Name: isolation and expansion of adult canine hippocampal neural precursors
Uploaded: 2016-11-29T15:00:00.000+00:00
Duration: 9 min 37 s
Description: Watch this Scientific Journal Video about Isolation and Expansion of Adult Canine Hippocampal Neural Precursors at JoVE.com

This work was supported by the National Health and Medical Research Council (NHMRC) of Australia grants (#568969 and 1004152).

ニューサウスウェールズ州、オーストラリアの法律によれば、 死後の脳組織は、研究とは無関係の理由のために安楽死させた成犬から取得しました。 培養培地の調製<ol><li> 100mlの蒸留水にゼラチン粉末を0.1gを添加し、溶解するまで37℃で攪拌し、0.1％のゼラチン溶液を調製します。 15分間のUV照射により溶液を滅菌します。このメディアは、その後1ヶ月まで4℃で保存することができます。 </li><li> DMEMの500ミリリットル（4.5グラム/ LのD-グルコース）にF-12の167ミリリットルを追加することで、ダルベッコ改変イーグル培地（DMEM）と栄養素混合F12の1の比率：3を準備します。 （最終濃度1％のための）ペニシリン/ストレプトマイシン溶液を6.7ミリリットルを追加します。このメディアは、最大1ヶ月間、4℃で暗所に保存することができます。 </li><li> DMEM（4.5グラム/ LのD-グルコース）、440ミリリットルを組み合わせることによって血清培地を500mlを調製5 mlのL-アラニル-L-グルタミンジペプチド（200ミリモル）、ペニシリン/ストレプトマイシン5mlの胎児bovin 50mlのE血清（FBS）。このメディアは、最大1ヶ月間、4℃で暗所に保存することができます。 </li><li>完全増殖培地が幹細胞の神経から成る（NSC）基礎培地および増殖神経補足-A（NS-A）、2％FBS、2 / mlのヘパリン、20 ngの/ mlの上皮成長因子（EGF）および10ng / mLの塩基性線維芽細胞増殖因子（bFGF）。このメディアは、脳の解剖の直前に、新鮮な、そして水浴中で37℃まで暖かくしてください。 </li></ol> 2.脳の抽出<ol><li>ポビドンヨードを使用して、犬の頭の背側表面を滅菌することにより6時間の死後内の抽出を開始します。 </li><li>メスを使用して、基礎となる咬筋を露出するように、頭蓋の全体背面全体に、矢状正中線に沿って縦切開を行います。このカットの長さは、犬種や年齢によって異なります。 </li><li>頭蓋の吻側および尾側の国境では、追加の5作る - 両側に10センチメートル垂直カットを、渡します皮膚が完全に反映させることができるようにするために、それぞれの目と耳の後ろる。 </li><li>メスを用いて、頭蓋骨の矢状稜への愛着から咬筋を分離し、頭蓋から任意の残りの結合組織をこすり。 </li><li>振動骨を使用すると、円周線で頭蓋骨のキャップをカットしました。プローブまたはメスを用いて硬膜と、残りの添付ファイルを切断してから、慎重に脳の背側表面を露出させるために頭蓋骨のキャップを外します。 注意：骨の厚さは、頭蓋骨内の異なる領域で変化するので注意があまりにも深く浸透し、下層の脳に損傷を与えないように注意が必要。 </li><li>上部頸椎の間にメスを挿入することにより、脊髄を切断。 </li><li>優しく脳を持ち上げて片手で、慎重に頭蓋窩から脳を解放し、その腹側に位置する接続神経や血管を切断するためにメスを使用しています。静かにBを持ち上げます頭蓋骨の外とDPBSの容器に雨。 注意：犬の脳の大嗅球は、前頭蓋窩の中に深く延びてもよいです。脳を除去する際にこれらの構造を保存するように注意を払う必要があります。 </li></ol> 3.海馬解剖<ol><li> 150ミリメートルペトリ皿の上に任意の血液を除去し、下に背側に配置するダルベッコのリン酸緩衝生理食塩水（DPBS）で脳をすすぎます。 </li><li>ゆっくりと湿った脳ナイフとブレードの全長を利用した単一のスライスを使用して、ミッド矢状面を通って長手方向に脳を二等分します。これは、組織に損傷を与える可能性がある、追加の下方圧力を適用するか、ソーイングモーションを使用しないでください。 </li><li>各半球の内側の面を上に配置し、メスと細かいピンセットを用いて、海馬を解剖し、35mm組織培養皿に移します。 </li></ol>神経前駆細胞の単離4 <ol><li>トンをミンチ約1mm 3の小片に手術用メスの刃を使用して、問題のサンプル。 </li><li> 、15mlチューブに組織を移す0.1％トリプシンEDTAを2mlを加え、37℃の水浴中で7分間インキュベートします。 </li><li>チューブに血清培地を4mlを添加することにより酵素反応を停止させます。 7分間100×gで遠心分離することによって、懸濁液をペレット化し、上清を除去します。 </li><li> DPBS300μlの中でペレットを再懸濁し、機械的に軽く滑らかなホモジネートが形成されるまで、千μlのピペットチップを使用して、上下にピペッティングにより解離しました。 </li><li>チューブに血清培地の14ミリリットルを追加し、40μmのセルストレーナーを介して細胞懸濁液を渡します。 