The authors thank members of the Renee Reijo Pera laboratory for help during development of this protocol and during preparation of this manuscript. This work is supported by California Institute for Regenerative Medicine (CIRM) shared laboratory (CL-00518).

培養培地の調製<ol><li>以下を組み合わせることにより、マウス胚線維芽細胞（MEF）培地の調製：445ミリリットルDMEM 50mlのウシ胎児血清（FBS）、および5mlの100×ペニシリン/アンピシリンストック溶液。せいぜい14日間4℃で濾過滅菌メディアを保管してください。 </li><li>以下を組み合わせることにより血清含有HPSC培地を調製する：385ミリリットルDMEM / F12 100mlのノックアウト血清代替物（KSR）を5ml 100×非必須アミノ酸ストック溶液を5ml 100×ペニシリン/アンピシリンストック溶液5mlの100倍ストック溶液をメルカプトエタノール、および10ng / mlのbFGF。せいぜい10日間4℃で濾過滅菌メディアを保管してください。 </li><li> 400ミリリットルmTeSR1基礎培地、100ミリリットル5倍サプリメント、および5ml 100×ペニシリン/アンピシリンストック溶液：次のように組み合わせることにより、mTeSR1無血清培地を準備します。せいぜい10日間4℃での媒体に保管してください。 </li><li> 10倍のコラゲナーゼIVストック溶液を準備します。0.5グラムコラゲナーゼIVパワーを秤量し、滅菌50ミリリットルDMEM / F12、フィルターでそれを溶かす。アリコートを作成し、数ヶ月のために-20℃でそれらをストック。 </li><li> 0.1％ゼラチン溶液を調製：（牛の皮膚から、タイプB）0.5gのゼラチン電力を計量し、脱イオン水で溶解する。週間室温でオートクレーブ滅菌溶液を保管してください。 </li><li>下記を組み合わせてKSRの分化培地を調製する：410ミリリットルDMEM、75ミリリットルKSRを、5mlのストック溶液をメルカプトエタノール100X非必須アミノ酸ストック溶液を5ml 100×ペニシリン/アンピシリンストック溶液を5mlの100倍。せいぜい10日間4℃で濾過滅菌メディアを保管してください。 注：KSRは分化効率に影響を与える可能性がロット、によって異なります。それは差別化のための最高のものを見つけるために、KSRの数バッチをテストすることをお勧めします。 </li><li>次のように組み合わせることにより、N2分化培地を準備します。98ミリリットルのDMEM、1ミリリットル100×N2サプリメント、1mlの100Xペニシリン/アンピシリンストック溶液。 4で濾過滅菌培地にしてく​​ださいせいぜい10日間°C。 </li><li> 10ミリリットル50倍B27サプリメント、5ミリリットルの原液をグルタマックス100X、および5ml 100×ペニシリン/アンピシリンストック溶液、480ミリリットル神経基礎培地：次のように組み合わせることにより、B27分化培地を準備します。せいぜい10日間4℃で濾過滅菌メディアを保管してください。 </li></ol> MEFフィーダー細胞上でのhESCとhiPSCs 2。文化たH9 hESCをWiCell研究所から入手し、hiPSCsは山中のレトロウイルス媒介形質導入を通じてReijoペラ研究室で確立されたOCT3 / 4、SOX2、KLF4、およびc-MYC 18因子 。 <ol><li>少なくとも1日細胞継代の前に、6ウェルプレートの1ウェル1 mlの0.1％ゼラチンを添加することによって、いくつかのゼラチン板を準備し、少なくとも30分間37℃でプレートをインキュベートする。 </li><li>インキュベーション後、プレートから吸引ゼラチン、および種子照射後1.6×10 5細胞の密度でプレートにMEF細胞を不活性化/ウェルMEF培地中の細胞をカウントする血球計数器を用いて。 