Bu çalışma Overstreet Vakfı parasal destek verdi.

1. Diseksiyon için Başlamadan Önce Temel Set Up: <ol><li> Tam MGK orta Uygun hacmi, 09:01 oranında NeuroCult MGK Bazal Orta ve NeuroCult MGK Silahların Yayılmasını Önleme Takviyeler sırasıyla karıştırılarak hazırlanır. </li><li> Orta, 37 ° C su banyosunda ısıtılır. </li><li> Antibiyotik yüksek konsantrasyonda (% 10) Soğuk Hepes-tamponlu asgari temel orta (HEM), diseksiyon ve yıkama amacı için hazırlanmıştır. Alternatif olarak, antibiyotik takviyesi ile MGK bazal orta de bu amaç için kullanılan olabilir. </li><li> 25-30 ml antibiyotik içeren soğuk HEM ​​embriyoların toplanması için 50 mL steril tüpler içine sürülür. </li><li> Birkaç 10cm plastik Petri kutularına Diseksiyon sırasında embriyoların ve beyinleri tutmak için gerekli olan ve aynı zamanda doku disseke tutun. </li><li> Embriyolar (büyük makas, küçük sivri makas, büyük forseps, küçük kavisli bir forseps) veya embriyonik beyin diseksiyon (küçük forseps, kıvrımlı ince forseps, 45 ° açılı ince forseps, küçük makas) kaldırmak için gerekli cerrahi aletler kullanılarak sterilize edilir cam boncuk sterilizatör 250 ° C veya diğer mevcut otoklav yöntemleri. </li><li> Diseksiyon mikroskobu,% 70 alkol ile sildi ve laminer akış veya PC2 kaput içinde kurulur. </li></ol> 2. E14 Fare Beyin ve Mikro-disseksiyon Hasat: <ol><li> Hamile fare evlendirilen bir süre günde bir kurumsal onaylı hayvan protokolü göre 14. gebelik uyuşturulduktan. </li><li> Servikal dislokasyon hayvanın acı ve sıkıntı zarar vermez emin olmak için yapılır. </li><li> Anestezi fare, emici bir kağıt mendil sırtını üzerine koydu ve karın alan sterilize etmek için% 70 etanol ile yıkanır. </li><li> Cilt karın üzerinde büyük bir forseps kullanarak kavradı ve sonra, cilt ve altta yatan fasya, karın boşluğu ve rahim boynuzları ortaya çıkarmak için büyük bir makas ile kesilir. </li><li> Uteri küçük forseps ve makas ile kaldırılır ve 50 ml konik tüp içeren soğuk HEM ​​aktarılır. </li><li> Rahim dokuları Kaputun aktarılır ve daha sonra kan ve saç gibi olası kirleri çıkarmak için bir veya iki kez taze steril soğuk HEM ​​yeterli hacmi ile durulanır. </li><li> Rahim dokuları sonra 10cm Petri kabı soğuk HEM ​​içeren bir transfer edilir. </li><li> Uterus boynuzları küçük kavisli bir forseps ve makas kullanılarak açılır ve soğuk HEM ​​içeren yeni bir 10cm çanak, embriyolar transfer edilir. </li><li> Embriyoların başkanları servikal spinal kord düzeyinde ayrılmış ve başka bir Petri kabı soğuk HEM ​​içeren transfer. </li><li> Petri kabı kafatası beyin kaldırmak için diseksiyon mikroskop altında aktarılır. </li><li> Kafaları başkanlarının dorsal tarafı yukarı bakacak şekilde kaudal tarafında ince kavisli bir forseps ile yapılmaktadır. Kullanarak mikro-makas, gözleri üzerinde ilk yatay bir kesim yapılmış ve daha sonra başın arkasına doğru alında orta hatta devam etti. Deri ve kafatası kesti ve altta yatan bir beyin hasarı değil emin olun. </li><li> Beyin kafatası kesik bölümünün kenarları kavisli bir forseps kullanarak geriye doğru hareket basarak kaldırılır. Bu prosedür tüm beyinleri kafatası kaldırılıncaya kadar devam eder. </li><li> Beyin dorsal tarafına bir kesim koku ampuller ganglion eminences maruz yarımkürenin arkasına uzanan her yarımkürenin korteks ile gerçekleştirilir microscissors kullanarak yukarı ve ardından bakacak şekilde kavisli bir forseps kullanarak sabit tutulur. </li><li> Bir yandan kavisli bir forseps ve diğer 45 ° açılı forseps kullanarak, korteks kesme flep yayılmış ve ganglion eminences disseke vardır. </li><li> Disseke doku steril bir 10cm Petri kabı yerleştirilir ve tüm beyinleri mikro-disseke kadar bu işlem tekrarlanır. </li><li> Doku parçaları MGK orta 1 ml 1 ml pipet kullanılarak toplanan ve 15 ml santrifüj tüpüne aktarılır. </li><li> Doku sonra iyice ayrışmış, ama nazikçe pipet tüpün dibine basarak ve süspansiyon yukarı ve aşağı pipetleme. Bu şekilde tek bir hücre içine yığınları kırık. Pipetleme sütlü gibi bir süspansiyon elde edilir, böylece 3-4 kez yapılır. Daha sonra, süspansiyon 1-2 dakika kadar