterility Mice: A Possible Role of β-Tubulin Glutamylation in Neurodegeneration." Cells 10, no. 1 (January 14, 2021): 155. http://dx.doi.org/10.3390/cells10010155.
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Ataxia and Male Sterility (AMS) is a mutant mouse strain that contains a missense mutation in the coding region of Nna1, a gene that encodes a deglutamylase. AMS mice exhibit early cerebellar Purkinje cell degeneration and an ataxic phenotype in an autosomal recessive manner. To understand the underlying mechanism, we generated neuronal stem cell (NSC) lines from wild-type (NMW7), Nna1 mutation heterozygous (NME), and Nna1 mutation homozygous (NMO1) mouse brains. The NNA1 levels were decreased, and the glutamylated tubulin levels were increased in NMO1 cultures as well as in the cerebellum of AMS mice at both 15 and 30 days of age. However, total β-tubulin protein levels were not altered in the AMS cerebellum. In NMO1 neurosphere cultures, β-tubulin protein levels were increased without changes at the transcriptional level. NMO1 grew faster than other NSC lines, and some of the neurospheres were attached to the plate after 3 days. Immunostaining revealed that SOX2 and nestin levels were decreased in NMO1 neurospheres and that the neuronal differentiation potentials were reduced in NMO1 cells compared to NME or NMW7 cells. These results demonstrate that the AMS mutation decreased the NNA1 levels and increased glutamylation in the cerebellum of AMS mice. The observed changes in glutamylation might alter NSC properties and the neuron maturation process, leading to Purkinje cell death in AMS mice.
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Hong, Xin, Kevin K. Nelson, Ana C. deCarvalho, and Steven N. Kalkanis. "Heparanase expression of glioma in human and animal models." Journal of Neurosurgery 113, no. 2 (August 2010): 261–69. http://dx.doi.org/10.3171/2009.9.jns09682.
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Object Mammalian heparanase has been shown to function in tumor progression, invasion, and angiogenesis. However, heparanase expression in gliomas has not been well analyzed. To clarify its expression in gliomas, human glioma tissues and glioma animal models were investigated. Methods The expression of heparanase mRNA was determined in 33 resected human glioma tissues by semiquantitative real-time polymerase chain reaction. Heparanase expression was verified with a Western blot assay and immunohistochemistry (IHC) staining. Primary neurospheres from human glioblastoma multiforme (GBM) were developed in vitro. Heparanase expression in murine astrocytoma and human primary neurosphere animal models was examined using IHC. Results The authors found that heparanase mRNA is greatly increased in gliomas including oligodendroglioma (9 samples), anaplastic astrocytoma (11 samples), and GBM (13 samples) as compared with healthy brain mRNA (3 samples). Note, however, that no significant difference was observed among the 3 tumor groups. Increased heparanase expression was also found in tumor tissues on Western blotting. Immunohistochemistry staining demonstrated that heparanase was expressed by neovessel endothelial cells, infiltrated neutrophils, and in some cases, by neoplastic cells. Heparanase-expressing cells, including GBM tumor cells and neovessel endothelial cells, exhibited decreased expression of CD44, a cell adhesion molecule on the cell membrane that is important for regulating tumor invasion. In addition, heparanase-expressing tumor cells showed an elevated density of the cell proliferation marker Ki 67, as compared with its density in non–heparanase-expressing tumor cells, suggesting that heparanase expression is correlated with enhanced tumor proliferation. Two animal glioma models were tested for heparanase expression. Both murine astrocytoma cells (Ast11.9-2) and cultured primary human GBM neurospheres expressed heparanase when grown in animal brain tissue. Conclusions Glioma tissues contain increased levels of heparanase. Multiple cell types contribute to the expression of heparanase, including neovessel endothelial cells, tumor cells, and infiltrated neutrophils. Heparanase plays an important role in the control of cell proliferation and invasion. Animal models using Ast11.9-2 and primary neurospheres are suitable for antitumor studies targeting heparanase.
