Bu çalışma Overstreet Vakfı parasal destek verdi.

1. Hazırlayan: Hücre Kaplama Başlamadan Önce olun gerekiyor Öğeler: <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> NeuroCult NCFC Serum-Alerjik Orta Sitokinler olmadan bir kısım çözülmüş. </li><li> Orta, 37 ° C su banyosunda ısıtılır. </li><li> Epidermal büyüme faktörü (EGF), 10 mg / ml% 0.2 ve heparin konsantrasyonu temel fibroblastik büyüme faktörü (b-FGF) Stok çözümleri önceden hazırlanır. </li><li> Deney boyutuna bağlı olarak, birkaç 35mm doku kültürü yemekleri plaka hücreleri ve bir 150-200cm plastik Petri kabı da yinelenen 35mm yemekleri tutmak için gerekli olan ve üçüncü bir su 35mm çanak ihtiyaç vardır. </li></ol> 2. Cep Hazırlanışı: Denemenizi bağlı olarak, hücrelerin embriyonik veya erişkin bir kaynak (birincil ayrışmış bir doku ya da ayrışmış neurospheres) hazırlanabilir. Yetişkin / embriyonik fare merkezi sinir sistemi (MSS) dokular disseke veya daha sonra 1,2 ve daha önce açıklandığı gibi embriyonik türetilen / yetişkin neurospheres ayrıştırmaları: <ol><li> Tek hücre süspansiyonu 40 mikron boyutlu gözenekli filtre geçirilir şekilde ayrışmış olmayan kümeleri kaldırmak. </li><li> 10μl hücre süspansiyonu, hücre sayımı gerçekleştirmek için Tripan mavi 90μl ile karıştırılır Not: Diğer uygun hücre dilüsyonları de kullanılabilir . </li><li> Eğer birincil embriyonik veya yetişkin elde edilen sinir hücreleri kullanarak, tam MGK orta 6,5 x 10 5 hücre / ml &#39;lik bir konsantrasyon hücre süspansiyonu sulandırmak. Hücreleri, yetişkin ya da embriyonik sinir hücrelerinden elde edilen ayrışmış neurospheres kullanıyorsanız, tam MGK orta 2.2 x 10 5 hücre / ml &#39;lik bir konsantrasyon hücre süspansiyonu sulandırmak . </li></ol> 3. Yarı-katı N-CFCA Orta Kaplama Hücreler: <ol><li> Aşağıdaki bileşenlerin uygun hacmi tekerrür sayısına bağlı olarak, sipariş karıştırılır. Burada çoğaltır iki için gerekli miktarı karıştırın veya yinelenen yemekleri. Çoğaltır ek numaraları için, tablo 1&#39;e bakınız. <ul><li> Sitokinler olmadan NeuroCult NCFC Serum-Alerjik Orta 1.7 mL </li><li> 330 mcL NeuroCult MGK Silahların Yayılmasını Önleme Takviyeler </li><li> 6.6 mcL EGF (10μg/mL) </li><li> Penisilin / Streptomisin 32 mcL </li><li> B-FGF (10μg/mL) 3.3 mcL hücreleri kültür yetişkin sinir hücrelerinden elde edilen sadece gerekli. </li><li> Heparin (% 0.2) 3.3 mcL hücreleri kültür yetişkin sinir hücrelerinden elde edilen sadece gereklidir. </li><li> Hücre süspansiyonu 25μL (2.2 ayrışmış neurospheres veya 6.5 x 10 5 hücre / mL birincil hücreler ayrışmış MSS dokusu x 10 5 hücre / mL hücreleri) Not: son hücre sayıları kaplama, 35 mm kültürü çanak başına yaklaşık 2500 hücre olmalıdır neurosphere edilen hücreler ve birincil hücreler için 35 mm kültür çanak başına 7500 hücreleri. Bazı durumlarda, nihai hücre kaplama yoğunluğu bir hücre titrasyon deneyi yaparak ayarlanması gerekir. 