Name: es cell-derived neuroepithelial cell cultures
Uploaded: 2006-11-30T00:00:00.000+00:00
Duration: 7 min 45 s
Description: Watch this Scientific Journal Video about ES Cell-derived Neuroepithelial Cell Cultures at JoVE.com

1 단계 <ol><li> 플레이트 mitomycin - C의 α - 멤 미디어 젤라틴 코팅 (30 분 0.01 %) 6 잘 접시에 70,000 / cm2의 밀도에 MS5 세포 (2.5 시간 10 μg / ML) ggrowth - 저해. </li><li> 세포 부착과 monolayer를 (야간 성장 이상) 형성되면, SRM로 전환합니다. </li><li> 수동으로 27과 주사기를 사용하여 ES 세포 배양 ½ G 바늘에서 ES 세포 식민지를 분리. </li><li> MS5 전지 (6 자 판 당 보통 2-3 식민지)에 저밀도에서 1 ML 파란색 팁 및 플레이트와 함께 신중하게 식민지를 Tritrurate. </li></ol> 참고 : ES 세포도 collagenase 또는 트립신 효소를 사용하여 전파에서 촬영하실 수 있습니다. 2 단계 <ol><li> 로제트 함유 식민지 양식까지 14~21일에 대한 MS5 모이통에서 ES 세포를 성장. </li></ol> 참고 : 변경 미디어 자주. rosettes은 식민지와 300-500 NG / ML 노긴는 SRM (3)에 추가하는 경우에는 자신의 형성이 크게 향상 될 수의 바깥 가장자리에 일반적으로있다. 차별 프로토콜에서는, SRM은 N2 - A 매체와 교환 수 있으며, 세포 사양 (2-5) 홍보 성장 또는 기타 요인과 함께 보충. 3 단계 <ol><li> 격리하고 27로 rosettes을 선택 ½ G 바늘을 현미경. </li></ol> 참고 : 절단 및 MS5 stromal 세포를 채취하지 마십시오. 식민지가 rosettes 모여있다면, 어떤 사람은 전체 식민지를 분리하실 수 있습니다. 4 단계 <ol><li> rosettes의 클러스터를 대기음, 폴리 - L - ornithine (0.001 %)과 fibronectin (1 μg / ML) 또는 laminin (1 μg / ML) 코팅 6 우물에 1 ML 파란색 팁 및 플레이트 그들과 함께 신중하게 tritrurate N2 - A 미디어는 일반적으로 bFGF (20 NG / ML)과 보완. rosettes는 개혁과 확장됩니다. </li></ol> 참고 : 셀 생존을 향상시키기 위해, 하나는 플레이트 방울의 작은 클러스터는 세포 밀도를 증가 수 있습니다. 이 단계는 또한 세포 사양 (2-5) 촉진 추가 성장이나 다른 요소와 N2 - 미디어를 보완하여 수정할 수 있습니다.

<ol>
	<li>Kawasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+midbrain+dopaminergic+neurons+from+ES+cells+by+stromal+cell-derived+inducing+activity.">Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.</a> Neuron. 28, 31-40 (2000).</li><li>Perrier, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+midbrain+dopamine+neurons+from+human+embryonic+stem+cells.">Derivation of midbrain dopamine neurons from human embryonic stem cells.</a> Proc Natl Acad Sci U S A. 101, 12543-12548 (2004).</li><li>Pruszak, J., Isacson, O., Sullivan, S., Cowan, C., Eggan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+embryonic+stem+cells+into+dopaminergic+neurons.">Directed differentiation of human embryonic stem cells into dopaminergic neurons.</a> Human Embryonic Stem Cells: The Practical Handbook. , (2007).</li><li>Pruszak, J., Sonntag, K. C., Aung, M. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+yield+of+neuroepithelial+precursors+and+midbrain-like+dopaminergic+neurons+from+human+embryonic+stem+cells+using+the+bone+morphogenic+protein+antagonist+noggin.">Enhanced yield of neuroepithelial precursors and midbrain-like dopaminergic neurons from human embryonic stem cells using the bone morphogenic protein antagonist noggin.</a> Stem Cells. 25, 411-418 (2007).</li><li>Kawasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+dopaminergic+neurons+and+pigmented+epithelia+from+primate+ES+cells+by+stromal+cell-derived+inducing+activity.">Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity.</a> Proc Natl Acad Sci U S A. 99, 1580-1585 (2002).</li><li>Ko, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+neural+precursors+as+a+continuous,+stable,+and+on-demand+source+for+human+dopamine+neurons.">Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons.</a> J Neurochem. 103, 1417-1429 (2007).</li><li>Hong, S., Kang, U. J., Isacson, O., Kim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+precursors+derived+from+human+embryonic+stem+cells+maintain+long-term+proliferation+without+losing+the+potential+to+differentiate+into+all+three+neural+lineages,+including+dopaminergic+neurons.">Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons.</a> J Neurochem. , (2007).</li><li>Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+subtype+specification+from+embryonic+stem+cells.">Neural subtype specification from embryonic stem cells.</a> Brain Pathol. 16, 132-142 (2006).</li><li>Pankratz, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+neural+differentiation+of+human+embryonic+stem+cells+via+an+obligated+primitive+anterior+stage.">Directed neural differentiation of human embryonic stem cells via an obligated primitive anterior stage.</a> Stem Cells. 25, 1511-1520 (2007).</li><li>Lazzari, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+derivation+of+neural+rosettes+from+cloned+bovine+blastocysts:+a+model+of+early+neurulation+events+and+neural+crest+specification+in+vitro.">Direct derivation of neural rosettes from cloned bovine blastocysts: a model of early neurulation events and neural crest specification in vitro.</a> Stem Cells. 24, 2514-2521 (2006).</li><li>Pomp, O., Brokhman, I., Ben-Dor, I., Reubinoff, B., Goldstein, R. 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The authors have nothing to disclose.