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Okwara, Chike K., Roza Vaez Ghaemi, Charlotte Yu, Melissa Le, Vikramaditya G. Yadav, and John M. Frostad. "The Mechanical Properties of Neurospheres." Advanced Engineering Materials 23, no. 8 (May 2, 2021): 2100172. http://dx.doi.org/10.1002/adem.202100172.
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Giri, R. K., R. Young, R. Pitstick, S. J. DeArmond, S. B. Prusiner, and G. A. Carlson. "Prion infection of mouse neurospheres." Proceedings of the National Academy of Sciences 103, no. 10 (February 22, 2006): 3875–80. http://dx.doi.org/10.1073/pnas.0510902103.
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Herva, Maria Eugenia, Aroa Relaño-Ginés, Ana Villa, and Juan María Torres. "Prion infection of differentiated neurospheres." Journal of Neuroscience Methods 188, no. 2 (May 2010): 270–75. http://dx.doi.org/10.1016/j.jneumeth.2010.02.022.
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Ganapathy, Kavina, Sowmithra Sowmithra, Ramesh Bhonde, and Indrani Datta. "By Changing Dimensionality, Sequential Culturing of Midbrain Cells, rather than Two-Dimensional Culture, Generates a Neuron-Glia Ratio Closer to in vivo Adult Midbrain." Cells Tissues Organs 201, no. 6 (2016): 445–63. http://dx.doi.org/10.1159/000446424.
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The neuron-glia ratio is of prime importance for maintaining the physiological homeostasis of neuronal and glial cells, and especially crucial for dopaminergic neurons because a reduction in glial density has been reported in postmortem reports of brains affected by Parkinson's disease. We thus aimed at developing an in vitro midbrain culture which would replicate a similar neuron-glia ratio to that in in vivo adult midbrain while containing a similar number of dopaminergic neurons. A sequential culture technique was adopted to achieve this. Neural progenitors (NPs) were generated by the hanging-drop method and propagated as 3D neurospheres followed by the derivation of outgrowth from these neurospheres on a chosen extracellular matrix. The highest proliferation was observed in neurospheres from day in vitro (DIV) 5 through MTT and FACS analysis of Ki67 expression. FACS analysis using annexin/propidium iodide showed an increase in the apoptotic population from DIV 8. DIV 5 neurospheres were therefore selected for deriving the differentiated outgrowth of midbrain on a poly-L-lysine-coated surface. Quantitative RT-PCR showed comparable gene expressions of the mature neuronal marker β-tubulin III, glial marker GFAP and dopaminergic marker tyrosine hydroxylase (TH) as compared to in vivo adult rat midbrain. The FACS analysis showed a similar neuron-glia ratio obtained by the sequential culture in comparison to adult rat midbrain. The yield of β-tubulin III and TH was distinctly higher in the sequential culture in comparison to 2D culture, which showed a higher yield of GFAP immunopositive cells. Functional characterization indicated that both the constitutive and inducible (KCl and ATP) release of dopamine was distinctly higher in the sequential culture than the 2D culture. Thus, the sequential culture technique succeeded in the initial enrichment of NPs in 3D neurospheres, which in turn resulted in an optimal attainment of the neuron-glia ratio on outgrowth culture from these neurospheres.
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Bleau, Anne-Marie, Brian M. Howard, Lauren A. Taylor, Demirkan Gursel, Jeffrey P. Greenfield, H. Y. Lim Tung, Eric C. Holland, and John A. Boockvar. "New strategy for the analysis of phenotypic marker antigens in brain tumor–derived neurospheres in mice and humans." Neurosurgical Focus 24, no. 3-4 (March 2008): E28. http://dx.doi.org/10.3171/foc/2008/24/3-4/e27.