150 koloniler istatistiksel analizler için, kültür 21 gün sonra tespit edilen kolonilerin toplam sayısı en az 50 aralığında olmalıdır. </li></ul></li><li> Içeren hücreler orta 1.3 mL soğuk Kollajen çözüm hücre süspansiyonu aktarılır ve herhangi bir kabarcıkları tanıtan önlemek için yumuşak pipetleme iyice karıştırılır ve sonra hafifçe karıştırılır. </li><li> Karma çözüm her 35mm kültür çanak (~ 1.5 mL / çanak) merkezi dağıtılır ve yemekleri karışımı yemeğin yüzeyi üzerinde eşit biçimde yayılmasına izin dairesel hareketlerle hafifçe kullanarak uçlu. . </li><li> Yinelenen 35 mm kültür kaplarına 100 mm Petri plaka yerleştirilir. Yeni 35 mm çanak kapağı çıkarılır ve açık bir tabak da aynı 100 mm Petri kabı yerleştirilir. Steril su kuluçka dönemi sırasında optimum nemi korumak için, bu açık 35 mm çanak eklenir. Kare biyoassay levhalar (245 mm) 35 mm yemekleri daha çoğaltmak kurulmuş olduğunda kullanılır. Yine, 2 ya da steril su içeren 3 adet açık, 35 mm yemekleri içerir. </li><li> Plakalar 37 kuvöz ayarlamak için transfer ° C,% 5 CO2 ve% 95 nem. Kollajen congeals artan sıcaklık ve jel oluşumu tarafından yaklaşık bir saat içinde gerçekleşir. Kültürleri, bu süre içinde rahatsız olmamalıdır. </li><li> Kültüre hücreleri 21 gün (21 gün sonra koloni boyutu farklar açıkça ayırt edilebilir) boyunca inkübe edilir. </li></ol> 4. Yenileme Orta hazırlanması ve Kültür Besleme: N-CFCA kültürleri bir süre (21 gün) uzun süre inkübe olarak, kültürler uygun tam NeuroCult ile beslenmelidir ikmal orta aşağıdaki gibi taze olarak hazırlanır: <ul><li> NeuroCult MGK Bazal Orta 4.5 mL 0.5 m karışıkL NeuroCult MGK Silahların Yayılmasını Önleme Destekleri ve büyüme faktörleri sonra eklenir: </li><li> 250 mcL 10μg/mL EGF (0.5 mikrogram / ml EGF nihai konsantrasyonu vermek için) </li><li> 10μg/mL b-FGF (0.25μg/mL b-FGF final konsantrasyon vermek için) 125 mcL hücreleri kültür yetişkin sinir hücrelerinden elde edilen sadece gerekli. </li><li> % 0.2 Heparin 125 mcL, kültür hücreleri, yetişkin sinir hücrelerinden elde edilen sadece gerekli . </li><li> Uygun NeuroCult Yenileme Orta (embriyonik veya erişkin hücreler için) 60 mcL tüm NCFC Testi kültürü inkübasyon (21 gün) boyunca her 7 günde bir her NCFC Testi çanak merkezine eklenir. </li></ul> 5. N-CFC Testi Türetilmiş Kolonileri Puanlama ve Bona Fide NSC&#39;lerde ve Sinir Progenitör Hücreler Frekans hesaplanması: <ul><li> Her 35 mm kültür çanak ızgara boyutu ile 2.0 mm x 2.0 mm ızgara puanlama tabağına yerleştirilir ve sonra her iki yemekleri mikroskop aşamasında yerleştirilir. </li><li> Objektif lens, her bir çanak taranır ve koloniler kendi boyutlarına göre puanlanır (5X 2.5X) düşük güç kullanma. </li><li> Kolonileri iki ana kategoriye ayrılır: <ol><li> 2 mm çapında daha az </li><li> ≥ 2 mm çaplı </li></ol></li></ul> 6. Temsilcisi Sonuçlar: Kaplama 7 gün sonra (Şekil 1) - neurosphere tayininde, N-CFC tayininde kaplama hücreleri prolifere hücre küçük koloniler 3 gün içinde yapmak için başlayın. Iki hafta içinde, farklı boyutlarda koloni ayırt edilebilir. 