、7分間100×gで遠心分離することによって、懸濁液をペレット上清を除去し、完全増殖培地中に再懸濁します。 </li></ol> 5.ニューロスフェアの文化<ol><li>分離で得られた細胞懸濁液から、トリパンブルー色素排除およびHEMOを使用して生細胞の数を数えますサイトメーター。 </li><li> 6ウェルプレートのコーティングされていないウェルに1×10 5細胞/ cm 2で完全増殖培地と種子中の細胞懸濁液を希釈します。 </li><li> 37℃、5％CO 2及び14のための&gt; 95％の湿度でインキュベート- 28日、7日ごとに成長培地の50％を置き換えます。定期的にニューロスフェアの直径を測定します。直径100μmのを超えて成長させた場合、細胞死および自発的分化は、神経球内で発生する可能性があります。 </li></ol> 6.ニューロスフェアパサージュ<ol><li>浮遊培養として継代に、ニューロスフェアは、直径が約100μmに達すると、単一の管にメディアを含むすべての神経球を兼ね備えています。 7分間100×gで遠心分離することによってペレット化し、上清を除去します。 </li><li> 0.1％トリプシンEDTA 1mlにペレットを再懸濁し、37℃の水浴中で7分間インキュベートします。穏やかdissociatiを確実にするために、200μlのピペットチップを使用して、下の100倍をピペットニューロスフェアの上。 </li><li> 7分間、100×gでチューブを遠心分離し、血清培地を5mlを加えることにより酵素反応を停止させます。 </li><li> 、上清を除去し1mlの完全増殖培地を細胞ペレットを再懸濁し、トリパンブルー色素排除および血球計を用いて生存細胞の数を数えます。 </li><li> 6ウェルプレートのコーティングされていないウェルに1×10 5細胞/ cm 2で完全増殖培地と種子中の細胞懸濁液を希釈します。 7日ごとに成長培地の50％を置き換える、28日- 14を37℃、5％CO 2、&gt; 95％の湿度でインキュベートします。 </li></ol> 7.接着培養神経前駆細胞の<ol><li>接着培養のためのニューロスフェアを処理する前に1時間37℃のインキュベーターで組織培養皿及び硬化の表面を覆うように、0.1％ゼラチン溶液の十分な量を追加します。 </li><li>浮動ニューロスフェア培養物から、単一のチューブの中にすべてのメディアを組み合わせます。 neurペレット100×gで遠心分離によってospheres 7分間、上清を除去します。 </li><li> 0.1％トリプシンEDTA 1mlにペレットを再懸濁し、37℃の水浴中で7分間インキュベートします。穏やかに200μlのピペットチップを使用して、下の100倍をピペット。 </li><li> 7分間100×gで酵素反応および遠心分離機を停止する血清培地の5ミリリットルを追加します。上清を捨て、完全増殖培地1ミリリットル中に細胞ペレットを再懸濁し、トリパンブルー色素排除し、血球計数器を用いて生細胞の数を数えます。 </li><li>完全増殖培地中の細胞懸濁液を希釈し、組織培養フラスコからゼラチンを除去し、1×10 4細胞/ cm 2で細胞を播種。 </li><li> 37℃、5％CO 2、&gt; 95％の湿度で、細胞をインキュベートします。培養物が約80％コンフルエンス（後約7日）に達するまで、3日ごとに新鮮な、温め、完全増殖培地で培地を交換してください。 </li></ol> 8. Neurアルコロニー形成アッセイ<ol><li>神経コロニー形成細胞（NCFC）アッセイ培地成分を組み合わせる：NCFC無血清培地、増殖NS-A、およびコラーゲン溶液（3 mg / mlで）。 2 / mlのヘパリン、20 ngの/ mlのEGF、および10ng / mLのbFGFでこのメディアを補完します。 </li><li>海馬組織解離に続いて、水浴中で37℃に予熱し、アッセイ培地を形成する神経コロニー中で細胞を再懸濁します。 35mmの培養皿に1.1×10 5細胞/ mlのクローン密度で細胞を播種。 </li><li> 37℃、5％CO 2、&gt; 95％の湿度でこの半固体のコラーゲンマトリックス中で細胞をインキュベートします。 28日間培養し、7日ごとに成長培地の50％を置き換えます。 </li></ol>神経前駆細胞の分化9. <ol><li>差異のために、細胞を処理する前に24時間37℃のインキュベーター中で組織培養皿及び硬化の表面を覆うように5μg/ cm 2のラミニン溶液の十分な量を加えまする。 注：ラミニン溶液を除去し、播種直前にDPBSで二回コーティングされた表面をすすぎます。 </li><li>継代すると、DMEM-F12で1×10 4細胞/ cm 2で細胞懸濁液をシード（3：1）の上に20 ngの/ mlのEGFおよび40 ngの/ mlのbFGFを添加した培地（予め温めた37℃まで）ラミニンは、培養皿をコーティングしました。 </li><li> 37℃、5％CO 2、&gt; 95％の湿度で、細胞をインキュベートし、細胞を3日間増殖することを可能にします。 </li><li>接着性神経前駆細胞を区別するために、3日後（3：1）分化培地は、DMEM-F12を含有する培地を交換媒体は、10ng / mLの脳由来神経栄養因子（BDNF）を補充し°C、37に予め温めました。 </li><li>分化の間に3日毎に培地の50％を置き換えます。 28日 - 分化は、約21をかけて徐々に発生します。 注意：細胞が分化し始めたら、一つだけtiにおけるメディアの50％を置き換えます空気への暴露のような私は、細胞の健康に有害な影響を有することができます。 </li></ol>