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Object Brain tumor stem cells (TSCs) hypothetically drive the malignant phenotype of glioblastoma multiforme (GBM), and evidence suggests that a better understanding of these TSCs will have profound implications for treating gliomas. When grown in vitro, putative TSCs grow as a solid sphere, making their subsequent characterization, particularly the cells within the center of the sphere, difficult. Therefore, the purpose of this study was to develop a new method to better understand the proteomic profile of the entire population of cells within a sphere. Methods Tumor specimens from patients with confirmed GBM and glioma models in mice were mechanically and enzymatically dissociated and grown in traditional stem cell medium to generate neurospheres. The neurospheres were then embedded in freezing medium, cryosectioned, and analyzed with immunofluorescence. Results By sectioning neurospheres as thinly as 5 μm, the authors overcame many of the problems associated with immunolabeling whole neurospheres, such as antibody penetration into the core of the sphere and intense background fluorescence that obscures the specificity of immunoreactivity. Moreover, the small quantity of material required and the speed with which this cryosectioning and immunolabeling technique can be performed make it an attractive tool for the rapid assessment of TSC character. Conclusions This study is the first to show that cryosectioning of neurospheres derived from glioma models in mice and GBM in humans is a feasible method of better defining the stem cell profile of a glioma.
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Peng, Chunyang, Yajiao Li, Li Lu, Jianwen Zhu, Huiyu Li, and Jingqiong Hu. "Efficient One-Step Induction of Human Umbilical Cord-Derived Mesenchymal Stem Cells (UC-MSCs) Produces MSC-Derived Neurospheres (MSC-NS) with Unique Transcriptional Profile and Enhanced Neurogenic and Angiogenic Secretomes." Stem Cells International 2019 (December 18, 2019): 1–15. http://dx.doi.org/10.1155/2019/9208173.
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Cell therapy has emerged as a promising strategy for treating neurological diseases such as stroke, spinal cord injury, and various neurodegenerative diseases, but both embryonic neural stem cells and human induced Pluripotent Stem Cell- (iPSC-) derived neural stem cells have major limitations which restrict their broad use in these diseases. We want to find a one-step induction method to transdifferentiate the more easily accessible Umbilical Cord-Derived Mesenchymal Stem Cells (UC-MSCs) into neural stem/progenitor cells suitable for cell therapy purposes. In this study, UC-MSCs were induced to form neurospheres under a serum-free suspension culture with Epidermal Growth Factor- (EGF-) and basic Fibroblast Growth Factor- (bFGF-) containing medium within 12 hours. These MSC-derived neurospheres can self-renew to form secondary neurospheres and can be readily induced to become neurons and glial cells. Real-time PCR showed significantly upregulated expression of multiple stemness and neurogenic genes after induction. RNA transcriptional profiling study showed that UC-MSC-derived neurospheres had a unique transcriptional profile of their own, with features of both UC-MSCs and neural stem cells. RayBio human growth factor cytokine array analysis showed significantly upregulated expression levels of multiple neurogenic and angiogenic growth factors, skewing toward a neural stem cell phenotype. Thus, we believe that these UC-MSC-derived neurospheres have amenable features of both MSCs and neural stem/progenitor cells and have great potential in future stem cell transplantation clinical trials targeting neurological disorders.
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HOPKINS, TRACY M., JOHN VENNEMEYER, CHAMINDA JAYASINGHE, VESSELIN SHANOV, and SARAH K. PIXLEY. "CARBON NANOTUBE RIBBON AND THREAD SUPPORT ATTACHMENT AND DIFFERENTIATION OF NEURAL STEM CELLS." Nano LIFE 01, no. 01n02 (March 2010): 89–97. http://dx.doi.org/10.1142/s1793984410000080.