14 gün sonra büyüyen kolonilerin çoğunluğu durdurmak için eğiliminde iken, bazı koloniler boyutu artmaya devam. 1 mm çapında, 3) 1 - 2 mm çapında ve 4) ≥ 2mm çapında en az 0,5 mm çapında, 2) 0.5) 1: 21 gün, koloniler dört kategoriden birine sınıflandırılır olabilir. Uygulamada, 2 mm çapından daha küçük koloniler progenitör türetilmiştir olarak adlandırılır (Şekil 2) ve MGK (Şekil 3) türetilen koloniler çapı ≥ 2mm adlandırılır. Kaplama toplam hücre ortalama çapı ≥ 2mm kolonilerin sayısı, uzun vadeli kendini yenileyen ve çok potansiyel yetenekleri ile gerçek bona fide sinir kök hücrelerinin sıklığı temsil eder. Paralel bir NSA deneyde 7-8 gün sonra belirli bir hücre popülasyonu frekans oluşturan toplam neurosphere N-CFCA deneyde 21 gün sonra aynı hücrede nüfus sıklığı toplam koloni oluşturan benzer olması tahmin ediliyor. N-CFCA Ancak, her bir hücrenin tam proliferatif potansiyeli gösterebilir kadar, daha müsamahakâr bir durum sağlar. <table border="1"><tbody><tr><td> Bileşen </td><td> 2 çoğaltır </td><td> 3 çoğaltır </td><td> 4 çoğaltır </td></tr><tr><td> Sitokinler olmadan NeuroCult NCFC Serum-Alerjik Orta </td><td> 1700 </td><td> 2550 </td><td> 3400 </td></tr><tr><td> NeuroCult MGK Silahların Yayılmasını Önleme Takviyeler </td><td> 330 </td><td> 495 </td><td> 660 </td></tr><tr><td> EGF (10 mg / ml) </td><td> 6,6 </td><td> 9,9 </td><td> 13,2 </td></tr><tr><td> Sadece yetişkin hücreler için bFGF (10 mg / ml), </td><td> 3,3 </td><td> 4,95 </td><td> 6,6 </td></tr><tr><td> Heparin sadece yetişkin hücreler için Çözüm (% 0.2), </td><td> 3,3 </td><td> 4,95 </td><td> 6,6 </td></tr><tr><td> Penisilin / Streptomisin (1:100) </td><td> 32 </td><td> 48 </td><td> 64 </td></tr><tr><td> Az Hücreler: 2.2 x 10 5 kültürlü hücre / ml VEYA 6.5 x 10 5 birincil hücre / ml </td><td> 25 </td><td> 37,5 </td><td> 50 </td></tr><tr><td> Kollajen Çözüm </td><td> 1300 </td><td> 1950 </td><td> 2600 </td></tr></tbody></table> Tablo 1 eksiksiz bir N-CFC tahlil kültür Bileşenleri. <img src="/files/ftp_upload/2639/2639fig1.jpg" alt="Şekil 1" /> Şekil 1. geçit bir E14 fare NSC&#39;lerde N-CFCA kültür Temsilcisi koloniler 7 gün sonra kaplama. Koloniler farklı morfoloji ve boyutunu görüntülemek olabilir. Orijinal büyütme; 4x <img src="/files/ftp_upload/2639/2639fig2.jpg" alt="Şekil 2" /> Şekil 2 N-CFCA pasaj kültürünün farklı boyutları kaplama 21 gün sonra bir E14 fare NSC&#39;lerde Temsilcisi progenitör elde edilen koloniler. Gösterildiği gibi, farklı koloniler morfoloji var ama boyutu az 2 mm. Orijinal büyütme; 4x <img src="/files/ftp_upload/2639/2639fig3.jpg" alt="Şekil 3" /> Şekil 3. Temsilcisi bona fide kök hücre kaplama 21 gün sonra geçiş bir E14 fare NSC&#39;lerde N-CFCA kültüründe koloni türetilmiştir. Kök hücre elde edilen koloniler farklı morfoloji gösterebilir (bkz.video) ama çapı ≥ 2mm. Orijinal büyütme; 4x

