This work was supported by the Brain Research Program through the National Research Foundation (NRF), funded by the Korean Ministry of Science, ICT, and Future Planning (Grants NRF-2015M3C7A1028790); and by a grant from the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (HI14C3347).

材料および培養培地の調製<ol><li> SCZの解剖および解離のために、アルミホイルで脳マトリックス、両刃カミソリの刃、および鉗子をラップしてから、オートクレーブでそれらを滅菌します。 </li><li>全マウスの脳を洗浄するために、冷PBS緩衝液の50ミリリットルを準備します。 </li><li>解剖顕微鏡を設定し、脳（オートクレーブ処理ハサミやピンセット）の解剖とSCZ（1 mlシリンジ、30 G針、脳マトリックス、および細かい鉗子）を単離するために必要な手術器具を準備します。 </li><li> N2培地の調製： <ol><li> F-12 / DMEM（+ L-グルタミン、+炭酸水素ナトリウム）2％B27、1％N2サプリメント、および1％ペニシリン - ストレプトマイシンを含む培地を準備します。 注：成長培地は、N2培地および成長因子（20 ngの/ mLの精製された上皮成長因子（EGF）および20 ng / mLの塩基性線維芽細胞増殖因子（bFGF2））からなります。 </li></ol></li><li>消化緩衝液を準備します（40単位/ mlのパパイン、2.4単位/ ml dispasE II、および2％のペニシリン - ストレプトマイシンPBS中）組織消化のために。 </li><li>コーティングプレートとカバースリップの調製： <ol><li> （; 0.01％PLO）とラミニン（DH 2 Oに溶解し10μg/ mlの）ポリ-L-オルニチンを準備します。免疫染色のために、単層または18ミリメートルカバースリップとしてSCZ-aNSCsのメンテナンスのためのコート6ウェルプレートに、4℃で一晩PLOでそれらをインキュベートします。そしてO.は、プレートとカバースリップは、最後の洗浄後に乾燥することができDH 2でそれらを3回洗浄します。 </li><li>次に、4℃で一晩ラミニンでプレートをインキュベート。そして、DH 2 Oでそれらを3回洗浄 注意：細胞付着に影響を与えるであろう、ラミニンを乾燥させないでください。 注：コーティング溶液を3回再利用することができます。 </li></ol></li></ol> 2.単離と大人SCZの解離<ol><li>文化の準備に先立ち、氷上で脳マトリックスと両刃カミソリを配置します。 脳マトリックスを凍結しないでください、理由BR注：アインは、それに添付可能性があります。 </li><li> CO 2窒息または頚椎脱臼により（8週齢）のマウスを生け贄に捧げます。 <ol><li> 70％エタノールを噴霧した後、鋭利なハサミで頭部を切断。ヘッドを固定化するために、しっかりと頭の両側を保持。尾 - 吻側方向に正中線でハサミで皮膚をカット。これは、頭蓋骨から皮膚の完全な除去を促進します。 </li><li>最初の（ラムダに後方）頭蓋骨の尾の部分を削除してから、背側正中線の位置で頭蓋骨と脳の間に鉗子を挿入します。左（または右）頭頂骨をつかみ、慎重にはがし。他の頭頂骨の除去のために、この手順を繰り返します。 注：視神経と脳から髄膜の除去の切断はそれが簡単に頭蓋骨から脳をデタッチすることを可能にします。 </li><li>冷PBS緩衝液（25ミリリットル）に脳を移し、余分な血液を除去するために、それを2回すすいでください。 </li></ol></li><li>氷とミリアンペアに脳マトリックスに脳を置きますKE冠状カットは1mm厚のスライスを得ることができます。 35mmのプラスチック製ペトリ皿に冷PBSに脳の後部に配置されSCZ領域を含む脳切片（1mm）で転送します。 注意：冠状切片の平行な面を得るために、脳の亀裂は、脳マトリックスの正中線に配置されることを確実にします。セクション内の不要な変動を回避することが重要です。 注：ブレグマ2〜3ミリの後部に脳スライスを入手。これは、SVZからSCZの分離を可能にします。 </li><li>低倍率で解剖顕微鏡下で、曲がった30G針6,9と皮質と海馬の白質領域からSCZをマイクロ解剖。そして、SCZ上記の皮質領域を削除します。冷PBSせずに氷の上で35ミリメートルのプラスチックペトリ皿にスライスから解剖SCZ領域を配置します。 注：成熟した神経細胞を含む余分な地域の汚染がaNSCsとニューロスフェア形成bの生存能力に影響を与える可能性がありますecause彼らはANSC培養条件下で細胞死を起こします。 </li><li>曲がった30 G針を使用して、すぐに小片に切開した組織を切ります。 注：長い時間がSCZ組織を切開するために必要とされている場合は、チョッピングの前に冷PBSで解剖した組織を浸します。 </li><li>再懸濁消化緩衝液1mlで刻んだ組織を、消化緩衝液2mlを含有する15mlチューブに組織を転送し、そして37℃の水浴中で30分間それをインキュベートします。 注：十分に混合するためにチューブを10分ごとに振ります。 </li></ol> 3.梁下ゾーン由来の成体神経幹細胞培養<ol><li>消化された組織を分離し、その後5分間、145×gでチューブを遠心分離する穏やかにチューブをタップします。消化緩衝液を洗い流し、ゆっくりP1000ピペットを用いて試料溶液を5回の最大をピペットするために予め温めておいたN2培地1mlで再懸濁消化された組織、上清を捨てます。 