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BMPRⅡ^(+)neural precursor cells isolated and characterized from organotypic neurospheres:an in vitro model of human fetal spinal cord development

中国神经科学学会第九届全国学术会议暨第五次会员代表大会

作者： Chun ZHOU,Tun-An CHEN,Liang YI,Min-Hui XU,Chun-Xia LUO,Fei LI,Mei LI,Jun HU,Ming-Yue ZHAO, Hua FENG~* Department of Neurosurgery,Southwest Hospital,Third Military Medical University,Chongqing,China

＜正＞Glioma-initiating cells（GICs） have been proven to play key roles in initiation and progress of malignant ***,a mass of pervious in vivo studies were based on the heterograft model which is implanting GICs derived from primary human glioma specimens into immunodeficient *** this in vivo model could duplicate the microenvironment of malignancy and evaluate the biological features of GICs accurately is unclear. Here,we compared the biological features of neurosphere-like tumor cells derived from murine glioma cell line GL261（GL261-NS） and neurosphere-like tumor cells of primary human glioma specimens（P-NS） in vitro and in vivo. We found that GICs enriched in GL261-NS,GL261-NS and P-NS both exhibited similar increased GICs potential and drug resistance in ***,GL261-NS in syngeneic graft model showed different enhanced tumorigenicity and better duplicated the primary human glioma histologically relative to P-NS in nude mice which were lack of immunocytes and ***,the biological features of P-NS were different among individual largely,although the patients were all diagnosed as *** studies conclude that implantation of GL261-NS into C57/BL6 mice is a more reliable and stable syngeneic graft model for in vivo study on GICs relative to implantation of P-NS into heterogenous immunodeficient mice.

关键词： glioma stem cells glioma C57/BL6 mice neurosphere

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Neural Regeneration Research 2024年第2期19卷 447-457页

作者： Michael W.Weible II Michael D.Lovelace Hamish D.Mundell Tsz Wai Rosita Pang Tailoi Chan-Ling Bosch Institute Discipline of Anatomy and Histology(F13)University of SydneySydneyNSWAustralia School of Environment and Science Griffith UniversityNathanQLDAustralia Discipline of Medicine Nepean Clinical SchoolFaculty of Medicine and HealthUniversity of SydneyKingswoodNSWAustralia New South Wales Brain Tissue Resource Centre School of Medical SciencesFaculty of Medicine and HealthUniversity of SydneyCharles Perkins Centre(D17)SydneyNSWAustralia

Roof plate secretion of bone morphogenetic proteins(BMPs)directs the cellular fate of sensory neurons during spinal cord development,including the formation of the ascending sensory columns,though their biology is not well ***-ⅡBMP receptor(BMPRⅡ),the cognate receptor,is expressed by neural precursor cells during embryogenesis;however,an in vitro method of enriching BMPRⅡ^(+)human neural precursor cells(hNPCs)from the fetal spinal cord is *** was undertaken on intact second-trimester human fetal spinal cord using antibodies to BMPRⅡand leukemia inhibitory factor(LIF).Regions of highest BMPRⅡ^(+)immunofluorescence localized to sensory *** and meningeal-associated BMPRⅡ^(+)vascular cells were identified in both intact fetal spinal cord and cortex by co-positivity with vascular lineage markers,CD34/*** immunostaining identified a population of somas concentrated in dorsal and ventral horn interneurons,mirroring the expression of LIF receptor/CD118.A combination of LIF supplementation and high-density culture maintained culture growth beyond 10 passages,while synergistically increasing the proportion of neurospheres with a stratified,*** neurospheres were characterized by BMPRⅡ^(+)/MAP2ab^(+/–)/βⅢ-tubulin^(+)/nestin^(–)/vimentin^(–)/GFAP^(–)/NeuN^(–)surface hNPCs surrounding a heterogeneous core ofβⅢ-tubulin^(+)/nestin^(+)/vimentin^(+)/GFAP^(+)/MAP2ab^(–)/NeuN^(–)multipotent *** cultures from tripotential neurospheres contained neuronal(βⅢ-tubulin^(+)),astrocytic(GFAP+),and oligodendrocytic(O4+)lineage ***-activated cell sorting-sorted BMPRⅡ^(+)hNPCs were MAP2ab^(+/–)/βⅢ-tubulin^(+)/GFAP^(–)/O4^(–)in *** is the first isolation of BMPRⅡ^(+)hNPCs identified and characterized in human fetal spinal *** data show that LIF combines synergistically with high-density reaggregate cultures to support the organotypic reorganization of neurospheres,characterize