<ol>
	<li>Azari, H., Rahaman, M., Sharififar, S., Reynolds, B. A. <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>Azari, H., Sharififar, S., Rahaman, M., Ansari, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J Vis Exp. , (2011).</li><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>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. , (2008).</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>Louis, S. A., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosphere+and+Neural+Colony-Forming+Cell+Assays.">Neurosphere and Neural Colony-Forming Cell Assays.</a> Protocols for Neural Cell Culture. 10, 1-28 (2010).</li></ol>

Yazarlardan biri, Sharon A. Louis StemCell Technologies Inc, bu çalışmada kullanılan bazı reaktiflerin bir yapımcı ile bağlanmıştır.

Neurosphere tahlil 3,1,2, yetişkin ve embriyonik MSS dokusu gibi çeşitli kaynaklardan sinir kök hücreler izole etmek ve genişletmek için en yaygın yöntem olmakla birlikte, doğru nöral prekürsör hücrelerinin karışık bir nüfus MGK frekansını ölçmek değildir (kök ve atalarıdır) neurospheres sayısı ve bona fide kök hücreler 4 sayısı 1-1 arasında bir ilişki yok gibi. Bu sınırlama gidermek için, orijinal NSA nöral kök ve atalarıdır 5-7 üç hafta boyunca tam proliferasyon kapasitesi büyümeye izin verecek şekilde adapte olmuştur. Sıvı NSA aksine, yarı katı kollajen matriks koloniler tek kaplama hücreleri ve agregasyonu göç engeller N-CFCA koloniler aslında klonal, elde edilir. Bu testte tutarlı sonuçlar için bizim önerimiz: <ol><li> Orijinal hücre süspansiyonu tek bir hücre süspansiyonu olun. Ayrışmış olmayan kümeleri kaldırmak şekilde 40 mikron boyutlu gözenekli filtre ile tek hücre süspansiyonu iletin. </li><li> Kollajen çözüm buz üzerinde veya +4 ° C&#39;de buzdolabında tutun. Kollajen sıcaklığı artırarak congeals hücre süspansiyonu karışımı eklenebilir son öğe. </li><li> Her hafta kültürü beslemek için ihmal etmeyin. Her 7 günde bir büyüme faktörü (ler) içeren ikmal orta ekleme hücreleri genişletilmiş kültürü süre içinde yayılmaya devam etmektedir sağlayacaktır. </li><li> Son hücre kaplama yoğunluğu, primer veya kültür sinir hücreleri elde edilen hücreler için optimize edilmiştir, böylece kültürün 21 gün sonra tespit edilen koloniler toplam sayısı NCFC aralığında en az 50 - 200 koloniler. Bu aralık, istatistiksel analizler için yeterli sayıda kolonilerin sağlarken örnekleri çapında nadir MGK elde edilen koloniler&gt; 2mm algılanmasını sağlar. Başlangıç ​​hücre kaynağına bağlı olarak, önemli ölçüde daha az (&lt;50) ve kolonilerin sayısı daha yüksektir (&gt; 250), bazen elde edilir. Pek çok koloniler yanlış bir koloni sayımı sonucunda büyüme orta bileşenlerinin aşırı kalabalık anddepletion neden olurken, çok az koloniler, çapı tespit seviyelerinin altında koloniler&gt; 2mm neden olacaktır. Bu nedenle hücre kaplama yoğunluğu buna göre ayarlanır gerekecektir (yani toplam hücrelerin kaplama sayısı artan ya da azalan) </li></ol>

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>NeuroCult NSC Basal Medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05700</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NSC Proliferation Supplements</td>
<td>Medium supplement</td>
<td>STEMCELL</td>
<td>05701</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NCFC medium</td>
<td>Medium</td>
<td>STEMCELL</td>
<td>05720</td>
<td>&nbsp;</td>
<tr>
<td>0.05% 	trypsin-EDTA</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25300-062</td>
<td>&nbsp;</td>
<tr>
<td>Soybean trypsin inhibitor </td>
<td>Reagent</td>
<td>Sigma</td>
<td>T6522</td>
<td>&nbsp;</td>
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<td>Cell strainer </td>
<td>Sieve</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep </td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>T25 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>136196</td>
<td>&nbsp;</td>
<tr>
<td>T80 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>178905</td>
<td>&nbsp;</td>
<tr>
<td>Collagen</td>
<td>Reagent </td>
<td>STEMCELL</td>
<td>04902</td>
<td>&nbsp;</td>
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<td>EGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>2028-EG</td>
<td>&nbsp;</td>
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<td>b-FGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>3139-FB</td>
<td>&nbsp;</td>
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<td>Heparin</td>
<td>Growth factor</td>
<td>Sigma</td>
<td>H4784</td>
<td>Reconstituted in PBS</td>
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<tr>
<td>15 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>50 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>35 mm culture dishes</td>
<td>Culture ware</td>
<td>STEMCELL</td>
<td>27100</td>
<td>&nbsp;</td>
<tr>
<td>Gridded scoring dishes</td>
<td>Culture ware</td>
<td>STEMCELL</td>
<td>27500</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

neural-colony forming cell assay: an assay to discriminate bona fide neural stem cells from neural progenitor cells