ayrışmış olmayan kümeleri çökeltileri yerleşmek için izin verilir. </li><li> Hemen hemen tüm hücre süspansiyonu diğer tüpe aktarılır ve daha sonra başka bir 1 ml MGK orta açıklandığı gibi kalan tek bir hücre kümeleri ve ayrışmış eklenir. </li><li> Tüplerin içeriği 700Rpm (110g), oda sıcaklığında 5 dakika boyunca toplanmış ve santrifüj edilir. </li><li> Süpernatant aşağı gerçek pelet kapalı vakumlu, ve hücrelerin tam MGK orta 1 mL yeniden süspanse. </li><li> Orta hafifçe aşağı yukarı pipetlenir.homojen bir tek hücre süspansiyonu var. </li><li> Hücre süspansiyonu 10 mcL bir hücre sayımı gerçekleştirmek için 90 tripan mavi mcL ile karıştırılır. </li><li> Son olarak, hücreler 2x10 5 hücre / mL 20ng / ml epidermal büyüme faktörü (EGF) ile desteklenmiş tam MGK orta yoğunlukta kaplanmıştır. 5 ml orta, T25, T75 ve T175 şişeler için 40 ml 20 ml için kullanılır. </li><li> 37 ° C,% 5 ile nemlendirilmiş bir inkübatör CO 2 inkübe Neurospheres 5-7 gün içinde oluşur . 6-7 gün içinde, küreler 150-200 mikron arasında ölçmelidir ve altkültürü (geçit) hazır olacaktır. </li></ol> 3. Embriyonik NSC&#39;lerde Pasajlanması ve genişletilmesi: <ol><li> Neurospheres altkültürü (150-200 mikron çapında) için hazır olduğunuzda, orta askıya küreler, şişeler kaldırılır uygun bir boyut steril doku kültürü tüp yerleştirilir ve 700 rpm (110 g) 5 dakika santrifüj oda sıcaklığında bekletin. </li><li> Süpernatant atılır ve küreler 1 mL% 0.05 tripsin-EDTA yeniden süspanse. </li><li> Hücre süspansiyonu daha sonra 2-3 dakika süreyle 37 ° C su banyosunda inkübe edilir, sonra soya tripsin inhibitörü eşit miktarda tripsin faaliyeti durdurmak için kullanılır. </li><li> Hücre süspansiyonu tripsin tamamen inaktive olduğundan emin olmak için hafifçe aşağı yukarı pipetlenir ve. </li><li> Hücre süspansiyonu, 5 dakika süreyle 700 rpm (110g) santrifüj edilir. Sonra süpernatantı kaldırılır ve hücrelerin tam MGK orta 1 mL yeniden süspanse. </li><li> Hücre süspansiyonu 10 mcL bir hücre sayımı gerçekleştirmek için 90 tripan mavi mcL ile karıştırılır. </li><li> Hücreleri uygun bir boyut doku kültürü şişesi 20ng / ml EGF ile desteklenmiş tam MGK orta 5x10 4 hücre / mL konsantrasyonda kaplanmıştır. 5 ml orta, T25, T75 ve T175 şişeler için 40 ml 20 ml için kullanılır. </li><li> 37 ° C,% 5 ile nemlendirilmiş bir inkübatör CO 2 inkübe İkincil neurospheres 5-7 gün içinde oluşur . </li></ol> 4. Temsilcisi Sonuçlar: Birincil embriyonik MGK kültürü, hücrelerin çoğunluğunu hipertrofik olacak ve kaplama üzerine doku kültürü yemekleri ekleyin. Hücrelerin çoğunluğu ya ölür ya da 2-3 gün sonra, ayırt ederken, proliferatif hücreler substrat (Şekil 1) ayırmak hücrelerinin küçük kümeler olun. Kültürün ilk 48 saat içinde büyük sfero agrega oluşumu primer küreler için yanlış olmamalıdır. Agrega oluşumu ağırlıklı olarak enkaz ve kültürü olmayan ayrışmış doku kümeleri tutarlar bağlıdır. Gerçek neurospheres faz parlak ve boyutunu artırır (Şekil 2) gibi daha küresel hale. Küçük microspikes sağlıklı ve canlı küre (Şekil 3) dış yüzeyinde görünür. 5-7 gün sonra, küreler yuvarlak olabilir ama sıkıştırılmış değil; ve çapı 150 ila 200 mikron ölçmelidir. Neurospheres (9-10 gün sonra kültür) çok büyük büyümek için izin verilirse, onlar agrega form veya küre merkezinde (video) hücre ölümü nedeniyle koyu renkli olabilir. Büyük neurospheres sonunda in situ (substrat bağlama ve çevre doğru göç) ayırt etmek için başlayabilir. Aynı zamanda büyük neurospheres ayırmak zordur ve bunları altkültürü. <img src="/files/ftp_upload/2457/2457fig1.jpg" alt="Şekil 1" /> Şekil 1. İlköğretim E14 MGK kültür kaplama 3 gün sonra. Oklar NSC&#39;lerde prolifere kümeleri göstermektedir. Orijinal büyütme; 20x <img src="/files/ftp_upload/2457/2457fig2.jpg" alt="Şekil 2" /> Şekil 2 İlköğretim E14 MGK kültür kaplama 7 gün sonra. Orijinal büyütme; 10x <img src="/files/ftp_upload/2457/2457fig3.jpg" alt="Şekil 3" /> Şekil 3 Passage bir E14 neurospheres 5 gün sonra kaplama. Küreler çevresindeki mikro-ani (oklar) dikkat edin. Orijinal Büyütme; 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>