<ol>
	<li>Kheirbek, M. A., Hen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+vs+ventral+hippocampal+neurogenesis:+implications+for+cognition+and+mood.">Dorsal vs ventral hippocampal neurogenesis: implications for cognition and mood.</a> Neuropharmacol. 36 (1), 373 (2011).</li><li>Tanti, A., Rainer, Q., Minier, F., Surget, A., Belzung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+environmental+regulation+of+neurogenesis+along+the+septo-temporal+axis+of+the+hippocampus.">Differential environmental regulation of neurogenesis along the septo-temporal axis of the hippocampus.</a> Neuropharmacol. 63 (3), 374-384 (2012).</li><li>Eriksson, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+the+adult+human+hippocampus.">Neurogenesis in the adult human hippocampus.</a> Nat Med. 4 (11), 1313-1317 (1998).</li><li>Bull, N. D., Bartlett, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+mouse+hippocampal+progenitor+is+neurogenic+but+not+a+stem+cell.">The adult mouse hippocampal progenitor is neurogenic but not a stem cell.</a> J Neurosci. 25 (47), 10815-10821 (2005).</li><li>Kempermann, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+and+how+physical+activity+promotes+experience-induced+brain+plasticity.">Why and how physical activity promotes experience-induced brain plasticity.</a> Front Neurosci. 4 (189), 1-9 (2010).</li><li>Golmohammadi, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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 (4), 979-987 (2008).</li><li>Lowe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+and+precursor+cell+differences+in+the+dorsal+and+ventral+adult+canine+hippocampus.">Neurogenesis and precursor cell differences in the dorsal and ventral adult canine hippocampus.</a> Neurosc. Lett. 593, 107-113 (2015).</li><li>Sasaki, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+identification+of+dorsal+hippocampus+homologue+in+human+brain.">MRI identification of dorsal hippocampus homologue in human brain.</a> Neuroreport. 15 (14), 2173-2176 (2004).</li><li>Powell, E. W., Hines, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Hippocampus. , 41-59 (1975).</li><li>Jung, M. -. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Canine+hippocampal+formation+composited+into+three-dimensional+structure+using+MPRAGE.">Canine hippocampal formation composited into three-dimensional structure using MPRAGE.</a> J Vet Med Sci. 72 (7), 853-860 (2010).</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 (5052), 1707-1710 (1992).</li><li>Ray, J., Raymon, H. K., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+culturing+of+precursor+cells+and+neuroblasts+from+embryonic+and+adult+central+nervous+system.">Generation and culturing of precursor cells and neuroblasts from embryonic and adult central nervous system.</a> Methods Enzymol. 254, 20-37 (1995).</li><li>Oliver-Dela Cruz, J., Ayuso-Sacido, A. <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+from+mammalian+brain:+isolation+protocols+and+maintenance+conditions.">Neural stem cells from mammalian brain: isolation protocols and maintenance conditions.</a> INTECH Open Access Publisher. , (2012).</li><li>Pacey, L., Stead, S., Gleave, J., Tomczyk, K., Doering, 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+cell+culture:+neurosphere+generation,+microscopical+analysis+and+cryopreservation.">Neural stem cell culture: neurosphere generation, microscopical analysis and cryopreservation.</a> Nat Protoc. , (2006).</li><li>Azari, H., Rahman, 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. (45), e2393 (2010).</li><li>Walker, T. L., Kempermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+mouse,+two+cultures:+isolation+and+culture+of+adult+neural+stem+cells+from+the+two+neurogenic+zones+of+individual+mice.">One mouse, two cultures: isolation and culture of adult neural stem cells from the two neurogenic zones of individual mice.</a> J Vis Exp. (84), e51225 (2014).</li><li>Lim, J. -. H., Koh, S., Olby, N. J., Piedrahita, J., Mariani, C. L. <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+characterization+of+neural+progenitor+cells+from+adult+canine+brains.">Isolation and characterization of neural progenitor cells from adult canine brains.</a> Am J Vet Res. 73 (12), 1963-1968 (2012).</li><li>Herranz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneously+Arising+Canine+Glioma+as+a+Potential+Model+for+Human+Glioma.">Spontaneously Arising Canine Glioma as a Potential Model for Human Glioma.</a> J Comp Pathol. 154 (2), 169-179 (2016).</li><li>Walton, R. M., Parmentier, T., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neural+precursor+cell+regions+in+the+rostral+subventricular+zone,+hippocampal+subgranular+zone+and+cerebellum+of+the+dog+(Canis+lupus+familiaris).">Postnatal neural precursor cell regions in the rostral subventricular zone, hippocampal subgranular zone and cerebellum of the dog (Canis lupus familiaris).</a> Histochem Cell Biol. 139 (3), 415-429 (2013).</li><li>Walton, R. M., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormalities+in+neural+progenitor+cells+in+a+dog+model+of+lysosomal+storage+disease.">Abnormalities in neural progenitor cells in a dog model of lysosomal storage disease.</a> J Neuropathol Exp Neurol. 66 (8), 760-769 (2007).</li><li>Milward, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+transplantation+of+multipotential+populations+of+epidermal+growth+factor-responsive,+neural+progenitor+cells+from+the+canine+brain.">Isolation and transplantation of multipotential populations of epidermal growth factor-responsive, neural progenitor cells from the canine brain.