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

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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 understood.Type-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 absent.Immunofluorescence 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 columns.Parenchymal and meningeal-associated BMPRⅡ^(+)vascular cells were identified in both intact fetal spinal cord and cortex by co-positivity with vascular lineage markers,CD34/CD39.LIF 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,cytoarchitecture.These 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 precursors.Dissociated cultures from tripotential neurospheres contained neuronal(βⅢ-tubulin^(+)),astrocytic(GFAP+),and oligodendrocytic(O4+)lineage cells.Fluorescence-activated cell sorting-sorted BMPRⅡ^(+)hNPCs were MAP2ab^(+/–)/βⅢ-tubulin^(+)/GFAP^(–)/O4^(–)in culture.This is the first isolation of BMPRⅡ^(+)hNPCs identified and characterized in human fetal spinal cords.Our data show that LIF combines synergistically with high-density reaggregate cultures to support the organotypic reorganization of neurospheres,characterized by surface BMPRⅡ^(+)hNPCs.Our study has provid

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

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High-frequency magnetic stimulation attenuates beta-amyloid protein 1-42 neurotoxicity in organotypic hippocampal slices

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Neural Regeneration Research 2010年第18期5卷 1365-1372页

作者： Don-Kyu Kim Young Chul Yoon Soo Ahn Chae Kyung Mook Seo Tai Ryoon Han Si-Hyun Kang Department of Physical Medicine and Rehabilitation Chung-Ang University College of Medicine Department of Neurology Chung-Ang University College of Medicine Department of Pediatrics Chung-Ang University College of Medicine Department of Physical Medicine and Rehabilitation Seoul National University

Repetitive transcranial magnetic stimulation （rTMS） has been utilized as a therapeutic tool for neurodegenerative disorders including Alzheimer＇s disease. However, the precise mechanisms of its clinical effects remain unknown. β-amyloid （Aβ） exhibits direct neurotoxic effects and is closely related to neuronal degeneration in Alzheimer＇s disease. Therefore, it has been hypothesized that the neuroprotective effects of rTMS are related to the mechanisms of protection against Aβ neurotoxicity. organotypic hippocampal slices were prepared from 8-day old, Sprague Dawley rats. The tissue slices were exposed to 100 μmol/L Al3142 since day 12 in vitro with and without high-frequency （20 Hz） magnetic stimulation. Magnetic stimulation efficacy was evaluated by measuring neuronal nuclei （NeuN） protein expression and by observing cultures following propidium iodide fluorescence staining and bromodeoxyuridine （BrdU） immunohistochemistry. Lactate dehydrogenase activity was detected in the culture media to evaluate hippocampal neuronal damage. Our results demonstrated that high-frequency magnetic stimulation significantly reversed the reduction of NeuN protein expression because of Aβ1-42 exposure （P 〈 0.05） and significantly reduced the number of damaged cells in the hippocampal slices （P 〈 0.05）. However, lactate dehydrogenase levels and anti-BrdU staining results did not reveal any statistical differences These findings indicate that high-frequency magnetic stimulation might have protective effect on hippocampal neurons from Aβ1-42 neurotoxicity.

关键词： organotypic hippocampus amyloid beta-protein magnetic stimulation nerve degeneration/metabolism nerve degeneration/pathology organ culture techniques rats, Sprague Dawiey

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A benchtop brain injury model using resected donor tissue from patients with Chiari malformation

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Neural Regeneration Research 2023年第5期18卷 1057-1061页

作者： Jacqueline A.Tickle Jon Sen Christopher Adams David N.Furness Rupert Price Viswapathi Kandula Nikolaos Tzerakis Divya M.Chari Aston Pharmacy School College of Health and Life SciencesAston UniversityBirminghamUK School of Medicine Faculty of Medicine and Health SciencesKeele UniversityStaffordshireUK School of Life Sciences Keele UniversityStaffordshireUK Department of Neurosurgery Royal Stoke University HospitalStoke-on-TrentUK

