이 작품은 오버 재단 기금에 의해 지원되었다.

1. 기본은 절개로 진행하기 전에 설정 : <ol><li> 전체 NSC 매체의 적절한 볼륨이 각각 9시 1분 비율 NeuroCult NSC 기초 중간 및 NeuroCult NSC 확산 보조 식품을 혼합하여 준비가되어 있습니다. </li><li> 매체는 37 ° C의 물을 욕조에 워밍업이다. </li><li> 항생제의 높은 농도 (10 %)와 추위 HEPES 버퍼 최소 필수 매체 (앙)은 절개와 세탁 목적을 위해 준비가되어 있습니다. 또한, 항생제의 보완과 함께 NSC 기초 매체는 또한 이러한 목적으로 사용할 수 있습니다. </li><li> 항생제를 포함하는 감기 앙의 25-30 ML은 배아를 수집 무균 50 ML 튜브에 적절합니다. </li><li> 여러 10cm 플라스틱 배양 접시는 절개시 태아와​​ 두뇌를 개최하는 데 필요한 또한 해부 조직을 개최합니다. </li><li> 배아 (대형 가위, 작은 지적 가위, 큰 집게, 작은 곡선 포셉) 또는 배아 뇌 해부를위한 것이 (작은 포셉, 곡선 좋은 포셉, 45 ° 직각 좋은 포셉, 작은 가위)를 제거하기 위해 필요한 수술 도구는 사용 소독 아르 250 유리 구슬 살균기 ° C 또는 기타 가능한 압력솥 방법. </li><li> 해부 현미경은 70 % 알코올로 닦아 및 층류 또는 PC2 후드 내부에 설정됩니다. </li></ol> 2. E14 마우스 두뇌와 마이크로 절개를 수확 : <ol><li> 임신 마우스를 성관계 시간이 하루에 하나의 기관 승인 동물 프로토콜에 따라 태동 14 일 anesthetized입니다. </li><li> 자궁 경부 전위는 동물이 고통과 고통을 경험하게하지 않도록하기 위해 수행됩니다. </li><li> anesthetized 마우스는 흡수성 티슈에 미치는 뒷면에 마련이고, 복부는 지역을 소독하기 위해 70 %의 에탄올로 씻어서 있습니다. </li><li> 복부 이상의 피부는 큰 집게를 사용하여 파악하고, 다음 피부와 기본 근막은 복강과 자궁 뿔을 폭로 큰 가위로 절단됩니다. </li><li> uteri 작은 집게와 가위로 제거되고 50 ML 원뿔 튜브 함유 추위 앙을로 전송됩니다. </li><li> 자궁 조직이 후드로 전송 후 혈액과 머리카락과 같은 가능한 오염 물질을 제거하는 신선한 무균 추위 앙의 충분한 볼륨 한두 번 씻어서 있습니다. </li><li> 자궁 조직은 다음 차가운 앙을 포함 10cm 페트리 접시로 전송됩니다. </li><li> 자궁 뿔은 작은 곡선 집게와 가위를 사용하여 개설하고 배아는 추위 앙을 포함하는 새로운 10cm 요리로 전송됩니다. </li><li> 태아의 머리가 자궁 경부 척수 수준에서 분리되어 추위 앙을 포함하는 다른 페트리 접시로 전송됩니다. </li><li> 페트리 접시는 두개골에서 뇌를 제거하는 해부 현미경으로 전송됩니다. </li><li> 머리는 머리의 등 부분의 면이 위쪽 직면만큼 꼬리 측면에서 멋진 곡선 포셉 개최하고 있습니다. 를 사용하여 마이크로 가위, 먼저 수평 상처가 눈 위에서 만든 다음 머리 뒤쪽으로 이마의 정중선에서 계속됩니다. 피부와 두개골을 통해 절감과 기본 뇌 손상하지 않도록하십시오. </li><li> 두뇌는 곡선 집게를 사용하여 역방향 움직임에 절단 부분의 가장자리를 추진하여 두개골에서 제거됩니다. 모든 두뇌는 두개골에서 제거되기 전까지는이 절차는 계속됩니다. </li><li> 두뇌는 지속 등의 측면이 상처가 후각 구근에서 ganglionic eminences을 폭로하는 북반구의 뒷면에 걸친 각 반구의 피질을 통해 수행되는 microscissors를 사용하는 이상하고 직면되도록 곡선 집게를 사용하여 개최됩니다. </li><li> 한손에 곡선 포셉와 다른 45 ° 직각 집게를 사용하여 cortices의 컷 펄럭이 확산되고 ganglionic eminences 밖을 해부하고 있습니다. </li><li> 해부 조직은 멸균 10cm 페트리 접시에 배치되며, 모든 머리가 마이크로 해부 때까지이 절차가 반복됩니다. </li><li> 조직 조각은 1 ML 피펫 팁의 NSC 매체 1 ML을 사용하여 수집하고 15 ML의 원심 관으로 전송됩니다. </li><li> 조직은 철저히 다음 dissociated이지만, 부드럽게 튜브의 바닥에 피펫 팁을 누르고 정지를 pipetting 아래로. 이 방법은 대단히 짧은 시간은 단일 세포로 헤어 졌 수 있습니다. Pipetting는 우유 같은 현탁액이 달성되도록 3-4 회를 실시합니다. 