Neurosphere assay (NSA), izole, genişletmek ve aynı zamanda nöral kök hücreler (NSC&#39;lerde) sıklığını hesaplamak için en sık kullanılan yöntemlerden biridir. Ayrıca, bu serum serbest kültür sistemi kök hücrelerin çeşitli tümörler ve normal dokuları genişletmek ve sıklığını belirlemek için istihdam edilmiştir. Neurosphere oluşumu ve NSC&#39;lerde arasında bire bir ilişki yok son zamanlarda kanıtlanmıştır. Bu NSA gibi şu anda uygulanan nöral prekürsör hücrelerin embriyonik ve yetişkin memelilerin beyin hem izole karışık bir nüfus NSC&#39;lerde frekans olduğundan yük olduğunu göstermektedir. Bu video neredeyse yarı-katı test tabanlı bir roman kollajen gösterir, sinir koloni böylece uzun vadeli proliferatif potansiyeli dayalı, progenitör hücrelerden kök ayırt etmek yeteneğine sahiptir ve hücre çalışması (N-CFCA) oluşturan bir yöntem sağlar MGK frekans numaralandırmak. N-CFCA, koloniler &lt;2mm atalarıdır türetilmiş ise ≥ 2 mm çapında koloniler MGK tüm fonksiyonel kriterleri karşılayan hücreler türetilmiştir. N-CFCA prosedürü birincil ve kültürlü bir yetişkin ya da embriyonik fare merkezi sinir sistemi hücreleri dahil olmak üzere farklı kaynaklardan hazırlanan hücreler için kullanılabilir. Burada geçit embriyonik gün 14 farelerin beyin üretilen N-CFCA gerçekleştirmek için bir neurospheres hazırlanan hücreleri kullanır. Kültürlerin çoğalması orta ile kaplı hücreler tam proliferatif potansiyeli sergilemek için izin ve daha sonra nöral progenitör ve iyi niyetli sinir kök hücrelerinin sıklığı sırasıyla &lt;2mm kolonilerin sayısı sayarak hesaplanır üç hafta boyunca her yedi günde bir yenileniyor ve ≥ 2mm başlangıçta kaplama hücrelerinin sayısı referans olanlar.

Watch this Scientific Journal Video about Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells at JoVE.com

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

Bu video protokolü iyi niyetli nöral kök hücreler nöral prekürsör hücrelerin koloni oluşturan nöral hücre çalışması ile oluşan karışık bir nüfus nasıl ayrımcılık ve numaralandırmak göstermektedir.

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). ...

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Research

JoVE Journal

Neuroscience

22.0K Görüntüleme.  University of Florida. Bu video protokolü iyi niyetli nöral kök hücreler nöral prekürsör hücrelerin koloni oluşturan nöral hücre çalışması ile oluşan karışık bir nüfus nasıl ayrımcılık ve numaralandırmak göstermektedir.

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

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 ...

Nörobilim

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

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 span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

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 ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Bozulmamış Duyu Organ Öncü Hücreler Canlı hücre Görüntüleme Drosophila Pupa

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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 ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

İnsan Pluripotent dan Sinir Crest Progenitör Hücreler besleyici ücretsiz Derivasyon Kök Hücre

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Hücre Yetişkin Fare subventricular Bölgesinden Nöral Kök ve Progenitör Hücreler Sıralama ve Hücre Döngüsü Dynamics Canlı görüntüleme

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

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. ...

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.

Sıçan gözlerinin içine insan retina Pigment epitel hücrelerinin Trans-scleral Subretinal nakli için rafine bir protokol geliştirilmesi

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Primer Nöral Kök ve Progenitor Hücrelerinin Hücre Yüzey Belirteçlerinin Sialoglycan'ın Metabolik Etiketlemesi ile Belirlenmesi

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...