注：過粉砕狭いとピペットチップは、細胞の生存及びその後の成長を減少させることができます。 </li><li> 5分間、145×gでチューブを遠心。上清を廃棄した後、N2培地1ml中に細胞ペレットを再懸濁します。 </li><li>非コート6ウェルプレート中のN2培地1mlを調製し、2mlの最終容量を作成するために懸濁された細胞を1ml加えます。 </li><li>各ウェルにEGF（20ng / ml）およびbFGFを（20 ngの/ ml）を加えます。穏やかに播種した細胞を添加した成長因子を混合するために手で6ウェル培養皿を振ります。 37℃、5％CO 2インキュベーター中で6ウェルプレートを保管してください。 </li><li> 8日間毎日各ウェルにEGF（20ng / ml）およびbFGFを（20 ngの/ ml）を加えます。すべての三日目は、培地のおおよそ2ミリリットルの容量を維持するために、N2培地200μlのを追加します。 </li></ol>ニューロスフェアとして、また単層培養へのNSCの4継代<ol><li>ニューロスフェアを収集し、新しい15ミリリットルコニカルチューブに移します。 注：ニューロスフェアの数（&gt;直径50μm）PERならびに単一マウス脳のSCZからは、SVZ（190.5±6.33）9のそれよりも少なかった64.3±7.31でした。 </li><li>単一細胞に神経球を解離し、37℃の水浴中で5分間解離緩衝液0.5 mlのニューロスフェアを培養します。一次ニューロスフェアは、単一の細胞に分離し、神経球または単層培養物のいくつかの継代にわたって維持することができます。 SCZ-aNSCsは、単層培養形式で&gt; 10回継代することができるので、単層としてSCZ-aNSCsを展開すると、神経球よりも優れているが、&lt;ニューロスフェア培養形式で5回：注意してください。 注意：SCZ-aNSCs強い凝集を示し、単一細胞にそれらを分離することは困難です。このように、ニューロスフェア培養形式は、ルーチン細胞増殖のために推奨されません。 </li><li>ゆっくり5分間145×gで上下P1000ピペットで5倍未満と遠心チューブを試料溶液をピペット。上清および再suコマンドを破棄N2培地1mlの神経球を過ごします。 注：細胞の生存及びその後の成長を減少させることができ、狭いピペットチップで粉砕にわたり。 </li><li>細胞懸濁液（10μl）を0.4％トリパンブルー溶液の1混合し、次いで血球上の生細胞の数を数える：細胞をカウントするには、1を作ります。 PLO /ラミニンで6ウェルプレートに塗布した後、各ウェルのためのN2培地2mlで2.5×10 5細胞/ mlで細胞をプレート。 注：細胞密度の変化は、それらの条件と分化能に影響を与えることができます。 </li><li> 5日間の増殖因子の毎日の治療とSCZ-aNSCs（2ミリリットル、20 ngの/ ml）を維持し、それらの通路それら。 </li></ol>梁下ゾーン由来の成体神経幹細胞の分化5. <ol><li> SCZ-aNSCsの分化のための成長因子を有する1ミリリットルN2（20 ngの/ ml）の中の1×10 5細胞/ mlとPLO /ラミニンでコーティングした18ミリメートルカバースリップ上にプレートaNSCs。 </li><li>次の日、細胞がしっかりとカバースリップに結合している場合には、増殖因子を除去するためにN 2で成長培地を交換します。 </li><li> 6日後、細胞破片を除去し、免疫染色のためにそれらを修正するためにPBS 1mlで分化した細胞を洗浄します。 注：BrdUを増殖細胞の新たに合成されたDNAに組み込むことができます。そのため、必要に応じて、BrdUを（10μg/ ml）を、細胞の固定前に細胞を生きて追加することができます。 </li></ol> 6.免疫染色成体神経幹細胞および分化した前駆細胞<ol><li>免疫染色のために、PBSで細胞を洗浄し、室温で20分間、4％PFAで固定し。 注意：PFAは非常に有毒です。皮膚や眼との接触を避けます。 </li><li> 、4％PFAを削除し、PBSで3回を固定した細胞を洗い流し、その後、4°Cuntil彼らは免疫染色のために必要とされるでそれらを格納します。 </li><li> 1×P溶液（3％ウシ血清アルブミン及び0.1％トリトンX-100を遮断してカバースリップ上で細胞をインキュベート室温で少なくとも30分間BS）。 </li><li>新鮮なブロッキング溶液中で一次抗体を準備し、4℃で一晩サンプルをインキュベートします。 <ol><li> aNSCsを免疫染色<ol><li>抗ネスチンおよび抗BrdU抗体を用いてaNSCsを染色。固定する前に、aNSCsへのBrdUの20μgのを追加し、2時間のためにそれらをインキュベートします。ブロッキング工程を実施する37°Cbeforeで20分間2 N HClを用いて変性ステップを実行します。 </li></ol></li><li>分化した前駆細胞を免疫染色： <ol><li>差別化前駆細胞を標識する抗O4、抗BIIIチューブリン、および抗グリア線維酸性タンパク質（GFAP）抗体を使用してください。 </li></ol></li></ol></li><li> PBSで試料を3回洗浄し、蛍光色素（1：500）にコンジュゲート二次抗体でそれらをインキュベートし、室温で30分間ブロッキング溶液中。その後、PBSで試料を3回洗浄します。 注：プライマリのホストと一致し使用した二次抗体抗体。 Hoechest33343（1：2,000）は、核染色のために使用されます。 </li><li>スライドガラスにマウントソリューションを追加し、取り付けを進めます。複数の波長で共焦点顕微鏡でサンプルを観察し、画像：FITC（488 nm）と、のCy3（543 nm）で、Cy5の（647 nm）であり、Hoechest33343（405 nm）を。 </li></ol>