그런 다음, 정지가 아닌 dissociated 대단히 짧은 시간이 precipitates만큼 1~2분을 받아들여야 허용됩니다. </li><li> 거의 모든 세포 현탁액은 다른 튜브로 전송됩니다 그리고 NSC 매체의 또 다른 한 ML은 설명된대로 남아있는 대단히 짧은 시간 및 단일 세포에 dissociated에 추가됩니다. </li><li> 튜브의 내용은 700rpm (110g)에서, 실온에서 5 분 풀링 및 centrifuged 있습니다. </li><li> 뜨는은 실제 펠렛 아래로 떨어져 진공 청소기로 청소를하고, 세포가 완전한 NSC 매체 1 ML에 resuspended 있습니다. </li><li> 매체는 부드럽게하기 위해 아래 pipetted 있으며단일 단일 세포 현탁액 있습니다. </li><li> 세포 현탁액의 10 μL는 셀 카운트를 수행하는 trypan 파랑 90 μL와 혼합됩니다. </li><li> 마지막으로, 세포가 20ng / ML 표피 성장 인자 (EGF)와 보충 전체 NSC 매체에 2x10 5 셀 / ML의 밀도에 도금입니다. 5 ML 매체는 T175 flasks에 대한 T75 40 ML에 대한 T25, 20 ML에 사용됩니다. </li><li> 37 ° humidified 인큐베이터에서 C 5 % CO 2에서 incubated 때 Neurospheres는 5~7일를 형성하고 있습니다. 6-7일에서 분야는 150-200 미크론 사이에 측정해야하며 subculture (통로)을 준비합니다. </li></ol> 3. Passaging 및 배아 NSCs의 확장 : <ol><li> neurospheres가 subculture (직경 150-200 μm의)에 대한 준비가되면, 중지 분야와 매체는 flasks에서 삭제 적절한 크기 무균 조직 배양 튜브에 배치하고,시 5 분 700 RPM (110g)에 centrifuged입니다 상온. </li><li> 뜨는은 삭제되고 분야는 0.05 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) 1 ML에 resuspended 있습니다. </li><li> 세포 현탁액은 다음 2-3 분 37 ° C의 물을 욕조에 incubated이며, 다음 대두 트립신 억제제의 동등한 볼륨 트립신의 활동을 중지하는 데 사용됩니다. </li><li> 세포 현탁액은 부드럽게 트립신가 완전히 inactivated되었는지 확인하기 위해 아래 pipetted하고 있습니다. </li><li> 세포 현탁액은 5 분 700 RPM (110g)에 centrifuged입니다. 그런 다음, 표면에 뜨는이 제거되고 세포 전체 NSC 매체 1 ML에 resuspended 있습니다. </li><li> 세포 현탁액의 10 μL는 셀 카운트를 수행하는 trypan 파랑 90 μL와 혼합됩니다. </li><li> 세포는 적절한 크기의 조직 문화 플라스크에 20ng / ML EGF와 보충 전체 NSC 매체에 5x10 4 셀 / ML의 농도에 도금입니다. 5 ML 매체는 T175 flasks에 대한 T75 40 ML에 대한 T25, 20 ML에 사용됩니다. </li><li> 37 ° humidified 인큐베이터에서 C 5 % CO 2에서 incubated 때 보조 neurospheres는 5~7일를 형성하고 있습니다. </li></ol> 4. 대표 결과 : 기본 배아 NSC 문화에서, 세포의 대부분은 비대가 될 것이며, 도금시 조직 문화 요리에 첨부합니다. 세포의 대부분이 중 죽게하거나 2-3일 후 차별화되지만, proliferative 전지는 기판 (그림 1)에서 분리됩니다 세포의 작은 클러스터를합니다. 문화의 첫 48 시간에 큰 회전 타원체의 집계의 형성은 주요 분야에 대한 착각해서는 안됩니다. 집계 형성은 주로 파편과 문화의 비 dissociated 조직 대단히 짧은 시간의 양을에 따라 달라집니다. 진정한 neurospheres이 단계 밝고하고 크기 증가 (그림 2)와 같은 더 구형이된다. 작은 microspikes이 가능한 건강 분야 (그림 3)의 바깥쪽 표면에 나타납니다. 5~7일 후, 분야는 둥근 수 있지만 압축 안된다, 그리고 직경 150 사이 200 μm의 측정해야합니다. neurospheres가 (문화 9~10일 이후) 너무 커질 수있다면, 그들은 집계를 형성하거나 (비디오 참조) 분야의 중심에 있기 때문에 세포 죽음의 어두운 색상이 될 수도 있습니다. 대형 neurospheres 결국 (기판에 연결하여 주변위한 마이 그 레이션) 현장에서 차별 시작 있습니다. 그것은 또한 큰 neurospheres을 떼어 놓다하기 어려운 그들을 subculture. <img src="/files/ftp_upload/2457/2457fig1.jpg" alt="그림 1" /> 그림 1. 삼일 도금 후 기본 E14 NSC 문화. 화살표 NSCs의 proliferating 클러스터를 보여줍니다. 원본 확대, 20x <img src="/files/ftp_upload/2457/2457fig2.jpg" alt="그림 2" /> 그림 2. 칠일 도금 후 기본 E14 NSC 문화. 원본 확대, 10X <img src="/files/ftp_upload/2457/2457fig3.jpg" alt="그림 3" /> 그림 3. 도금 후 패시지 한 E14 neurospheres 오일합니다. 분야의 주변에있는 마이크로 스파이크를 (화살표) 참고. 원본 확대, 20x