</a> J Neurosci Res. 50 (5), 862-871 (1997).</li><li>Ray, J., Peterson, D. A., Schinstine, M., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+differentiation,+and+long-term+culture+of+primary+hippocampal+neurons.">Proliferation, differentiation, and long-term culture of primary hippocampal neurons.</a> Proc. Natl. Acad. Sci. 90 (8), 3602-3606 (1993).</li><li>Palmer, T., Ray, J., Gage, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-2-responsive+neuronal+progenitors+reside+in+proliferative+and+quiescent+regions+of+the+adult+rodent+brain.">FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain.</a> Mol Cell Neurosci. 6 (5), 474-486 (1995).</li><li>Zhu, G., Mehler, M., Mabie, P., Kessler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+progenitor+cell+responsiveness+to+cytokines.">Developmental changes in progenitor cell responsiveness to cytokines.</a> J. Neurosci. Res. 56 (2), 131-145 (1999).</li><li>Whittemore, S. R., Morassutti, D. J., Walters, W. M., Liu, R. -. H., Magnuson, 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=Mitogen+and+substrate+differentially+affect+the+lineage+restriction+of+adult+rat+subventricular+zone+neural+precursor+cell+populations.">Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations.</a> Exp. Cell. Res. 252 (1), 75-95 (1999).</li><li>Kennedy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmortem+survival+characteristics+of+rat+glial+cells+in+culture.">Postmortem survival characteristics of rat glial cells in culture.</a> J Neurol Neurosurg Psychiatry. 50 (6), 798-800 (1987).</li><li>Viel, J. J., McManus, D. Q., Cady, C., Evans, M. S., Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+and+time+interval+for+culture+of+postmortem+neurons+from+adult+rat+cortex.">Temperature and time interval for culture of postmortem neurons from adult rat cortex.</a> J. Neurosci. Res. 64 (4), 311-321 (2001).</li><li>Huat, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-1+enhances+cell+proliferation+and+survival+during+early+differentiation+of+mesenchymal+stem+cells+to+neural+progenitor-like+cells.">IGF-1 enhances cell proliferation and survival during early differentiation of mesenchymal stem cells to neural progenitor-like cells.</a> BMC Neurosci. 15 (91), 1-13 (2014).</li><li>Supeno, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-1+acts+as+controlling+switch+for+long-term+proliferation+and+maintenance+of+EGF/FGF-responsive+striatal+neural+stem+cells.">IGF-1 acts as controlling switch for long-term proliferation and maintenance of EGF/FGF-responsive striatal neural stem cells.</a> Int. J. Med. Sci. 10 (5), 522-531 (2013).</li><li>Ahmed, S., 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=BDNF+enhances+the+differentiation+but+not+the+survival+of+CNS+stem+cell-derived+neuronal+precursors.">BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors.</a> J Neurosci. 15 (8), 5765-5778 (1995).</li><li>Wohl, C. 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=Retinoic+acid+enhances+neuronal+proliferation+and+astroglial+differentiation+in+cultures+of+CNS+stem+cell-derived+precursors.">Retinoic acid enhances neuronal proliferation and astroglial differentiation in cultures of CNS stem cell-derived precursors.</a> J Neurobiol. 37 (2), 281-290 (1998).</li><li>Araujo, D. M., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+effects+of+interleukin-4,-7+and-8+on+hippocampal+neuronal+cultures:+potential+involvement+of+glial-derived+factors.">Trophic effects of interleukin-4,-7 and-8 on hippocampal neuronal cultures: potential involvement of glial-derived factors.</a> Brain Res. 600 (1), 49-55 (1993).</li><li>Jensen, J. B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+the+neurosphere+culture+system.">Strengths and limitations of the neurosphere culture system.</a> Mol Neurobiol. 34 (3), 153-161 (2006).</li><li>Singec, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+actual+sensitivity+and+specificity+of+the+neurosphere+assay+in+stem+cell+biology.">Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology.</a> Nat Methods. 3 (10), 801-806 (2006).</li><li>Conti, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Niche-independent+symmetrical+self-renewal+of+a+mammalian+tissue+stem+cell.">Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.</a> PLoS Biol. 3 (9), 283 (2005).</li><li>Pollard, S. M., Conti, L., Sun, Y., Goffredo, D., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherent+neural+stem+(NS)+cells+from+fetal+and+adult+forebrain.">Adherent neural stem (NS) cells from fetal and adult forebrain.</a> Cereb Cortex. 16, 112-120 (2006).</li><li>Ma, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-extracellular+matrix+interactions+regulate+neural+differentiation+of+human+embryonic+stem+cells.">Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells.</a> BMC Dev Biol. 8 (90), 1-13 (2008).</li><li>Goffredo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Setting+the+conditions+for+efficient,+robust+and+reproducible+generation+of+functionally+active+neurons+from+adult+subventricular+zone-derived+neural+stem+cells.">Setting the conditions for efficient, robust and reproducible generation of functionally active neurons from adult subventricular zone-derived neural stem cells.</a> Cell Death Differ. 15 (12), 1847-1856 (2008).</li></ol>

