Neural stem cells  screen
210831332
Integrin avb5 Internalizes Zika Virus during Neural Stem Cells Infection and Provides a Promising Target for Antiviral Therapy
SUMMARY We perform a CRISPR Cas9 genome wide screen in glioblastoma stem cells and identify integrin avb5 as an internalization factor for Zika virus (ZIKV) Expression of avb5 is correlated with ZIKV susceptibility in various cells and tropism in developing human cerebral cortex. A blocking antibody against integrin avb5, but not avb3, efficiently inhibits ZIKV infection. ZIKV binds to cells but fails to internalize when treated with integrin avb5 blocking antibody. avb5 directly binds to ZIKV virions and activates focal adhesion kinase, which is required for ZIKV infection. Finally, avb5 blocking antibody or two inhibitors, SB273005 and cilengitide, reduces ZIKV infection and alleviates ZIKV induced pathology in human neural stem cells and in mouse brain. Altogether, our findings identify integrin avb5 as an internalization factor for ZIKV, providing a promising therapeutic target, as well as two drug candidates for prophylactic use or treatments for ZIKV infections.
Cell reports
Shaobo Wang Qiong Zhang Shashi Kant Tiwari Gianluigi Lichinchi Edwin H Yau Hui Hui Wanyu Li Frank B Furnari Tariq M Rana
13763010
Compounds with species and cell type specific toxicity identified in a 2000 compound drug screen of neural stem cells and rat mixed cortical neurons.
Human primary neural tissue is a vital component for the quick and simple determination of chemical compound neurotoxicity in vitro. In particular, such tissue would be ideal for high throughput screens that can be used to identify novel neurotoxic or neurotherapeutic compounds. We have previously established a high throughput screening platform using human induced pluripotent stem cell (iPSC) derived neural stem cells (NSCs) and neurons. In this study, we conducted a 2000 compound screen with human NSCs and rat cortical cells to identify compounds that are selectively toxic to each group. Approximately 100 of the tested compounds showed specific toxicity to human NSCs. A secondary screen of a small subset of compounds from the primary screen on human iPSCs, NSC derived neurons, and fetal astrocytes validated the results from >80% of these compounds with some showing cell specific toxicity. Amongst those compounds were several cardiac glycosides, all of which were selectively toxic to the human cells. As the screen was able to reliably identify neurotoxicants, many with species and cell type specificity, this study demonstrates the feasibility of this NSC driven platform for higher throughput neurotoxicity screens.
Neurotoxicology
Nasir Malik Anastasia G Efthymiou Karly M Mather Nathaniel Chester Xian-tao Wang Avindra Nath Mahendra S Rao Joseph P Steiner
204814859
Effects of Tianqijiangtang capsule on survival, self renewal and differentiation of hippocampal neural stem cells of embryonic rats cultured in high glucose medium.
OBJECTIVE This study aims to investigate the effects of Tianqijiangtang capsule on the survival, self renewal and differentiation of hippocampal neural stem cells (NSCs) of embryonic rats cultured in high glucose medium. MATERIALS AND METHODS A cell model of diabetic encephalopathy was established. Cell viability was assessed to screen the optimal concentration of glucose for the cell model of diabetic encephalopathy. Then, the effects of Tianqijiangtang capsule on the proliferation and differentiation of NSCs, and the expression of vascular endothelial growth factor (VEGF) and brain derived neurotrophic factor (BDNF) in the culture medium and cells were detected. RESULTS High glucose significantly reduced the ability of survival, proliferation and differentiation of NSCs, which was statistically significant, when compared to the control group (P 0.05 or 0.01) Tianqijiangtang capsule significantly enhanced the survival, proliferation and differentiation of NSCs cultured in high glucose medium, which was statistically significant, when compared with the high glucose group (P 0.05 or 0.01) The high glucose culture resulted in a significant decrease in VEGF and BDNF levels in culture medium and cells of NSCs. Tianqijiangtang capsule significantly increased the level of VEGF nuclear BDNF in cells and the culture medium, which was significantly higher, when compared to that in the high glucose group (P 0.05 or 0.01) CONCLUSION Tianqijiangtang capsule enhances the level of neurotrophic factor synthesized and secreted by hippocampal NSCs cultured with high glucose through the autocrine and paracrine pathway, promotes the NSC survival, replication and differentiation of new neurons and astrocytes, and reduces the degeneration and necrosis of nerve cells.