이 프로토콜은 SDIA을 사용하여 인간의 ES 세포에서 neuroepithelial 세포를 생성 및 분리의 여러 단계를 보여줍니다. 이 방법의 응용 프로그램은 매니폴드이며 (예 : 1, 2, 5-9) 지정 뉴런을 생산하기 위해 많은 프로토콜에서 사용되고 있습니다. rosettes는 앞쪽에 표현형 (2, 5, 10)과 신경 튜브 세포를 닮은 또한 신경 크레스트 progenitors (11, 12)를 포함하는 것으로 생각되고 있습니다. 그들이 정의된 문화 환경에있는 특정 요인에 의해 패턴 될 수 있기 때문에 또한, 그들은 소성의 특정 수준을 유지합니다. 따라서 SDIA 파생 신경 progenitors 그들 ES 세포 분화의 패러다임에서 다른 신경 세포 인구의 유도를위한 유용한 도구 제작의 중심과 주변 신경 시스템에서 다양한 세포 유형으로 상승을 줄 수 있습니다.

<table><tbody><tr><td>L-Glutamine</td><td></td><td>GIBCO, by Life Technologies</td><td>25030</td><td></td></tr><tr><td>alpha-MEM</td><td></td><td>GIBCO, by Life Technologies</td><td>12571</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td></td><td>GIBCO, by Life Technologies</td><td>15140</td><td></td></tr><tr><td>Knockout-DMEM</td><td></td><td>GIBCO, by Life Technologies</td><td>10829</td><td></td></tr><tr><td>Knockout serum replacement</td><td></td><td>GIBCO, by Life Technologies</td><td>10828</td><td></td></tr><tr><td>MEM nonessential amino acid solution</td><td></td><td>GIBCO, by Life Technologies</td><td>12383</td><td></td></tr><tr><td>DMEM/F12</td><td></td><td>GIBCO, by Life Technologies</td><td>11330</td><td></td></tr><tr><td>N2-A supplement</td><td></td><td>Stem Cell Technologies</td><td>07152</td><td></td></tr><tr><td>Mitomycin-C</td><td></td><td>Sigma-Aldrich</td><td>M0503</td><td></td></tr><tr><td>Gelatine type-A</td><td></td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>poly-L-ornithine</td><td></td><td>Sigma-Aldrich</td><td>P4957</td><td>0.01% solution</td></tr><tr><td>Laminin</td><td></td><td>Sigma-Aldrich</td><td>L-2020</td><td></td></tr><tr><td>Fibronectin</td><td></td><td>Sigma-Aldrich</td><td>F2006</td><td></td></tr><tr><td>Basic fibroblast growth factor (bFGF)</td><td></td><td>Invitrogen</td><td>13256</td><td></td></tr><tr><td>1 mL Syringe with 27 1/2 G needle</td><td></td><td>BD Biosciences</td><td>309623</td><td></td></tr><tr><td>N2-A media</td><td>medium</td><td></td><td></td><td>DMEM/F12 + 1% N2-A supplement </td></tr><tr><td>Serum replacement media (SRM)</td><td>medium</td><td></td><td></td><td>Knockout-DMEM + 20% Knockout serum replacement +1% MEM nonessential amino acid solution + 2 mM L-glutamine</td></tr><tr><td>&amp;alpha;-MEM media</td><td>medium</td><td></td><td></td><td>&amp;alpha;-MEM + 10% FBS + 2 mM L-glutamine + 1% penicillin/streptomycin</td></tr><tr><td>MS5</td><td>cell line</td><td></td><td></td><td>stromal cells </td></tr><tr><td>Microscope</td><td></td><td></td><td></td><td></td></tr><tr><td>6-well Plates</td><td></td><td></td><td></td><td>for tissue culture</td></tr></tbody></table>