Neurosphere tahlil sadeliği ve tekrarlanabilirlik nedeniyle nöral kök hücreler 1-5 izolasyonu ve genişleme için tercih edilen bir yöntemdir. Bu testte hem de in vitro ve in vivo çalışmalar için kullanılan olabilir farklılaşmamış MSS öncü hücreler, büyük ölçekli üretimi için paha biçilmez bir araçtır. Neurospheres iyi niyetli nöral kök hücreler ve daha sınırlı atalarıdır hem olabileceği vurgulanmalıdır. Bu nedenle, neurosphere oluşturan frekans hesaplanırken, sadece iyi niyetli NSC&#39;lerde herhangi bir sinir hücresinin nüfusu 6 olduğundan yük . Iyi niyetli NSC&#39;lerde sıklığını tahmin etmek, bu amaçla 7 için geliştirilmiştir Hücre Testi Şekillendirme Sinir Colony (N-CFCA) kullanılması tavsiye edilir. E14 NSC&#39;lerde tutarlı bir yüksek kalite neurosphere kültüre sahip olması için, bizim önerimiz: <ol><li> Embriyolar hasat başlamadan önce gerekli öğeleri hazırlamak için. </li><li> Diseksiyon boyunca soğuk HEM ​​veya bazal MGK orta embriyolar tutmak için. </li><li> 1 saat içinde mümkün olduğunca çabuk diseksiyon için, doku zamanla yumuşak ve yapışkan hale gelir ve incelemek için zor olabilir. </li><li> Alanında çok fazla büyümesine izin vermemek. Genellikle küre büyüklüğüne bağlı olarak, alt-kültür her 5-7 gün olmalıdır. </li><li> 2-3 dakika boyunca 150-200 mikron boyutunda küreler trypsinize. 3 dakikadan daha fazla tripsin bırakmak hücrelere zarar verir ve küre oluşturan etkinliği azalacak ve hücrelerin kültür kaplarına takın ve ayırt etmek eğiliminde olacaktır. </li></ol>

