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>

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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 border="0" cellpadding="0" cellspacing="0" width="1699">
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		<tr height="21">
			<td height="21" style="height:21px;width:337px;">1000 &#956;L filtered pipette tip</td>
			<td style="width:196px;">Axygen</td>
			<td style="width:252px;">TF1000</td>
			<td style="width:915px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 mm petri dish</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">351058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15mL centrifuge tubes</td>
			<td style="width:196px;">Greiner Bio One</td>
			<td style="width:252px;">188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 &#956;L filtered pipette tip</td>
			<td style="width:196px;">Axygen</td>
			<td style="width:252px;">TF200</td>
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			<td height="21" style="height:21px;">24 well culture plate</td>
			<td style="width:196px;">Greiner Bio One</td>
			<td style="width:252px;">662160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm tissue culture dish</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">353001</td>
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			<td height="21" style="height:21px;">40 &#181;m cell strainer</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">352340</td>
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			<td height="21" style="height:21px;">6 well culture plate</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">351146</td>
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			<td height="21" style="height:21px;width:337px;">B-27 Supplement (50X) serum free</td>
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			<td style="width:252px;">17504044</td>
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			<td height="21" style="height:21px;">Basic fibroblast growth factor (bFGF)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">13256029</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Brain derived neurotrophic factor (BDNF)</td>
			<td style="width:196px;">Millipore</td>
			<td style="width:252px;">GF029</td>
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			<td height="21" style="height:21px;">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&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (4.5g/L, D-glucose) 500mL</td>
			<td style="width:196px;">Life Technologies&#160;</td>
			<td style="width:252px;">11960044&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:337px;">DPBS</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal growth factor (EGF)</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">354001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">F-12 nutrient mixture (Ham) (1X) Liquid</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">31765035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">16141079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin from Porcine Skin Type A</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:252px;">G1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-alanyl-L-glutamine dipeptide (GlutaMAX)</td>
			<td>Life Technologies</td>
			<td style="width:252px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin sodium salt from (porcine)</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:252px;">H314950KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin (mouse)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">23017015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proliferation NS-A (NeuroCult)</td>
			<td>Stem Cell Technologies</td>
			<td style="width:252px;">05773</td>
			<td>Also available in the Neurocult NCFC Assay Kit Cat: 05740, and NS-A Prolieration Kit (Rat) Cat: 05771&#160; from Stem Cell Technologies.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NSC basal medium (Rat; NeuroCult)</td>
			<td>Stem Cell Technologies</td>
			<td style="width:252px;">5770</td>
			<td>Also available in the Neurocult NS-A Prolieration Kit (Rat) from Stem Cell Technologies. Cat: 05771&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Penicillin/Streptomycin (5000 U/mL)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">15070063</td>
			<td></td>
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			<td height="21" style="height:21px;">Povidone-iodine</td>
			<td style="width:196px;">Munipharma</td>
			<td style="width:252px;">Betadine</td>
			<td></td>
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			<td height="21" style="height:21px;width:337px;">Trypan blue (0.4%)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">15250061</td>
			<td></td>
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			<td height="21" style="height:21px;">Trypsin EDTA</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">25200056</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Class II biological safety cabinet</td>
			<td style="width:196px;">ThermoFisher Scientific</td>
			<td style="width:252px;">Safe 2020 1.2</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Brain knife (disposable)</td>
			<td style="width:196px;">Macroknife</td>
			<td style="width:252px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Cell culture incubator</td>
			<td style="width:196px;">ThermoFisher Scientific</td>
			<td style="width:252px;">HERAcell 150i</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Centrifuge</td>
			<td style="width:196px;">Hettich</td>
			<td style="width:252px;">Universal 320R</td>
			<td style="width:915px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 Forceps</td>
			<td style="width:196px;">Dumont</td>
			<td style="width:252px;"></td>
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			<td height="21" style="height:21px;width:337px;">Easypet Electric pipette</td>
			<td style="width:196px;">Eppindorf&#160;</td>
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			<td height="21" style="height:21px;">Hemocytometer</td>
			<td style="width:196px;">Boeco</td>
			<td style="width:252px;">Bright-Line Improved Neubauer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Manual pipettes</td>
			<td style="width:196px;">Eppindorf research</td>
			<td style="width:252px;"></td>
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			<td height="21" style="height:21px;">Oscillating bone saw&#160;</td>
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			<td style="width:196px;">Delta</td>
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			<td height="21" style="height:21px;">Water bath</td>
			<td style="width:196px;">Grant</td>
			<td style="width:252px;">JB Aqua 18 plus</td>
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</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

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

成体ラットおよびヒト脊髄の脳室周囲領域から神経幹/前駆細胞の単離

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

発生生物学

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

神経の分析のための成体マウスのV-SVZにおける細胞の安定的かつ効率的な遺伝子改変幹細胞自律と非自律効果

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

チック小脳スライスと顆粒細胞前駆体の空間的にターゲットを絞ったエレクトロポレーションの ex vivo培養

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

大人のゼブラフィッシュ脳由来神経球の単離および培養

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Basicおよび前臨床研究のための密閉型細胞培養系でヒト多能性および神経幹細胞を培養します

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

単離およびヒト胎盤組織由来の間葉系幹/間質細胞の増殖

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

マウスの破骨細胞前駆体におけるNotchシグナル伝達の刺激

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

人間の成人および胎児心標本由来分離とプライマリの心外膜細胞の培養

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

導出とイヌの卵巣間葉系幹細胞の分化

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

大人の神経前駆細胞における P2X7 受容体を貪食、細孔の形成、カルシウム流入を調査するためのリアルタイム生細胞フローサイトメトリー

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...