<ol>
	<li>Reynolds, B. A., Weiss, S. <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+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Morshead, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+in+the+adult+mammalian+forebrain:+a+relatively+quiescent+subpopulation+of+subependymal+cells.">Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.</a> Neuron. 13, 1071-1082 (1994).</li><li>Craig, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+growth+factor+expansion+of+endogenous+subependymal+neural+precursor+cell+populations+in+the+adult+mouse+brain.">In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain.</a> J Neurosci. 16, 2649-2658 (1996).</li><li>Golmohammadi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+the+frequency+and+distribution+of+stem+and+progenitor+cells+in+the+adult+mouse+brain.">Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.</a> Stem Cells. 26, 979-987 (2008).</li><li>Azari, H., Rahman, M., Sharififar, S., BA, R. e. y. n. o. l. d. s. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J Vis Exp. , (2010).</li><li>Reynolds, B. A., Rietze, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+and+neurospheres--re-evaluating+the+relationship.">Neural stem cells and neurospheres--re-evaluating the relationship.</a> Nat Methods. 2, 333-336 (2005).</li><li>Louis, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+neural+stem+and+progenitor+cells+in+the+neural+colony-forming+cell+assay.">Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.</a> Stem Cells. 26, 988-996 (2008).</li></ol>

관심 없음 충돌 선언하지 않습니다.

neurosphere 분석 때문에 간단하고 재현성의 신경 줄기 세포 1-5의 격리 및 확장을 위해 선택의 방법입니다. 이 분석은 체외에서 생체내 연구에서 모두 사용할 수 undifferentiated CNS 전구체 세포의 대규모 발전을위한 소중한 도구입니다. 그것은 neurospheres는 타고난 신경 줄기 세포 및 기타 제한된 progenitors 모두에서 발생 될 수 있다고 강조해야합니다. 따라서 neurosphere 성형 주파수를 계산하...