The use of live animal models for testing new therapies for brain and spinal cord repair is a controversial area. Live animal models have associated ethical issues and scientific concerns regarding the predictability of human responses. Alternative models that replicate the 3 D architecture of the central nervous system have prompted the development of organotypic neural injury models. However, the lack of reliable means to access normal human neural tissue has driven reliance on pathological or post-mortem tissue which limits their biological utility. We have established a protocol to use donor cerebellar tonsillar tissue surgically resected from patients with Chiari malformation(cerebellar herniation towards the foramen magnum, with ectopic rather than diseased tissue) to develop an in vitro organotypic model of traumatic brain injury. Viable tissue was maintained for approximately 2 weeks with all the major neural cell types detected. Traumatic injuries could be introduced into the slices with some cardinal features of post-injury pathology evident. Biomaterial placement was also feasible within the in vitro lesions. Accordingly, this ‘proof-of-concept’ study demonstrates that the model offers potential as an alternative to the use of animal tissue for preclinical testing in neural tissue engineering. To our knowledge, this is the first demonstration that donor tissue from patients with Chiari malformation can be used to develop a benchtop model of traumatic brain injury. However, significant challenges in relation to the clinical availability of tissue were encountered, and we discuss logistical issues that must be considered for model scale-up.

关键词： biomaterial Chiari malformation cerebellar slice human tissue injury model neuroregeneration organotypic traumatic brain injury

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New antioxidant SkQ1 is an effective protector of rat eye retinal pigment epithelium and choroid under conditions of long-term organotypic cultivation

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Advances in Aging Research 2012年第2期1卷 31-37页

作者： E. N. Grigoryan Y. P. Novikova O. S. Gancharova O.V. Kilina P. P. Philippov Belozersky Institute of Physico-Chemical Biology Lomonosov Moscow State University Moscow Russia Institute of Mitoengineering Lomonosov Moscow State University Moscow Russia Kol’tsov Institute of Developmental Biology Russian Academy of Sciences Moscow Russia

Cells have intrinsic mechanisms for cleaning harmful oxidants represented mainly by reactive oxygen species (ROS). Despite the antioxidant defense, ROS can cause serious damage to the retina that with age leads to various eye diseases and even blindness. Among numerous cell sites of ROS generation, mitochondrial electron transport is of crucial importance. Recently, for the purpose of cleaning ROS in the mitochondrial matrix, powerful mitochondria- targeted antioxidant “SkQ1” has been invented. We studied SkQ1 effects upon tissues of rat posterior eye cup that consisted: retinal pigment epithelium (RPE) ? choroidal coat ? scleral coat. The eye cups were isolated from the eyes of adult albino rats and cultivated in rotary tissue culture system in the presence of 20 nM SkQ1 or without this compound. After 7 days - 1 month in vitro eye cup samples were studied by immunohistochemistry, routine histology, morphometry, and digital image analysis. We have found that under chosen, “in vitro like in vivo” conditions 20 nM SkQ1 effectively reduced cell death in RPE and choroid, protected RPE from disintegration caused by cell phenotypic transformation and withdrawal from the layer, suppressed transmigration of choroidal coat cells. In the ex vivo model we used degenerative processes were more pronounced in the eye cup center where SkQ1 effect was most vivid. All this give us hopes for effectiveness of SkQ1 treatment of retinal central part that is very susceptible to light-induced over-oxidation injury and mostly suffering in many age-related diseases, AMD, in particular.

关键词： Rat Eye Retinal Pigment Epithelium Choroid organotypic Culturing in Vitro AMD Remodeling Antioxidant SkQ1 Cell Behavior Cell Death

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New antioxidant SkQ1 is an effective protector of rat neural retina under conditions of long-term organotypic cultivation

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Advances in Aging Research 2013年第2期2卷 65-71页