关键词： BMPRⅡ bone morphogenetic protein histotypic human spinal cord development leukemia inhibitory factor neurosphere organotypic reaggregate sensory columns

Ultrastructure of human neural stem/progenitor cells and neurospheres

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Neural Regeneration Research 2009年第5期4卷 365-370页

作者： Yaodong Zhao Tianyi Zhang Qiang Huang Aidong Wang Jun Dong Qing Lan Zhenghong Qin Department of Cerebral Surgery Second Affiliated Hospital of Soochow University Suzhou 215004 Jiangsu Province China Department of Electron Microscopy Jiangsu Province Key Laboratory of Neuroregenerafion Nantong 226001 Jiangsu Province China Laboratory of Aging and Neuro-disease of Soochow University Suzhou 215123 Jiangsu Province China

BACKGROUND： Biological and morphological characteristics of neural stem/progenitor cells （NSPCs） have been widely investigated. OBJECTIVE： To explore the ultrastructure of human embryo-derived NSPCs and neurospheres cultivated in vitro using electron microscopy. DESIGN, TIME AND SETTING： A cell biology experiment was performed at the Brain Tumor Laboratory of Soochow University, and Jiangsu Province Key Laboratory of Neuroregeneration, Nantong University between August 2007 and April 2008. MATERIALS： Human fetal brain tissue was obtained from an 8-week-old aborted fetus; serum-free Dulbecco＇s modified Eagle＇s medium/F12 culture medium was provided by Gibco, USA; scanning electron microscope was provided by Hitachi Instruments, Japan; transmission electron microscope was provided by JEOL, Japan. METHODS： NSPCs were isolated from human fetal brain tissue and cultivated in serum-free Dulbecco＇s modified Eagle＇s medium/F12 culture medium. Cells were passaged every 5-7 days. After three passages, NSPCs were harvested and used for ultrastructural examination. MAIN OUTCOME MEASURES： Ultrastructural examination of human NSPCs and adjacent cells in neurospheres. RESULTS： Individual NSPCs were visible as spherical morphologies with rough surfaces under scanning electron microscope. Generally, they had large nuclei and little cytoplasm. Nuclei were frequently globular with large amounts of euchromatin and a small quantity of heterochromatin, and most NSPCs had only one nucleolus. The Golgi apparatus and endoplasmic reticulum were underdeveloped; however, autophagosomes were clearly visible. The neurospheres were made up of NSPCs and non-fixiform material inside. Between adjacent cells and at the cytoplasmic surface of apposed plasma membranes, there were vesicle-like structures. Some membrane boundaries with high permeabilities were observed between some contiguous NSPCs in neurospheres, possibly attributable to plasmalemmal fusion between adjacent cells. CONCLUSION： A large n

关键词： Neural stem/progenitor cells neurosphere ultrastructure autophagosome cell junction

Growth and differentiation of neural stem cells in a three-dimensional collagen gel scaffold

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Neural Regeneration Research 2013年第4期8卷 313-319页

作者： Fei Huang Qiang Shen Jitong Zhao Department of Orthopedics Fourth Affiliated Hospital of Anhui Medical University Department of Orthopedics Shanghai First People's HospitalShanghai Jiao Tong University

Collagen protein is an ideal scaffold material for the transplantation of neural stem cells. In this study rat neural stern cells were seeded into a three-dimensional collagen gel scaffold, with suspension cultured neural stem cells being used as a control group. Neural stem cells, which were cultured in medium containing epidermal growth factor and basic fibroblast growth factor, actively expanded and formed neurospheres in both culture groups. In serum-free medium conditions, the processes extended from neurospheres in the collagen gel group were much longer than those in the suspension culture group. Immunofluorescence staining showed that neurespheres cultured in collagen gels were stained positive for nestin and differentiated cells were stained positive for the neuronal marker βIII-tubulin, the astrocytic marker glial fibrillary acidic protein and the oligodendrocytic marker 2＇,3＇-cyclic nucleotide 3＇-phosphodiesterase. Compared with neurospheres cultured in suspension, the differentiation potential of neural stem cells cultured in collagen gels increased, with the formation of neurons at an early stage. Our results show that the three-dimensional collagen gel culture system is superior to suspension culture in the proliferation, differentiation and process outgrowth of neural stem cells.