* 앙을 만들려면, 1M HEPES의 멤 and160ml의 110L 패킷을 혼합하고 증류수를 사용하여 8.75 L로 볼륨을 가져. 7.4에 대한 최종 PH를 설정하고 그것은 4 ° C. 저장을

establishing embryonic mouse neural stem cell culture using the neurosphere assay

mammalians에서 줄기 세포는 동물의 수명 동안 여러 조직과 장기의 세포 창 및 갱신을 유지하기 위해 undifferentiated 세포의 소스 역할을합니다. 그들은 잠재적인 노화 과정에서 부상과 질병의 과정에서 길을 잃었 세포를 바꿀 수 있습니다. 신경 줄기 세포 (NSCs)의 존재가 처음 소설 혈청 무료 문화 시스템, neurosphere 분석 (NSA)를 사용하여 성인 포유류의 중추 신경계 (CNS)에 레이놀즈와 바이스 (1992)에 의해 설명되었다. 이 분석을 사용하여, 그것은 또한 배아 CNS의 다른 지역에서 NSCs을 분리하고 확장 가능합니다. 체외 확장의 NSCs에서 이들은 multipotent 있으며 CNS의 세 가지 주요 세포 유형 상승을 줄 수 있습니다. NSA가 수행하는 비교적 간단한데 동안 안정적이고 일관된 결과를 달성하기 위해 필요합니다 여기 증명 절차에주의. 이 비디오는 사실상 NSA가 14 마우스 뇌 조직을 생성하고 배아 날로부터 NSCs을 확장하는 방법을 보여줍니다 하나는 재현성 neurosphere 문화를 달성할 수 있도록 기술적인 세부 사항을 제공합니다. 프로 시저는 단일 세포 현탁액을 얻기 위해 NSC 매체에서 수확한 조직의 ganglionic eminences, 분리를 수확, 뇌 microdissection을 E14 마우스 배아를 수확하고, 마지막으로 NSA의 문화에 세포를 도금이 포함되어 있습니다. 문화 5-7일 후 결과 기본 neurospheres은 더 이후의 실험에 대한 NSCs의 수를 확장 passaged 있습니다.

Watch this Scientific Journal Video about Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay at JoVE.com

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

이 비디오 프로토콜은 배아 일 14 마우스 뇌의 ganglionic eminences에서 신경 줄기 세포의 분리 및 확장에 대한 neurosphere 분석의 응용 프로그램을 보여줍니다.

Neurosphere 분석을 사용하여 마우스 배아 신경 줄기 세포 문화 구축

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

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Neuroscience

24.5K 조회수.  Shiraz University of Medical Sciences, Shiraz, Iran. 이 비디오 프로토콜은 배아 일 14 마우스 뇌의 ganglionic eminences에서 신경 줄기 세포의 분리 및 확장에 대한 neurosphere 분석의 응용 프로그램을 보여줍니다.

비디오: Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

배아 척수에서 뉴런의 Commissural 해부와 문화

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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신경과학

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

절연 및 Neurosphere 어세를 사용하여 성인 마우스 신경 줄기 세포의 확장

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

3D 신경 줄기 세포 문화 번역하는 단백질 : 하이브리드 시각화 방법론

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

세포 어세를 형성 신경 콜로니 : 신경 전구 세포로부터 타고난 신경 줄기 세포를 차별 어세이

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

Neurotropic 마우스 간염 바이러스에 감염된 마우스의 척수 코드에 마우스 신경 줄기 세포의 이식 외과

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neuroblast 분석 : 유동세포계측법을 사용하여 차별화 신경 줄기 세포에서 자손의 연결 전구 세포의 생성과 강화에 대한 분석

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

배아 Murine 신경 튜브에서 신경 크레스트 세포의 분리 및 문화

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

신경 줄기 세포의 열거 클론 세이를 이용하여

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

마우스 뇌량 밑 영역에서의 분리 및 성인 신경 줄기 세포의 문화

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.