The authors have nothing to disclose.

ここで説明するプロトコルは、細胞生存率を最大にするための好ましい培養条件を維持するように最適化されています。抽出、単離および拡張中に撮影速度とケアが非常に重要です。接着単層の拡大を確立するための重要なステップは、一次ニューロスフェアの有効解離です。通路に続いて、十分に解離したニューロスフェアは、二次浮遊ニューロスフェアを生成することができます。培地?...

Regional variations in the rate of neurogenesis have been observed along the dorsoventral axis of the rodent hippocampus1,2. Furthermore, the rates of hippocampal neurogenesis also show distinct inter-species variation, with precursor cell turnover in the subgranular zone shown to be significantly lower in adult humans than in rodents3-5. Inter-species differences in hippocampal structural anatomy may be relevant here, as it has been postulated that neural stem cell distribution along the murine ventricular neuraxis may be influenced by cephalic flexures during embryological development6. To date, the rodent brain remains the most popular system in which to study adult neurogenesis. However, the brain of the domestic dog (Canis familiaris), with a size and structural organization intermediate between that of humans and rodents7, represents a valuable yet highly underused animal model. The canine hippocampus in particular embodies this structurally intermediate nature8-10 and can provide a unique perspective on intrinsic variations in neural precursor cell populations. With many closer parallels to the human brain, the canine model may also offer insight into the biology of adult human neurogenesis.
In vitro culture assays have become a key tool for the study of neural precursors and the cellular and biomolecular processes of adult neurogenesis. The neurosphere assay and adherent monolayer culture represent the two predominant systems for expanding neural precursor cells in vitro11-13. Protocols for brain extraction, hippocampal microdissection or neural precursor culture assays have been well documented for the rodent model14-16. However, for the adult canine brain they remain comparatively few17,18, focused instead on fetal or neonatal tissue19-21.
In our published study7 we investigated regional variations in neurogenesis and neural precursor cell populations across the dorsoventral axis of the adult canine hippocampus. Although highly dependent on breed, adulthood in canines is reached between 1 and 3 years of age. Here, we present detailed methods for the extraction, isolation and culture of neural precursor cells from the canine hippocampus. We provide systematic protocols for the expansion of neural precursor cells as both floating neurospheres and as an adherent monolayer culture, and for their subsequent differentiation into mature neural cell types.

<table><tbody><tr><td>1,000 &amp;mu;l filtered pipette tip</td><td>Axygen</td><td>TF1000</td><td></td></tr><tr><td>150 mm Petri dish</td><td>BD Biosciences</td><td>351058</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>Greiner Bio One</td><td>188271</td><td></td></tr><tr><td>200 &amp;mu;l filtered pipette tip</td><td>Axygen</td><td>TF200</td><td></td></tr><tr><td>24 well culture plate</td><td>Greiner Bio One</td><td>662160</td><td></td></tr><tr><td>35 mm tissue culture dish</td><td>BD Biosciences</td><td>353001</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>BD Biosciences</td><td>352340</td><td></td></tr><tr><td>6 well culture plate</td><td>BD