American journal of translational research
Chun-Yong Han Jing Liu Feng Wan Mo Tian Yan-li Zhang Qing-hu He Yin-Chu Si
3453699
Activation of neural stem cells from quiescence drives reactive hippocampal neurogenesis after alcohol dependence
ABSTRACT Neural stem cell driven adult neurogenesis contributes to the integrity of the hippocampus. Excessive alcohol consumption in alcoholism results in hippocampal degeneration that may recover with abstinence. Reactive, increased adult neurogenesis during abstinence following alcohol dependence may contribute to recovery, but the mechanism driving reactive neurogenesis is not known. Therefore, adult, male rats were exposed to alcohol for four days and various markers were used to examine cell cycle dynamics, the percentage and number of neural progenitor cell subtypes, and the percentage of quiescent versus activated progenitors. Using a screen for cell cycle perturbation, we showed that the cell cycle is not likely altered at 7 days in abstinence. As the vast majority of Bromodeoxyuridine positive cells were co labeled with progenitor cell marker, Sox2, we then developed a quadruple fluorescent labeling scheme to examine Type 1, 2a, 2b and 3 progenitor cells simultaneously. Prior alcohol dependence indiscriminately increased all subtypes at 7 days, the peak of the reactive proliferation. An evaluation of the time course of reactive cell proliferation revealed that cells begin proliferating at 5 days post alcohol, where only actively dividing Type 2 progenitors were increased by alcohol. Furthermore, prior alcohol increased the percentage of actively dividing Sox2+ progenitors, which supported that reactive neurogenesis is likely due to the activation of progenitors out of quiescence. These observations were associated with granule cell number returning to normal at 28 days. Therefore, activating stem and progenitor cells out of quiescence may be the mechanism underlying hippocampal recovery in abstinence following alcohol dependence. HighlightsReactive neurogenesis after alcohol dependence is confirmed with NeuroD1.Recruitment of progenitors out of quiescence likely drives reactive neurogenesis.Reactive neurogenesis begins earlier than original reports via Type 2a progenitors.Reactive neurogenesis is not due to alterations in cell cycle.Type 1 neural stem cell activation has implications for alcoholic neuropathology.
Neuropharmacology
Dayna M Hayes Chelsea Rhea Geil Nickell Kevin Y Chen Justin A McClain Megan M Heath M Ayumi Deeny Kimberly Nixon
5028126
Validation and bioinformatic analysis of propofol induced differentially expressed microRNAs in primary cultured neural stem cells.
Propofol, a widely used intravenous anesthetic, was previously considered as a neuroprotective agent. Recently, however, accumulating evidence suggests that it may cause neurotoxicity, especially in the development of neural stem cells (NSCs) The potential mechanisms contributing to propofol induced neurotoxicity during neurogenesis, such as those involving microRNAs (miRNAs) are still unknown. In this study, a total of 27 differentially expressed miRNAs were identified in our initial screen and 6 miRNAs were validated by qRT PCR. Three miRNAs were up regulated (miR 377 5p, miR 194 3p and miR 143 5p) and three were down regulated (miR 3583 3p, miR 466b 5p and miR 410 5p) Following gene ontology and KEGG pathway enrichment analysis, Gabbr1, Canca1b and Gabbr2, which are enriched in the GABAergic synapse pathway, were selected as genes potentially playing a role in propofol induced neurotoxicity. Gabbr1 and Cacna1b, which are targeted by miRNAs that are up regulated following propofol exposure, showed decreased expression at the mRNA and protein levels. Gabbr2, targeted by miRNAs that were down regulated following treatment with propofol, was up regulated at both the levels of mRNA and protein expression. The two clusters of miRNAs that show differential expression following propofol exposure may act in a synergistic manner to regulate several genes simultaneously during the development of NSCs. Our results may contribute to clarify the molecular mechanism and provide potential therapeutic targets for propofol induced neurotoxicity.
Gene
Yan Li Youtan Liu Jun Fan Quan Zhou Xiuling Song Zhiyong Peng Zaisheng Qin Taoliangshan Tao
46784917
Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis.
Human fetal brain neural stem cells are a unique non genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.
Journal of visualized experiments JoVE
Erica L McGrath Junling Gao Ping Wu
44171030
Generation of H1 PAX6WT/EGFP reporter cells to purify PAX6 positive neural stem/progenitor cells.
Neural conversion from human pluripotent cells (hPSCs) is a potential therapy to neurological disease in the future. However, this is still limited by efficiency and stability of existed protocols used for neural induction from hPSCs. To overcome this obstacle, we developed a reporter system to screen PAX6+ neural progenitor/stem cells using transcription activator like effector nuclease (TALEN) We found that knock in 2 A EGFP cassette into PAX6 exon of human embryonic stem cells H1 with TALEN based homology recombination could establish PAX6WT/EGFP H1 reporter cell line fast and efficiently. This reporter cell line could differentiate into PAX6 and EGFP double positive neural progenitor/stem cells (NPCs/NSCs) after neural induction. Those PAX6WT/EGFP NPCs could be purified, expanded and specified to post mitotic neurons in vitro efficiently. With this reporter cell line, we also screened out 1 NPC specific microRNA, hsa miR 99a 5p, and 3 ESCs enriched miRNAs, hsa miR 302c 5p, hsa miR 512 3p and hsa miR 518 b. In conclusion, the TALEN based neural stem cell screening system is safe and efficient and could help researcher to acquire adequate and pure neural progenitor cells for further application.