* HEM yapmak için, 110L paket 1M Hepes MEM and160ml karıştırın ve distile su kullanılarak 8.75 L hacmi getirmek. 7.4 nihai PH ayarlama ve 4 saklamak ° C

establishing embryonic mouse neural stem cell culture using the neurosphere assay

Memelilerde, kök hücreleri farklılaşmamış hücreler, hayvanın ömrü boyunca, farklı doku ve organlarda hücre doğuşu ve yenileme korumak için bir kaynak olarak görür. Bu potansiyel yaşlanma sürecini veya yaralanma ve hastalık sürecinde kaybolur hücreleri değiştirebilirsiniz. Nöral kök hücrelerin varlığı (NSC&#39;lerde) ilk roman serum serbest bir kültür sistemi, neurosphere assay (NSA) ile yetişkin memelilerin merkezi sinir sistemi (MSS) Reynolds ve Weiss (1992) tarafından tarif edilmiştir. Bu testte, aynı zamanda, farklı bölgelerinden embriyonik MSS NSC&#39;lerde izole etmek ve genişletmek için uygulanabilir. Bu in vitro genişletilmiş NSC&#39;lerde multipotent ve MSS üç önemli hücre tiplerinin ortaya çıkmasına neden olabilir. NSA gerçekleştirmek için nispeten basit olmakla birlikte, güvenilir ve tutarlı sonuçlar elde etmek için gerekli Burada gösterilen usul dikkat. Bu video pratik NSA 14-fare beyin dokusu oluşturmak ve embriyonik günden itibaren NSC&#39;lerde genişletmek gösterir ve teknik detaylar bir tekrarlanabilir neurosphere kültürleri elde edebilirsiniz sağlar. Bu prosedür, tek bir hücre süspansiyonu elde etmek için MGK orta hasat doku ganglion eminences, disosiyasyon hasat, beyin mikrodiseksiyon E14 fare embriyoları, hasat ve son olarak NSA kültür hücreleri kaplama içerir. Kültür 5-7 gün sonra, ortaya çıkan birincil neurospheres daha sonraki deneyler için NSC&#39;lerde sayısını artırmak için geçişli.

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

Bu video protokolü, embriyonik gün 14-fare beyninde ganglion eminences nöral kök hücrelerin izolasyonu ve genişleme için neurosphere testinin uygulama göstermektedir.

Neurosphere Testi kullanarak Embriyonik Fare Nöral Kök Hücre Kültürü oluşturulması

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 ...

establishing-embryonic-mouse-neural-stem-cell-culture-using

Nanyang Technological University

Research

JoVE Journal

Neuroscience

24.6K Görüntüleme.  Shiraz University of Medical Sciences, Shiraz, Iran. Bu video protokolü, embriyonik gün 14-fare beyninde ganglion eminences nöral kök hücrelerin izolasyonu ve genişleme için neurosphere testinin uygulama göstermektedir.

Video: 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.

Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

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

Nörobilim

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.

Neurosphere Testi Yetişkin Fare Nöral Kök Hücre İzolasyonu ve Genişleme

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 Nöral Kök Hücre Kültürü yerelleştirme Protein: Hybrid Görselleştirme Metodolojisi

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.

Hücre Testi Şekillendirme Sinir-Koloni: Sinir Progenitör Hücreler Bona Fide Sinir Kök Hücreler Ayırım Bir Testi

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.

Spinal Kordonlar nörotropik Mouse Hepatit Virüs ile Etkilenmiş Fare Fare Nöral Kök Hücre Transplantasyon Cerrahi

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

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Neurosphere Testi İnsan Glioblastoma Multiforme Tümör Hücreleri İzolasyon ve Genişleme

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

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 Test: Akım Sitometri Kullanımı Farklılaşan Nöral Kök Hücre Döl nöronal Progenitör Hücrelerinin üretilmesi ve zenginleştirilmesi için bir Testi

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.

Embriyonik Mürin Nöral Tüp çıkan sinirsel Crest Hücre İzolasyonu ve Kültür

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

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

F9 NADİR-LacZ Hücre tabanlı Muhabir Test kullanma E8.5 Embriyolar ve Neurosphere Kültürleri Bağıl Retinoik Asit Düzeylerinin Kantitatif Ölçümü

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

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.

Nöral Kök Hücre Sayım klonal Tahliller Kullanma

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