Biosciences</td><td>351146</td><td></td></tr><tr><td>B-27 Supplement (50x) serum free</td><td>Life Technologies&amp;nbsp;</td><td>17504044</td><td></td></tr><tr><td>Basic fibroblast growth factor (bFGF)</td><td>Life Technologies</td><td>13256029</td><td></td></tr><tr><td>Brain derived neurotrophic factor (BDNF)</td><td>Millipore</td><td>GF029</td><td></td></tr><tr><td>Collagen solution</td><td>Stem Cell Technologies</td><td>04902</td><td>Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740&amp;nbsp;</td></tr><tr><td>DMEM (4.5 g/L, D-glucose) 500 ml</td><td>Life Technologies</td><td>11960044</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190250</td><td></td></tr><tr><td>Epidermal growth factor (EGF)</td><td>BD Biosciences</td><td>354001</td><td></td></tr><tr><td>F-12 nutrient mixture (Ham) (1x) Liquid</td><td>Life Technologies</td><td>31765035</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Life Technologies</td><td>16141079</td><td></td></tr><tr><td>Gelatin from Porcine Skin Type A</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>L-alanyl-L-glutamine dipeptide (GlutaMAX)</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>Heparin sodium salt from (porcine)</td><td>Sigma-Aldrich</td><td>H314950KU</td><td></td></tr><tr><td>Laminin (mouse)</td><td>Life Technologies</td><td>23017015</td><td></td></tr><tr><td>NCFC serum free medium (NeuroCult)</td><td>Stem Cell Technologies</td><td>5720</td><td>Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740&amp;nbsp;</td></tr><tr><td>Proliferation NS-A (NeuroCult)</td><td>Stem Cell Technologies</td><td>05773</td><td>Also available in the Neurocult NCFC Assay Kit Cat: 05740, and NS-A Prolieration Kit (Rat) Cat: 05771&amp;nbsp; from Stem Cell Technologies.</td></tr><tr><td>NSC basal medium (Rat; NeuroCult)</td><td>Stem Cell Technologies</td><td>5770</td><td>Also available in the Neurocult NS-A Prolieration Kit (Rat) from Stem Cell Technologies. Cat: 05771&amp;nbsp;</td></tr><tr><td>Penicillin/Streptomycin (5,000 U/ml)</td><td>Life Technologies</td><td>15070063</td><td></td></tr><tr><td>Povidone-iodine</td><td>Munipharma</td><td>Betadine</td><td></td></tr><tr><td>Trypan blue (0.4%)</td><td>Life Technologies</td><td>15250061</td><td></td></tr><tr><td>Trypsin EDTA</td><td>Life Technologies</td><td>25200056</td><td></td></tr><tr><td>Class II biological safety cabinet</td><td>ThermoFisher Scientific</td><td>Safe 2020 1.2</td><td></td></tr><tr><td>Brain knife (disposable)</td><td>Macroknife</td><td></td><td></td></tr><tr><td>Cell culture incubator</td><td>ThermoFisher Scientific</td><td>HERAcell 150i</td><td></td></tr><tr><td>Centrifuge</td><td>Hettich</td><td>Universal 320R</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Dumont</td><td></td><td></td></tr><tr><td>Easypet Electric pipette</td><td>Eppindorf&amp;nbsp;</td><td></td><td></td></tr><tr><td>Hemocytometer</td><td>Boeco</td><td>Bright-Line Improved Neubauer</td><td></td></tr><tr><td>Manual pipettes</td><td>Eppindorf research</td><td></td><td></td></tr><tr><td>Oscillating bone saw&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Scalpel blades (No.21)</td><td>Paramount</td><td></td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td>Delta</td><td></td><td></td></tr><tr><td>Water bath</td><td>Grant</td><td>JB Aqua 18 plus</td><td></td></tr></tbody></table>