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Carbon nanotubes (CNTs) have properties that promise an exciting role in nervous tissue repair. CNTs are strong, extremely light weight, biocompatible and electrically conductive. A relatively novel form of CNT material, multiple strands of CNTs spun into thread, accentuates the linear geometry of CNTs while retaining electrical conductivity. We propose that CNT thread, which is strong, pliable and readily manipulated, has the potential to support longitudinal growth of nerves after injury, in nervous system prostheses. Towards understanding how CNT materials support nervous tissue regeneration, we examined the in vitro interactions between CNT materials and neural stem cell-containing neurospheres, prepared from newborn mouse cortices. Intact neurospheres attached to pieces of as-grown arrays of CNTs, CNT "ribbon" material pulled from the arrays, and CNT thread. Stem cells differentiated into both neurons and glia under these conditions. Processes and cells aligned with the longitudinal axis of the CNT materials, both with and without coatings. For comparisons, dissociated neurosphere cells were plated on CNT thread, polypropylene surgical suture thread and commercial carbon fibers. Initial cell attachment (within seconds) and attachment at 24 h was greater on CNT thread than on the other fiber types. Stem cells on CNT threads differentiated into neurons and astrocytes over several days, on all fibers. The presence of serum greatly aided the health and spreading of both neurons and astrocytes. These findings demonstrate that CNT materials, in particular the thread form, are viable preparations for neural cell attachment, outgrowth and differentiation.
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Capetian, Philipp, Lorenz Müller, Jens Volkmann, Manfred Heckmann, Süleyman Ergün, and Nicole Wagner. "Visualizing the Synaptic and Cellular Ultrastructure in Neurons Differentiated from Human Induced Neural Stem Cells—An Optimized Protocol." International Journal of Molecular Sciences 21, no. 5 (March 2, 2020): 1708. http://dx.doi.org/10.3390/ijms21051708.
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The size of the synaptic subcomponents falls below the limits of visible light microscopy. Despite new developments in advanced microscopy techniques, the resolution of transmission electron microscopy (TEM) remains unsurpassed. The requirements of tissue preservation are very high, and human post mortem material often does not offer adequate quality. However, new reprogramming techniques that generate human neurons in vitro provide samples that can easily fulfill these requirements. The objective of this study was to identify the culture technique with the best ultrastructural preservation in combination with the best embedding and contrasting technique for visualizing neuronal elements. Two induced neural stem cell lines derived from healthy control subjects underwent differentiation either adherent on glass coverslips, embedded in a droplet of highly concentrated Matrigel, or as a compact neurosphere. Afterward, they were fixed using a combination of glutaraldehyde (GA) and paraformaldehyde (PFA) followed by three approaches (standard stain, Ruthenium red stain, high contrast en-bloc stain) using different combinations of membrane enhancing and contrasting steps before ultrathin sectioning and imaging by TEM. The compact free-floating neurospheres exhibited the best ultrastructural preservation. High-contrast en-bloc stain offered particularly sharp staining of membrane structures and the highest quality visualization of neuronal structures. In conclusion, compact neurospheres growing under free-floating conditions in combination with a high contrast en-bloc staining protocol, offer the optimal preservation and contrast with a particular focus on visualizing membrane structures as required for analyzing synaptic structures.
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Hiramatsu, Satoe, Asuka Morizane, Tetsuhiro Kikuchi, Daisuke Doi, Kenji Yoshida, and Jun Takahashi. "Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application." Journal of Parkinson's Disease 12, no. 3 (April 5, 2022): 871–84. http://dx.doi.org/10.3233/jpd-212934.
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Background: Pluripotent stem cell (PSC)-derived dopaminergic (DA) neurons are an expected source of cell therapy for Parkinson’s disease. The transplantation of cell aggregates or neurospheres, instead of a single cell suspension has several advantages, such as keeping the 3D structure of the donor cells and ease of handling. For this PSC-based therapy to become a widely available treatment, cryopreservation of the final product is critical in the manufacturing process. However, cryopreserving cell aggregates is more complicated than cryopreserving single cell suspensions. Previous studies showed poor survival of the DA neurons after the transplantation of cryopreserved fetal ventral-mesencephalic tissues. Objective: To achieve the cryopreservation of induced pluripotent stem cell (iPSC)-derived DA neurospheres toward clinical application. Methods: We cryopreserved iPSC-derived DA neurospheres in various clinically applicable cryopreservation media and freezing protocols and assessed viability and neurite extension. We evaluated the population and neuronal function of cryopreserved cells by the selected method in vitro. We also injected the cells into 6-hydroxydopamine (6-OHDA) lesioned rats, and assessed their survival, maturation and function in vivo. Results: The iPSC-derived DA neurospheres cryopreserved by Proton Freezer in the cryopreservation medium Bambanker hRM (BBK) showed favorable viability after thawing and had equivalent expression of DA-specific markers, dopamine secretion, and electrophysiological activity as fresh spheres. When transplanted into 6-OHDA-lesioned rats, the cryopreserved cells survived and differentiated into mature DA neurons, resulting in improved abnormal rotational behavior. Conclusion: These results show that the combination of BBK and Proton Freezer is suitable for the cryopreservation of iPSC-derived DA neurospheres.