Biochemical and biophysical research communications
Wei Wu Juli Liu Zhenghui Su Zhonghao Li Ning Ma Ke Huang Tiancheng Zhou Linli Wang
13759095
Comparative neurotoxicity screening in human iPSC derived neural stem cells, neurons and astrocytes
Induced pluripotent stem cells (iPSC) and their differentiated derivatives offer a unique source of human primary cells for toxicity screens. Here, we report on the comparative cytotoxicity of 80 compounds (neurotoxicants, developmental neurotoxicants, and environmental compounds) in iPSC as well as isogenic iPSC derived neural stem cells (NSC) neurons, and astrocytes. All compounds were tested over a 24 h period at 10 and 100 mM, in duplicate, with cytotoxicity measured using the MTT assay. Of the 80 compounds tested, 50 induced significant cytotoxicity in at least one cell type; per cell type, 32, 38, 46, and 41 induced significant cytotoxicity in iPSC, NSC, neurons, and astrocytes, respectively. Four compounds (valinomycin, 3,3',5,5' tetrabromobisphenol, deltamethrin, and triphenyl phosphate) were cytotoxic in all four cell types. Retesting these compounds at 1, 10, and 100 mM using the same exposure protocol yielded consistent results as compared with the primary screen. Using rotenone, we extended the testing to seven additional iPSC lines of both genders; no substantial difference in the extent of cytotoxicity was detected among the cell lines. Finally, the cytotoxicity assay was simplified by measuring luciferase activity using lineage specific luciferase reporter iPSC lines which were generated from the parental iPSC line. This article is part of a Special Issue entitled SI: PSC and the brain.
Brain Research
Ying Pei Jun Peng Mamta Behl Nisha S Sipes Keith R Shockley Mahendra S Rao Raymond R Tice Xianmin Zeng
131778162
Genome wide CRISPR screen for Zika virus resistance in human neural cells
Significance Zika virus (ZIKV) is a neurotropic and neurovirulent arbovirus that has severe detrimental impact on the developing human fetal brain. We used a genome wide CRISPR Cas9 knockout screen to identify ZIKV host genes in human neural progenitors. The screen identified host factors involved in heparan sulfation, endocytosis, endoplasmic reticulum processing, Golgi function, and interferon activity. Our findings provide insights into host dependent mechanisms for ZIKV infection in the highly vulnerable human neural progenitor cells and identify molecular targets for potential therapeutic intervention. Zika virus (ZIKV) is a neurotropic and neurovirulent arbovirus that has severe detrimental impact on the developing human fetal brain. To date, little is known about the factors required for ZIKV infection of human neural cells. We identified ZIKV host genes in human pluripotent stem cell (hPSC) derived neural progenitors (NPs) using a genome wide CRISPR Cas9 knockout screen. Mutations of host factors involved in heparan sulfation, endocytosis, endoplasmic reticulum processing, Golgi function, and interferon activity conferred resistance to infection with the Uganda strain of ZIKV and a more recent North American isolate. Host genes essential for ZIKV replication identified in human NPs also provided a low level of protection against ZIKV in isogenic human astrocytes. Our findings provide insights into host dependent mechanisms for ZIKV infection in the highly vulnerable human NP cells and identify molecular targets for potential therapeutic intervention.
Proceedings of the National Academy of Sciences
Yun Li Julien Muffat Attya Omer Javed Heather R Keys Tenzin Lungjangwa Irene Bosch Mehreen Ali Khan Maria C Virgilio Lee Gehrke David M Sabatini Rudolf Jaenisch
214786267
Characterization and application of electrically active neuronal networks established from human induced pluripotent stem cell derived neural progenitor cells for neurotoxicity evaluation.
Neurotoxicity is mediated by a variety of modes of actions leading to disturbance of neuronal function. In order to screen larger numbers of compounds for their neurotoxic potential, in vitro functional neuronal networks (NN) might be helpful tools. We established and characterized human NN (hNN) from hiPSC derived neural progenitor cells by comparing hNN formation with two different differentiation media: in presence (CINDA) and absence (neural differentiation medium (NDM) of maturation supporting factors. As a NN control we included differentiating rat NN (rNN) in the study. Gene/protein expression and electrical activity from in vitro developing NN were assessed at multiple time points. Transcriptomes of 5, 14 and 28 days in vitro CINDA grown hNN were compared to gene expression profiles of in vivo human developing brains. Molecular expression analyses as well as measures of electrical activity indicate that NN mature into neurons of different subtypes and astrocytes over time. In contrast to rNN, hNN are less electrically active within the same period of differentiation time, yet hNN grown in CINDA medium develop higher firing rates than hNN without supplements. Challenge of NN with neuronal receptor stimulators and inhibitors demonstrate presence of inhibitory, GABAergic neurons, whereas glutamatergic responses are limited. hiPSC derived GABAergic hNN grown in CINDA medium might be a useful tool as part of an in vitro battery for assessing neurotoxicity.
Stem cell research
Laura Nimtz Julia Hartmann Julia Tigges Stefan Masjosthusmann Martin R Schmuck Eike Hagen Kessel Stephan Theiss Karl Kohrer Patrick Petzsch James Adjaye Claudia Wigmann Dagmar Wieczorek Barbara Hildebrandt Farina Bendt Ulrike Hubenthal Gabriele Brockerhoff Ellen Fritsche