作者： E. N. Grigoryan Y. P. Novikova O. V. Kilina P. P. Philippov Belozersky Institute of Physico-Chemical Biology Lomonosov Moscow State University Moscow Russia Institute of Mitoengineering Lomonosov Moscow State University Moscow Russia Kol’tsov Institute of Developmental Biology Russian Academy of Sciences Moscow Russia Belozersky Institute of Physico-Chemical Biology Lomonosov Moscow State University Moscow Russia Kol’tsov Institute of Developmental Biology Russian Academy of Sciences Moscow Russia Belozersky Institute of Physico-Chemical Biology Lomonosov Moscow State University Moscow Russia

During life human eye is constantly exposed to sunlight and artificial light, the sources of reactive oxygen species (ROS)—the main cause of age-related eye pathology. A novel mitochondria-targeted antioxidant SkQ1 has recently been invented to reduce mitochondrial ROS by cleaning the mitochondria matrix, “the dirtiest place in the cell” in respect of ROS production and accumulation. Earlier we studied SkQ1 effects upon retinal pigment epithelium and choroid in the rat eye posterior cups exposed to long-term 3D organotypic culturing. It was found that under in vitro conditions 20 nM SkQ1 effectively reduced cell death in retinal pigment epithelium and choroid and protected the tissues from disintegration and cell withdrawal. In the present study we used same ex vivo conditions to examine the effect of SkQ1 upon the rat neural retina kept in the content of the posterior eye cup. Eye cups were isolated and cultured in vitro during 7, 14, and 30 days under rotation in the presence and absence of 20 nM SkQ1 in the culture medium. Serial sections of cultivated eye cups were subjected to histology, computer morphometry and immunohistochemistry. Obtained results show that SkQ1 operates as a strong protective agent, preventing neuronal cell death and other degenerative processes in the neural retina. Cell rescue by SkQ1 was more vivid in the central part of the retina than at the periphery. That, in turn, suggests SkQ1 effectiveness in treatment of some age-related eye diseases when central part of the retina, including macula, is most susceptible to degeneration.

关键词： Rat Eye Neural Retina organotypic Culturing in Vitro Age-Related Ophthalmic Disorders Antioxidant SkQ1 Cell-Type Specific Proteins Cell Death

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Comparison between acute slices and organotypic slices in development of dentate granule cells in hippocampus of rat

Comparison between acute slices and organotypic slices in de...

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2008北京国际生理科学学术大会

作者： MAZZANTINI Mario EDWARDS Frances A Neuroscience Physiology and Pharmacology University College LondonGower St LondonWC1E 6BTUK

The hippocampal dentate gyrus is one of the few brain regions where neurogenesis occurs in adults.In recent years,organotypic slices have been widely used to study pharmacology,neurobiology of learning and memory as well as neurogenesis in hippocampus.As many studies are carried out in organotypic slices,it is essential to establish how connectivity develops over time in organotypic slices and compare it with better characterised preparations such as acute slices.This has been previously carried out in the CA1 region over the first 3 weeks in vitro(De Simoni,Griesinger & Edwards,J.Physiology 550,135-148,2003)and development was shown to be remarkably similar in the two types of slice preparations.Less is known however about other regions and to address this question in the dentate gyrus,which is the region of stem cell generation in adult mammals,we compared initially slices from postnatal day 14 rats(P14)rats in acute slices to organotypic slices after 7 d in vitro (DIV7).These stages have been shown to be equivalent in the previous CA1 study.Using whole-cell patch-clamp, we found that most of the measurable synaptic activity was GABAergic at the cell soma in both preparations.Thus to estimate the development of excitatory synapses,out in the dendrites,we used confocal microscopy to study morphology,including dendritic geometry and spine density.After one week in vitro we found that,in contrast to the CA1 study above,almost all factors measured in organotypic slices were significantly different from the acute slices.However,in both inhibitory synaptic activity and in density of excitatory synapses,both strengthening and weakening of different aspects of the system was seen.Thus,compared with acute slices,organotypic slices had significantly lower frequency but higher amplitude of spontaneous GABAergic synaptic currents.Similarly,despite lower spine density,the dendritic tree in organotypic slices was more complex than that in acute slices,suggesting changes in both systems may be homeostatically balanced.It seem