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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> JoVE (Journal of Visualized Experiments). , e51225 (2014).</li></ol>

The authors declare no conflicts of interest.

本論文では、成体マウスSCZからのNSCを生成し、様々なアプリケーションのためにそれらを維持するための詳細なプロトコルを記述します。 SCZ-のNSCを精製し、展開するために必要なin vitroの培養系を確立するための3つの重要なステップがあります。まず、SCZ領域が正確に他の潜在的な神経性領域（ 図1B）から解剖されていることを確認することが重要です。 SCZ領域を含む?...

NSCs have characteristics of self-renewal and multiple-lineage differentiation. To confirm these properties, a neurosphere culture system has widely been used. The neurosphere culture system was developed in the early 1990s and served as a standard stem cell culture system1. Depending on self-renewal potency, NSCs continuously proliferate and generate a cell mass in a suspension culture. The number of cells and the size of the neurosphere are considered to be closely associated with the proliferation properties of the NSCs. Monolayer cultures are also widely used for the maintenance and differentiation of NSCs. Compared to the neurosphere culture, the monolayer culture system provides better homogenous maintenance and expansion of NSCs2. These two well-developed culture systems have contributed to the characterization of aNSCs in vitro. 
NSCs reside in different brain regions, such as the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus3-5. The subcallosal zone (SCZ) of the caudal subcortical white matter is recognized as a novel neurogenic region6-8. It was recently reported that the SCZ-aNSCs have therapeutic potential in traumatic brain injury9. Compared to other neurogenic regions, the SCZ resides along the subcortical white matter. In the human brain, subcortical white matter occupies a larger region than in the mouse brain10. Therefore, an understanding of the characteristics of SCZ-aNSCs using an in vitro culture system is important in order to promote the potential use of these cells for neural regeneration. Precise dissection of the desired brain region is required to rule out possible contamination by unwanted regions containing active or quiescent NSCs. For instance, aNSCs in non-neurogenic regions can be activated and produce new neural cells in injured brains or during in vitro culturing11. To obtain NSCs from the SCZ, cells were collected from brain slices containing the SCZ. Then, a careful micro-dissection of the SCZ region was performed using a fine needle. To generate the neurosphere from the SCZ, micro-dissected SCZ tissue chunks were dissociated into single cells and then cultured as a suspension in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). After SCZ-aNSCs form neurospheres, they also can be maintained as neurospheres or monolayers for expansion. This procedure also demonstrates immunostaining processes with various markers for the detection of NSCs and their progeny after their expansion and differentiation in a monolayer culture. Here, a visual protocol of the SCZ-aNSC culture system is presented. This protocol contains detailed instructions for the micro-dissection of the SCZ region and for the maintenance and passaging of the cells.