isolation and expansion of adult canine hippocampal neural precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

in vitroで神経前駆アッセイ、神経発生および神経前駆細胞集団の使用によって特徴づけられ、大人の犬の海馬の背腹軸間で比較しました。文化の28日までに100ミクロンの直径に達し、分離の14日以内のニューロスフェアを浮動形成され孤立した海馬組織由来の神経前駆細胞。背側と腹側の分離株由来のニューロスフェアは、平均サイズに差は認められなかったし...

Watch this Scientific Journal Video about Isolation and Expansion of Adult Canine Hippocampal Neural Precursors at JoVE.com

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

犬の脳は大人の神経新生を研究する貴重なモデルです。ここで紹介する主要な脳組織から大人犬の海馬神経前駆細胞を単離および拡張するためのプロトコルです。

単離と大人の犬の海馬神経前駆体の拡大

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

isolation-and-expansion-of-adult-canine-hippocampal-neural-precursors

Research

JoVE Journal

Developmental Biology

7.0K ビュー.  University of Sydney. 犬の脳は大人の神経新生を研究する貴重なモデルです。ここで紹介する主要な脳組織から大人犬の海馬神経前駆細胞を単離および拡張するためのプロトコルです。 

ビデオ: Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

成人海馬ホッキョクジリス神経幹細胞の増殖と分化

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

神経科学

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.

ニューロスフェアのアッセイを使用して成体マウス神経幹細胞の単離と拡大

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 ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

マウスの海馬領域の歯状回における樹状樹枝状分岐の評価

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

大人の海馬神経新生を研究するために、同じホスト種由来の抗体による免疫組織化学および複数のラベル

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

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.

セル成体マウス脳室下帯から神経幹細胞および前駆細胞の選別とその細胞周期ダイナミクスのライブイメージング

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

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.

マウス梁下ゾーンから成体神経幹細胞の単離および培養

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

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

成人海馬神経前駆細胞のアッセイ回路特異的調節

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

出生後(P1-3)マウス神経起源ニッチからの神経球の分離と拡大

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

<meta charset="utf8"></head><body>マウス脳室内領域から単離されたNSCの効率的なヌクレオフェクション

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

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

<meta charset="utf8"></head><body>著者スポットライト:マウス脳室下ゾーン由来神経幹細胞培養における高効率遺伝子導入のためのヌクレオフェクションの改善

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

成体マウス脳室下領域からの神経幹細胞の単離、増殖、およびヌクレオフェクション

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