es cell-derived neuroepithelial cell cultures

ES 세포들이 새로운 치료법의 개발을위한 매력적인 도구를 만들어 모든 세균 레이어에서 세포로 차별화할 가능성이있다. 일반적으로 ES 세포의 분화 먼저 다음 증식과 성숙 세포 phenotypes로 차별화된 수있는 미숙 전구 세포를 생성하는 개념을 따르고 있습니다. 첫째, 성숙한 신경 세포에 미숙 neuroepithelial 엽 성의 전구 물질 둘째, 그들의 분화의 유도 및 확장 : 이것은 또한 신경 세포의 개발은 두 가지 주요 단계를 다음과하는, ES 세포 - 파생 neurogenesis 적용됩니다. ES 세포로부터 신경 progenitors을 생산하는 일반적인 방법은 neuroectoderm 포함한 모든 세균 레이어에서 세포의 분화를 보여 embryoid 몸 (EB) 형성에 따라 달라집니다. neuroepithelial 셀 개발을 유도하기 위해 대안과 더 효율적인 방법은 두개골 골수 - 유래 stromal 세포 (1) 공동 culturing의 ES 세포에 의해 얻을 수 stromal 세포 - 파생 유도 활동 (SDIA)를 사용합니다. , EB 형성과 SDIA 둘 다 신경 유사 것으로 생각되고 있습니다 로제트 같은 구조의 개발, 튜브 및 / 또는 신경 능선과 같은 progenitors를 공개. 신경 엽 성의 전구 물질은 정의 문화 조건을 사용하여 특정 뉴런과 glia 세포로 확장하고 더욱 차별, 격리 수 있습니다. 여기, 우리는 stromal 세포 라인 MS5 (2-5)와 함께 공동 문화 실험에서 세대와 같은 rosettes의 절연을 설명합니다.

Watch this Scientific Journal Video about ES Cell-derived Neuroepithelial Cell Cultures at JoVE.com

ES Cell-derived Neuroepithelial Cell Cultures

stromal 세포 - 파생 유도 활동 (SDIA)를 사용하여 배아 줄기 (ES) 세포 neuroepithelial 엽 성의 전구 물질의 유도.

ES 세포 - 파생 Neuroepithelial 셀 문화

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

es-cell-derived-neuroepithelial-cell-cultures

Nanyang Technological University

Research

JoVE Journal

Biology

13.5K 조회수.  Harvard Medical School. stromal 세포 - 파생 유도 활동 (SDIA)를 사용하여 배아 줄기 (ES) 세포 neuroepithelial 엽 성의 전구 물질의 유도.

비디오: ES Cell-derived Neuroepithelial Cell Cultures

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

마우스 신경 줄기 세포 엽 성의 전구 물질의 문화

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

연구

생물학

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

약 발견에 적합 EMT 조사를위한 효소 - 및 혈청이없는 신경 줄기 세포 배양 모델

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

발생학

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

폐기 인간 태아 두피 조직에서 신경 줄기 세포의 생성

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

신경과학

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

마우스 표피 신경 크레스트 줄기 세포 (에피 NCSC) 문화

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

문화의 차별화 된 인간 Neuroprogenitor 세포에서 신경 세포의 레이저 캡처 미세 절제

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

성인 쥐의 뇌의 기존 및 비 인습적 인 지역에서 신경 줄기 세포 성장

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

하나의 마우스, 두 문화 : 분리 및 성인 신경의 문화가 개인 마우스의 두 신경 인성 영역에서 줄기 세포

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

인간 배아 줄기 세포의 단세포 배양을 이용한 효율적인 신경 분화

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

인간 신경 전구 세포 유래 뉴런을 가진 Neurite 아웃growth 분석 및 신경 독성 평가

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

<meta charset="utf8"></head><body>쥐 뇌실하 영역에서 분리된 NSC의 효율적인 핵 분석

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>저자 스포트라이트: Improved Nucleofection for High-Efficiency Gene Delivery in Murine Subventricular Zone-Derived Neural Stem Cell Cultures

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...