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Stavely, Rhian, Sukhada Bhave, Wing Lam N. Ho, Minhal Ahmed, Weikang Pan, Ahmed A. Rahman, Jessica Ulloa, et al. "Enteric mesenchymal cells support the growth of postnatal enteric neural stem cells." Stem Cells 39, no. 9 (May 8, 2021): 1236–52. http://dx.doi.org/10.1002/stem.3388.
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Abstract Interplay between embryonic enteric neural stem cells (ENSCs) and enteric mesenchymal cells (EMCs) in the embryonic gut is essential for normal development of the enteric nervous system. Disruption of these interactions underlies the pathogenesis of intestinal aganglionosis in Hirschsprung disease (HSCR). ENSC therapy has been proposed as a possible treatment for HSCR, but whether the survival and development of postnatal-derived ENSCs similarly rely on signals from the mesenchymal environment is unknown and has important implications for developing protocols to expand ENSCs for cell transplantation therapy. Enteric neural crest-derived cells (ENCDCs) and EMCs were cultured from the small intestine of Wnt1-Rosa26-tdTomato mice. EMCs promoted the expansion of ENCDCs 9.5-fold by inducing ENSC properties, including expression of Nes, Sox10, Sox2, and Ngfr. EMCs enhanced the neurosphere-forming ability of ENCDCs, and this persisted after withdrawal of the EMCs. These effects were mediated by paracrine factors and several ligands known to support neural stem cells were identified in EMCs. Using the optimized expansion procedures, neurospheres were generated from small intestine of the Ednrb −/− mouse model of HSCR. These ENSCs had similar proliferative and migratory capacity to Ednrb +/+ ENSCs, albeit neurospheres contained fewer neurons. ENSCs derived from Ednrb −/− mice generated functional neurons with similar calcium responses to Ednrb +/+ ENSCs and survived after transplantation into the aganglionic colon of Ednrb −/− recipients. EMCs act as supporting cells to ENSCs postnatally via an array of synergistically acting paracrine signaling factors. These properties can be leveraged to expand autologous ENSCs from patients with HSCR mutations for therapeutic application.
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Khochenkova, Yu A., I. G. Dyrda, Yu S. Machkova, E. Sh Solomko, T. A. Sidorova, D. A. Khochenkov, and E. A. Avilova. "New approaches in 3D modeling of in vitro growth of primary cultures of malignant gliomas." Advances in molecular oncology 6, no. 4 (December 15, 2019): 69–74. http://dx.doi.org/10.17650/2313-805x-2019-6-4-69-74.