关键词： neural regeneration stem cells neural stem cells collagen gel scaffold central nervous system proliferation differentiation neurosphere photographs-containing paper neuroregeneration

Elimination of the geomagnetic field stimulates the proliferation of mouse neural progenitor and stem cells

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Protein & Cell 2016年第9期7卷 624-637页

作者： Jing-Peng Fu Wei-Chuan Mo Ying Liu Perry F. Bartlett Rong-Qiao He State Key Laboratory of Brain and Cognitive Science Institute of Biophysics Chinese Academy of Sciences Beijing 100101 China Queensland Brain Institute University of Queensland Brisbane QLD 4072 Australia University of the Chinese Academy of Sciences Beijing 100049 China Alzheimer's Disease Center Beijing Institute for Brain Disorders Capital Medical University Beijing 100069 China

Living organisms are exposed to the geomagnetic field （GMF） throughout their lifespan. Elimination of the GMF, resulting in a hypogeomagnetic field （HMF）, leads to central nervous system dysfunction and abnormal development in animals. However, the cellular mecha-nisms underlying these effects have not been identified so far. Here, we show that exposure to an HMF （〈200 nT）, produced by a magnetic field shielding chamber, pro- motes the proliferation of neural progenitor/stem cells （NPCslNSCs） from C57BL/6 mice. Following seven-day HMF-exposure, the primary neurospheres （NSs） were significantly larger in size, and twice more NPCslNSCs were harvested from neonatal NSs, when compared to the GMF controls. The self-renewal capacity and multi- potency of the NSs were maintalned, as HMF-exposed NSs were posltlve for NSC markers （Nestin and Sox2）, and could differentiate into neurons and astrocytelglial cells and be passaged continuously. In addition, adult mice exposed to the HMF for one month were observed to have a greater number of proliferative cells In the subventrlcular zone. These flndlngs indicate that continuous HMF-exposure increases the proliferation of NPCslNSCs, In vitro and in vlvo. HMF-dlsturbed NPCs/NSCs production probably affects brain development and function, which provides a novel clue for elucidating the cellular mechanisms of the *** response.

关键词： hypomagnetic field neural progenitor/stemcells neurosphere proliferation sternness multipotency

Sphere-forming corneal cells repopulate dystrophic keratoconic stroma:Implications for potential therapy

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World Journal of Stem Cells 2020年第1期12卷 35-54页

作者： Himanshu Wadhwa Salim Ismail Jennifer J McGhee Bert Van der Werf Trevor Sherwin Department of Ophthalmology Faculty of Medical and Health SciencesThe University of AucklandAuckland 1023New Zealand Department of Epidemiology and Biostatistics School of Population HealthFaculty of Medical and Health SciencesThe University of AucklandAuckland 1023New Zealand

BACKGROUND Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision *** extracted from peripheral corneas can form stem cell-enriched spheres,which have shown the potential to repopulate the normal peripheral corneal stroma in vitro upon sphere implantation but have not been previously studied in keratoconic *** To investigate the therapeutic potential of stem cell-enriched spheres formed from extracted peripheral human corneal cells when introduced to keratoconic *** Stem cell-enriched spheres were formed from extracts of normal cadaveric human peripheral corneal *** spheres were implanted into incisions created in full thickness and onto the surface of 10μm thin sections of keratoconic and normal stromal tissues in *** sections were used to maximise use of limited keratoconic tissue available for *** cells were stained with Calcein-AM and visualised with stereo and fluorescence microscopy to assess survival and behaviours between the time of implantation day 0 and 14 d(D14)from *** cells in implanted tissues were characterised for stem cell and differentiation markers using immunohistochemistry and droplet digital PCR to assess the potential implications of these characteristics in the use of spheres in keratoconus *** Spheres were successfully implanted into full-thickness central corneal tissue and onto the surface of 10μm thin en face tissue *** observable differences were seen in sphere migration,proliferation or differentiation in keratoconic tissue compared to normal between day 0 and *** stained positively with Calcein-AM up to *** migration increased from day 0 to D14,occurring radially in three dimensions from the sphere and in alignment with tissue *** proliferation marker,EdU,was detected at day *** spheres stained positively for putative stem cell markersΔNp63

关键词： Keratoconus Cell culture Immunohistochemistry Quantitative PCR Digital PCR Spheroid Holoclone neurosphere Regeneration

Comparative study on the stem cell phenotypes of C6 cells under different culture conditions

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Chinese Medical Journal 2011年第19期124卷 3118-3126页