<table><tbody><tr><td>&lt;strong&gt;Medium components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12&amp;nbsp;</td><td>Gibco</td><td>11320-033</td><td>+L-glutamin, +Sodium bicarbonate</td></tr><tr><td>Pen/Strep</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>N2 supplement</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>&lt;strong&gt;Growth factor&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D</td><td>233-FB</td><td></td></tr><tr><td>EGF</td><td>Gibco</td><td>PHG0313</td><td></td></tr><tr><td>&lt;strong&gt;Buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS (10x)</td><td>BIOSOLUTION</td><td>BP007a</td><td>1x dilution</td></tr><tr><td>HBSS</td><td>Gibco</td><td>14175-095</td><td></td></tr><tr><td>&lt;strong&gt;Dissociattion buffer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dispase II</td><td>Roche</td><td>04-942-078-001</td><td></td></tr><tr><td>Papain</td><td>Worthington</td><td>3126</td><td></td></tr><tr><td>Accutase</td><td>ICT</td><td>AT-104</td><td></td></tr><tr><td>&lt;strong&gt;Tool&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fine forceps</td><td>WPI</td><td>555229F</td><td></td></tr><tr><td>Scissors</td><td>Storz</td><td>E3321-C</td><td></td></tr><tr><td>Brain matrix (1 mm)</td><td>RWD</td><td>68707</td><td></td></tr><tr><td>Double-edged razor</td><td>DORCO</td><td>ST-300</td><td></td></tr><tr><td>30 G needle</td><td>SUNGSHIM</td><td>N1300</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15 ml tubes</td><td>SPL</td><td>50015</td><td></td></tr><tr><td>50 ml tubes</td><td>SPL</td><td>50050</td><td></td></tr><tr><td>35 mm dish</td><td>SPL</td><td>10035</td><td>Petri dish</td></tr><tr><td>100 mm dish</td><td>SPL</td><td>10090</td><td>Petri dish</td></tr><tr><td>Cover slip (18 mm)</td><td>Deckglaser</td><td>111580</td><td></td></tr><tr><td>12 well dish</td><td>SPL</td><td>32012</td><td>non-coating</td></tr><tr><td>6 well dish</td><td>SPL</td><td>32006</td><td>non-coating</td></tr><tr><td>&lt;strong&gt;Coating materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PLO</td><td>Sigma</td><td>P4957</td><td>0.01%</td></tr><tr><td>Laminin</td><td>Gibco</td><td>23017-015</td><td>10 mg/ml</td></tr><tr><td>&lt;strong&gt;Primary antibodies&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nestin</td><td>Millipore</td><td>MAB353</td><td>mouse (1:1,000)</td></tr><tr><td>EGFR</td><td>Abcam</td><td>ab2430</td><td>rabbit (1:1,000)</td></tr><tr><td>DCX</td><td>Santa Cruz</td><td>SC8066</td><td>goat (1:500)</td></tr><tr><td>Tuj1</td><td>Sigma</td><td>T2200</td><td>rabbit (1:2,000)</td></tr><tr><td>GFAP</td><td>Invitrogen</td><td>13-0300</td><td>rat (1:1,000)</td></tr><tr><td>O4</td><td>Millipore</td><td>MAB345</td><td>mouse (1:500)</td></tr><tr><td>BrdU</td><td>Abcam</td><td>ab6326</td><td>Rat (1:500)</td></tr><tr><td>&lt;strong&gt;Secondary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-mouse 488</td><td>Invitrogen</td><td>A21202</td><td>1:500</td></tr><tr><td>anti-mouse cy3</td><td>Jackson</td><td>715-165-151</td><td></td></tr><tr><td>anti-mouse 647</td><td>Jackson</td><td>715-606-150</td><td></td></tr><tr><td>anti-rabbit 488</td><td>Alexa</td><td>A21206</td><td></td></tr><tr><td>anti-rabbit cy3</td><td>Jackson</td><td>711-165-152</td><td></td></tr><tr><td>anti-rabbit 647</td><td>Jackson</td><td>711-605-152</td><td></td></tr><tr><td>anti-goat 488</td><td>Alexa</td><td>A11055</td><td></td></tr><tr><td>anti-goat cy3</td><td>Jackson</td><td>705-165-147</td><td></td></tr><tr><td>anti-goat 647</td><td>Invitrogen</td><td>A21447</td><td></td></tr><tr><td>anti-rat 488</td><td>Invitrogen</td><td>A21208</td><td></td></tr><tr><td>anti-rat cy3</td><td>Jackson</td><td>712-166-150</td><td></td></tr><tr><td>anti-rat 647</td><td>Jackson</td><td>712-605-153</td><td></td></tr><tr><td>&lt;strong&gt;Immunostaining materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BSA</td><td>Millipore</td><td>82-100-6</td><td>3% BSA with 0.1% Triton X-100 in PBS</td></tr><tr><td>Triton X-100</td><td>usb</td><td>22686</td><td></td></tr><tr><td>4% PFA</td><td>Biosesang</td><td>P2031</td><td></td></tr><tr><td>Hoechest33342</td><td>Life Technology</td><td>H3570</td><td>Dye for staining nuclei</td></tr></tbody></table>