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Background. The incidence of brain gliomas firmly occupies a leading position among all central nervous system tumors – 40–50 % of the cases detected, more than half of them are glioblastoma. Existing cell lines and cultivation methods do not reflect all the features of the three-dimensional (3D) organization of native glioblastoma. The use of temozolomide leads to the development of drug resistance and acute relapse, followed by a poor clinical outcome. The development of resistance is largely associated with the presence of tumor stem cells in the population and intratumoral heterogeneity. Obtaining 3D cultures from the primary material will allow us to save the stem cell pool and tumor-specific features.The study objective. Get a 3D model based on primary cell cultures, which allows you to save a heterogeneous population and the original phenotype of tumor cells.Materials and methods. We used U-87MG human glioma cells and GBM002 primary cell culture obtained from surgical material with a confirmed diagnosis of glioblastoma. Neurospheres were obtained from cell lines, the growth of which was monitored using the InCell Analyzer 6000 automatic cell analysis system. Flow cytometry was used to determine the CD133+ cell content. The expression of the receptor tyrosine kinases VEGFR1, VEGFR2 (endothelial growth factor type 1 and 2 receptors), FGFR2 (fibroblast growth factor receptor type 2) and the hypoxia marker HIF-1α (hypoxia inducible factor, 1α) in the neurospheres was evaluated using confocal microscopy.Results. GBM002 glioblastoma cells isolated from the surgical material formed neurospheres, while the number of CD133+ cells increased from 1–2 to 16–19 % compared with two-dimensional cultures. During long-term cultivation of cells with non-cytotoxic doses of temozolomide, it was found that such cells form smaller neurospheres compared to control cells. It was shown that the expression of receptor tyrosine kinases during cultivation of GBM002 glioblastoma cells in neurospheres differs from that in two-dimensional cultures. We found that in neurospheres, the expression of FGFR2 and VEGFR1, is significantly increased.Conclusion. 3D cultivation of primary cultures allows one to obtain a more heterogeneous population of tumor cells that reflects the spatial heterogeneity of cells, increase the pool of stem cells and recreate hypoxia conditions inside the brain micro-tumors.
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Clutter, Matt, Megan Romero, and Oren Becher. "DDIS-28. A HIGH THROUGHPUT SCREENING PLATFORM TO IDENTIFY RADIOSENSITIZERS FOR DIPG." Neuro-Oncology 21, Supplement_6 (November 2019): vi69. http://dx.doi.org/10.1093/neuonc/noz175.279.
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Abstract Diffuse intrinsic pontine glioma (DIPG) is an incurable brainstem tumor and the leading cause of death in children with brain cancer. Despite numerous clinical trials, no drugs have been found to prolong survival for DIPG patients, suggesting an urgent need to test therapeutics in preclinical models more predictive of clinical activity. To address this gap, we developed a genetically engineered mouse model incorporating the Histone H3.3 K27M mutation, p53 deletion, and PDGFR-α amplification, which co-occur in up to 40% of human DIPG. Here we report the results of a drug screen to identify radiosensitizers of DIPG cells isolated from our mouse model and cultured ex vivo as neurospheres. Although previous clinical trials combining radiotherapy with radiosensitizing agents failed to benefit DIPG patients, they incorporated general radiosensitizers. We hypothesize that searching for radiosynergy using 3-dimensional neurospheres derived from genetically defined primary cell DIPG models will enhance our ability to prioritize clinically relevant radiosensitizers. To identify candidates, we developed high throughput radiation and imaging protocols to quantify the number, size, and viability of neurospheres following treatment. We screened 1,280 FDA-approved drugs and 1,600 molecules with a history of clinical use. Two mechanistic classes of compounds were identified that sensitized DIPG neurospheres to radiotherapy, both targeting epigenetic factors. An HDAC1/3 inhibitor along with several different BET bromodomain inhibitors increased cell death 2–3 fold beyond the effect of radiation with minimal activity from the compounds alone. In addition to optimizing the dosing and timing of these compounds for animal studies, we are investigating whether radiosensitization occurs in H3.3 wildtype neurospheres. In the current molecular era of cancer, genetic features like the H3.3 K27M mutation could present an opportunity to develop therapeutics that preferentially radiosensitize diseased cells relative to normal cells. Such “precision radiosensitizers” would advance radiotherapy by enhancing tumor-specific toxicity while sparing bystander cells.
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Shehab-Eldeen, Noura, Mohamed Salama, Ahmed Lotfy, Mohamed Elgamal, Hussein Sheashaa, Mohamed Sobh, Mohamed Elsherbeeny, and Hossam Elmetwally. "179. Effect of papaverine on neurospheres." Toxicon 93 (January 2015): S56. http://dx.doi.org/10.1016/j.toxicon.2014.11.182.