关键词： hippocampus organotypic slice synaptic transmission

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Human Brain Slice Culture: A Useful Tool to Study Brain Disorders and Potential Therapeutic Compounds

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Neuroscience Bulletin 2019年第2期35卷 244-252页

作者： Xin-Rui Qi Ronald W.H.Verwer Ai-Min Bao Rawien A.Balesar Sabina Luchetti Jiang-Ning Zhou Dick F.Swaab Center for Translational Neurodegeneration and Regenerative Therapy Shanghai Tenth People's Hospital Affiliated to Tongji University School of Medicine Department of Neuropsychiatric Disorders Netherlands Institute for NeuroscienceAn Institute of the Royal Netherlands Academy of Arts and Sciences Department of Neurobiology Institute of NeuroscienceNHC and CAMS Key Laboratory of Medical NeurobiologyZhejiang University School of Medicine Key Laboratory of Brain Function and Diseases School of Life SciencesUniversity of Science and Technology of ChinaChinese Academy of Sciences

Investigating the pathophysiological mechanisms underlying brain disorders is a priority if novel therapeutic strategies are to be developed. In vivo studies of animal models and in vitro studies of cell lines/primary cell cultures may provide useful tools to study certain aspects of brain disorders. However, discrepancies among these studies or unsuccessful translation from animal/cell studies to human/clinical studies often occur, because these models generally represent only some symptoms of a neuropsychiatric disorder rather than the complete disorder. Human brain slice cultures from postmortem tissue or resected tissue from operations have shown that, in vitro, neurons and glia can stay alive for long periods of time, while their morphological and physiological characteristics, and their ability to respond to experimental manipulations are maintained. Human brain slices can thus provide a close representation of neuronal networks in vivo, be a valuable tool for investigation of the basis of neuropsychiatric disorders, and provide a platform for the evaluation of novel pharmacological treatments of human brain diseases.A brain bank needs to provide the necessary infrastructure to bring together donors, hospitals, and researchers who want to investigate human brain slices in cultures of clinically and neuropathologically well-documented material.

关键词： Alzheimer’s disease Brain bank Brain-derived neurotrophic factor Depression Electrical activity Human brain slice culture Neuropsychiatric disorders organotypic culture Postmortem human brain tissue Resected human brain tissue

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Cisplatin-induced vestibular hair cell lesion-less damage at high doses

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Journal of Otology 2018年第4期13卷 115-121页

作者： Dalian Ding Haiyan Jiang Jianhui Zhang Xianrong Xu Weidong Qi Haibo Shi Shankai Yin Richard Salvi Center for Hearing and Deafness University at BuffaloUSA Department of Otolaryngology Head and Neck Surgery The Third People’s Hospital of ChengduChina Air Force General Hospital BeijingChina Department of Otolaryngology Head and Neck Surgery Huashan HospitalFudan UniversityShanghaiChina Department of Otorhinolaryngology Affiliated Sixth People’s Hospital of Shanghai Jiaotong UniversityShanghaiChina Department of Audiology and Speech-Language Pathology Asia UniversityChina

Cisplatin, a widely used anticancer drug, damages hair cells in cochlear organotypic cultures at low doses, but paradoxically causes little damage at high doses resulting in a U-shaped dose-response function. To determine if the cisplatin dose-response function for vestibular hair cells follows a similar pattern, we treated vestibular organotypic cultures with doses of cisplatin ranging from 10 to 1000 μM. Vestibular hair cell lesions progressively increased as the dose of cisplatin increased with maximum damage occurring around 50-100 μM, but the lesions progressively decreased at higher doses resulting in little hair cell loss at 1000 μM. The U-shaped doseresponse function for cisplatin-treated vestibular hair cells in culture appears to be regulated by copper transporters, Ctrl, ATP7A and ATP7B, that dose-dependently regulate the uptake, sequestration and extrusion of cisplatin.