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.

未知の神経形成領域からaNSCsための培養システムを定義すると、これらの細胞を理解し、脳修復12におけるそれらの潜在的用途を開発するために不可欠です。異なる発生段階で、または異なる地域でのNSCが異なる3,4振る舞うことが知られています。最近、SCZ由来細胞は、SVZ由来細胞7-9と比較して、インビボおよび インビトロで神?...

Watch this Scientific Journal Video about Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone at JoVE.com

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

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 ...

isolation-culture-adult-neural-stem-cells-from-mouse-subcallosal

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

15.1K ビュー.  Korea University College of Medicine. 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. 

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

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 ...

神経科学

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

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

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

ニューロスフェアのアッセイを使用してマウス胎児神経幹細胞培養の確立

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

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

人間の毛包から神経堤幹細胞の単離および培養

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

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

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

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

胎児マウス神経管から神経堤細胞の単離および培養

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

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

大人の齧歯類の脳の従来の従来にない地域から神経幹細胞を増殖させる

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

マウスを一回、二つの文化：個々のマウスの二つの神経原性のゾーンから成体神経幹細胞の単離および培養

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

マウス萌芽期の神経幹細胞の分離と培養

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

<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 ...

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

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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

ニューロスフェアのアッセイを使用してマウス胎児神経幹細胞培養の確立

人間の毛包から神経堤幹細胞の単離および培養

胎児マウス神経管から神経堤細胞の単離および培養

大人の齧歯類の脳の従来の従来にない地域から神経幹細胞を増殖させる

マウスを一回、二つの文化：個々のマウスの二つの神経原性のゾーンから成体神経幹細胞の単離および培養

マウス萌芽期の神経幹細胞の分離と培養

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