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Zhdanov, V. P., and B. Kasemo. "Simulation of the growth of neurospheres." Europhysics Letters (EPL) 68, no. 1 (October 2004): 134–40. http://dx.doi.org/10.1209/epl/i2004-10170-1.
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Lee, You-Kyung, Su-Kyeong Hwang, Soo-Kyung Lee, Jung-eun Yang, Ji-Hye Kwak, Hyunhyo Seo, Hyunjun Ahn, et al. "Cohen Syndrome Patient iPSC-Derived Neurospheres and Forebrain-Like Glutamatergic Neurons Reveal Reduced Proliferation of Neural Progenitor Cells and Altered Expression of Synapse Genes." Journal of Clinical Medicine 9, no. 6 (June 16, 2020): 1886. http://dx.doi.org/10.3390/jcm9061886.
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Cohen syndrome (CS), a rare autosomal recessive disorder, has been associated with genetic mutations in the VPS13B gene, which regulates vesicle-mediated protein sorting and transport. However, the cellular mechanism underlying CS pathogenesis in patient-derived human neurons remains unknown. We identified a novel compound heterozygous mutation, due to homozygous variation of biparental origin and heterozygous variation inherited from the father, in the VPS13B gene in a 20-month-old female patient. To understand the cellular pathogenic mechanisms, we generated induced pluripotent stem cells (iPSCs) from the fibroblasts of the CS patient. The iPSCs were differentiated into forebrain-like functional glutamatergic neurons or neurospheres. Functional annotation from transcriptomic analysis using CS iPSC-derived neurons revealed that synapse-related functions were enriched among the upregulated and downregulated genes in the CS neurons, whereas processes associated with neurodevelopment were enriched in the downregulated genes. The developing CS neurospheres were small in size compared to control neurospheres, likely due to the reduced proliferation of SOX2-positive neural stem cells. Moreover, the number of SV2B-positive puncta and spine-like structures was significantly reduced in the CS neurons, suggesting synaptic dysfunction. Taking these findings together, for the first time, we report a potential cellular pathogenic mechanism which reveals the alteration of neurodevelopment-related genes and the dysregulation of synaptic function in the human induced neurons differentiated from iPSCs and neurospheres of a CS patient.
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Distler, Thomas, Ines Lauria, Rainer Detsch, Clemens M. Sauter, Farina Bendt, Julia Kapr, Stephan Rütten, Aldo R. Boccaccini, and Ellen Fritsche. "Neuronal Differentiation from Induced Pluripotent Stem Cell-Derived Neurospheres by the Application of Oxidized Alginate-Gelatin-Laminin Hydrogels." Biomedicines 9, no. 3 (March 5, 2021): 261. http://dx.doi.org/10.3390/biomedicines9030261.
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Biodegradable hydrogels that promote stem cell differentiation into neurons in three dimensions (3D) are highly desired in biomedical research to study drug neurotoxicity or to yield cell-containing biomaterials for neuronal tissue repair. Here, we demonstrate that oxidized alginate-gelatin-laminin (ADA-GEL-LAM) hydrogels facilitate neuronal differentiation and growth of embedded human induced pluripotent stem cell (hiPSC) derived neurospheres. ADA-GEL and ADA-GEL-LAM hydrogels exhibiting a stiffness close to ~5 kPa at initial cell culture conditions of 37 °C were prepared. Laminin supplemented ADA-GEL promoted an increase in neuronal differentiation in comparison to pristine ADA-GEL, with enhanced neuron migration from the neurospheres to the bulk 3D hydrogel matrix. The presence of laminin in ADA-GEL led to a more than two-fold increase in the number of neurospheres with migrated neurons. Our findings suggest that laminin addition to oxidized alginate—gelatin hydrogel matrices plays a crucial role to tailor oxidized alginate-gelatin hydrogels suitable for 3D neuronal cell culture applications.