作者： ZHANG Suo-jun YE Fei XIE Rui-fan HU Feng WANG Bao-feng WAN Feng GUO Dong-sheng LEI Ting Department of Neurosurgery and Neuro-Oncology Molecular Biology Laboratory Tongji Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan Hubei 430030 China

Background Glioma stem cell （GSC） hypothesis posits that a subpopulation of cells within gliomas have true clonogenic and tumorigenic potential. Significantly, a more controversial correlate to GSC is that cells in different culture conditions might display distinct stem cell properties. Considering these possibilities, we applied an approach comparing stem cell characteristics of C6 glioma cells under different culture *** C6 cells were cultured under three different growth conditions, i.e., adherent growth in conventional 10% serum medium, non-adherent spheres growth in serum-free medium, as well as adherent growth on laminin-coated flask in serum-free medium. Growth characteristics were detected contrastively through neurosphere formation assay and cell cycle analysis. Markers were determined by immunofluorescence, relative-quantitative reverse transcription （RT）-PCR,Western blotting and flow cytometry. Side population cells were analyzed via flow cytometry. Tumor models were detected by magnetic resonance imaging and hematoxylin ＆ eosin staining. Data analyses were performed with SPSS software （17.0）.Results C6 cells （C6-Adh, C6-SC-Sph and C6-SC-Adh） showed distinctive growth patterns and proliferation *** to suspending C6-SC-Sph, adherent C6-Adh and C6-SC-Adh displayed higher growth ratio. C6-SC-Sph and C6-SC-Adh showed enhanced capability of neurosphere formation and self-renewal. High side population ratio was detected in C6-SC-Sph and C6-SC-Adh. CD133 was not detected in all three kinds of cells. Conversely, Nestin and β-Ⅲ-tubulin were demonstrated positive, nonetheless with no statistical significance （P 〉0.05）. Interestingly, lower expression of glial fibrillary acidic protein was demonstrated in C6-SC-Sph and C6-SC-Adh. C6-Adh, C6-SC-Sph and C6-SC-Adh were all displayed in situ oncogenicity, while statistical difference of survival time was not *** C6 glioma cell line is endowed with some GSC phenotypes that

关键词： C6 glioma stem cell neurosphere CD133 side population laminin

Role of nestin in glioma invasion

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World Journal of Translational Medicine 2015年第3期4卷 78-87页

作者： Alex Lin Luigi Marchionni Jeffrey Sosnowski David Berman Charles G Eberhart Eli E Bar Department of Pathology Johns Hopkins University School of Medicine Department of Oncology Johns Hopkins University School of Medicine Department of Orthopedic Surgery Johns Hopkins University School of Medicine Department of Ophthalmology Johns Hopkins University School of Medicine Department of Neurological Surgery School of Medicine Case Western Reserve University Case Comprehensive Cancer Center Case Western Reserve University School of Medicine

AIM: To determine the role for the intermediate filament protein nestin in glioma invasion. METHODS: We examined the expression and function of nestin in gliomas(Grades Ⅱ-Ⅳ as defined by the World Health Organization). We determined nestin expression using Immunohistochemical methods. To elucidate nestin's biological function(s), we reduced m RNA levels by 61% and 87% in two glioblastomaderived neurosphere lines using short hairpin RNAs and determined the effect of reduced nestin expression on glioma cell proliferation and invasion using MTS and matrigel migration assays, respectively. We also utilized quantitative real time polymerase chain reaction assaysto determine the effect of reduced nestin expression on the expression of other markers associated with glioma stem cells and their differentiated progenies. RESULTS: We found a significant correlation between nestin immunoreactivity and astrocytoma tumor grade, with 36% of grade Ⅱ, 75% of grade Ⅲ, and 100% of grade Ⅳtumors expressing significant levels of the protein when assessed using immunohistochemistry. Reduction in nestin expression had no effect on cell growth in culture, but did retard the capacity of one line to migrate in-vitro on matrigel. Interestingly, in the line whose migration was not affected, m RNA levels of a second intermediate filament, synemin(also knowns as desmuslin), were elevated following introduction of sh RNA targeting nestin. As synemin was not induced in the line which required nestin for migration, it is a possibility that synemin may compensate for the loss of nestin in this process. CONCLUSION: Nestin expression is prominent in high-grade astrocytomas. Nestin is not required for cell growth but it may, however, be required for cell motility.

关键词： Nestin Stem cells Migration Glioma neurosphere