关键词： Cisplatin Ototoxicity Copper transporters Vestibular organotypic cultures

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Pancreatic cancer organotypics: High throughput, preclinical models for pharmacological agent evaluation

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World Journal of Gastroenterology 2014年第26期20卷 8471-8481页

作者： Stacey J Coleman Jennifer Watt Prabhu Arumugam Leonardo Solaini Elisabeta Carapuca Mohammed Ghallab Richard P Grose Hemant M Kocher Centre for Tumour Biology Barts Cancer Institute - a CR-UK Centre of ExcellenceQueen Mary University of LondonJohn Vane Science CentreCharterhouse SquareLondon EC1M 6BQUnited Kingdom Barts and the London HPB Centre The Royal London HospitalBarts Health NHS TrustLondon EC1M 6BQUnited Kingdom

Pancreatic cancer carries a terrible prognosis,as the fourth most common cause of cancer death in the Western world.There is clearly a need for new therapies to treat this disease.One of the reasons no effective treatment has been developed in the past decade may in part,be explained by the diverse influences exerted by the tumour microenvironment.The tumour stroma cross-talk in pancreatic cancer can influence chemotherapy delivery and response rate.Thus,appropriate preclinical in vitro models which can bridge simple 2D in vitro cell based assays and complex in vivo models are required to understand the biology of pancreatic cancer.Here we discuss the evolution of 3D organotypic models,which recapitulare the morphological and functional features of pancreatic ductal adenocarcinoma(PDAC).organotypic cultures are a valid high throughput preclinical in vitro model that maybe a useful tool to help establish new therapies for PDAC.A huge advantage of the organotypic model system is that any component of the model can be easily modulated in a short timeframe.This allows new therapies that can target the cancer,the stromal compartment or both to be tested in a model that mirrors the in vivo situation.A major challenge for the future is to expand the cellular composition of the organotypic model to further develop a system that mimics the PDAC environment more precisely.We discuss how this challenge is being met to increase our understanding of this terrible disease and develop novel therapies that can improve the prognosis for patients.

关键词： 3D organotypic model Pancreatic cancer Pancreatic

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Regenerative Properties of Fetal n. Raphe Cells in Modeling Injuries of Serotonergic Neurite-Glial Fibers in Vitro

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Journal of Pharmacy and Pharmacology 2017年第8期5卷 515-520页

作者： Vasylyeva I. G. Oleksenko N. P. Chopyk N. G. Galanta O. S. Shuba I. M., Tsyubko O. I. Snitsar N. D. Department neurobiochemistry SI "Institute ofNeurosurgeryn. "acad. A. Romodanov 04050 Ukraine

It is considered that renewal of growth in serotoninergic caudal fibers their sprouting to injury site and formation ofoutgrowth net is factor for locomotion recovery. The aim of our work was to study the influence of rat fetal neural cells enriched withserotonin-producing cells, on morphogenesis, gene expression of serotogenesis （Pet1, Nk2.2, Lmxlb, Tphl, Tph2, Sert） andserotonin level in organotypic n. raphe cultures in injury of neurite-glial fibers in vitro. Injury was modeled by the way of crossingneurite-glial fibers after their formation under long-term culture. The cell accumulation was observed in the injury zone when fetal n.raphe cells, previously enriched with serotonin-producing cells were added into organotypic culture of n. raphe with crossing fibersunder the culture conditions. As a result, crossing site was filled with these cells after 14 days of culture. The activation of expressionin cascade of regulatory genes-regulators of serotogenesis Pet1, Lmxlb, Tphl, Tph2, Sert and increased serotonin content in theculture material were also observed. The activation of gene expression of serotogenesis Pet1 and Sert, and increased serotonincontent by 1.7 times were revealed when conditioned medium from culture of fetal rat cells was used. An eviction of single cells wasobserved in control samples of organotypic n. raphe culture after injury. Crossing site was remained without signs of cell filling inthese samples at day 14 after culture. The activation of regulatory gene expression of serotogenesis Pet1, Nkx2.2, Lmxlb, Tphl,Tph2, Sert and restoration of serotonin content were also absent in the culture material.

关键词： Fetal rat nerve cells, serotonin, n. raphe, organotypic culture, suspension culture.

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