注：必要に応じて細胞継代の前に、早くも3日間としてMEFフィーダーを用意。 </li><li>継代細胞MEFフィーダープレーティングの翌日：hPSCsから吸引媒体は、1ミリリットル/ウェルの細胞に1 mg / mlのコラゲナーゼIVを追加し、1時間、37℃で細胞をインキュベートする。 </li><li>このインキュベーションの間、PBSで一回MEFフィーダーを洗い、その後、十分に1.5ミリリットル/の温水（37℃）血清含有HPSC培地を追加します。 </li><li>分化したコロニーが付着したままで未分化コロニーのほとんどは、プレートから離脱するように、1時間後、培養プレートを数回タップします。慎重に浮遊コロニーを収集し、15mlチューブにそれらを転送します。 </li><li>優しく温かい（37℃）DMEM / F12で一回プレートを洗浄し、同じ15ミリリットルチューブにDMEM / F12キーを転送します。 </li><li> 3分間179×gでチューブを遠心。 </li><li>管から培地を吸引し、ペレットを乱さないように注意しながら。戦争を追加M HPSC培地、ピペットで上下のセルを数回小さなクラスターにそれらを破壊し、それらをよく混合する。 </li><li> 1から新しいMEFフィーダー上に細胞を移す：3-1：6比率を。 </li><li>すべての5-6日間、毎日通路の細胞を培地に変更します。 </li></ol>分化のための細胞の調製<ol><li>細胞の調製の前に少なくとも一日には、いくつかのマトリゲルプレート（6ウェルプレートの一般的に3つのウェル）を準備します。 1時40分の冷（4℃）DMEM / F12でマトリゲルを希釈し、6ウェルプレートのウェルあたり1mlマトリゲルを加える。 4℃で一晩マトリゲル培養する。注：一つは、限り、一週間などのためにパラフィルムでマトリプレートを密封し、4℃でそれらをストックすることができます。 </li><li> 4日経過した後に細胞（手順2を参照）を使用します。プレートからのすべてに分化コロニーを削除します。培養プレートから培地を吸引し、十分に混合ACCUTASEにつき1を追加し、5分間、37℃で細胞をインキュベートする。 </li><li>インキュベーションの間、いくつかのゲルを調製6ウェルプレートの1ウェルに1ミリリットルのゼラチンを加えることによりプレートATIN。これらのプレートを配置し、マトリゲルプレートをインキュベーター中で一日の前に用意しました。 </li><li> 5分間のインキュベーション後、またはとき細胞は単一の細胞にそれらを作るために数回上下半浮動、ピペット細胞となっている。 </li><li> 15ミリリットルチューブに単一細胞を収集し、3分間258×gで遠心する。 </li><li> HPSC培養培地を含む血清を適当量の細胞を再懸濁し、および2μMの最終濃度までThiazovivinを追加する。 </li><li>トランスファ1でゼラチン被覆プレート上に細胞：1の比、及びMEFフィーダーを除去し、37℃で30分間細胞をインキュベートする。 </li><li> 15ミリリットルコニカルチューブに細胞を移し、HPSC媒体を含む適切な血清を追加し、血球計数器で細胞を数える。 </li><li> 3分間258×gで細胞を遠心。 </li><li>遠心分離機中、210μMの濃度を作るためにmTeSR1媒体を適切なThiazovivinを追加します。 </li><li>遠心分離機、Aの後メディアをspirate及び/ mlの培地1.8×10 5個の細胞が存在するようにThiazovivinで適切なmTeSR1媒体を追加します。 </li><li> 、インキュベーターからマトリプレートを取り出しマトリゲルを吸引し、6ウェルプレートの1ウェルに混合セルの2ミリリットルを追加します。 注：細胞密度は、表面積の3.6×10 4細胞/ cm 2であるべきである。 </li><li>バックインキュベーターにプレートを戻し、細胞を24時間、アタッチしましょう​​。 </li><li> 24時間細胞をプレーティングした後、古い培地を吸引し、ウェル当たり2ミリリットル新しいmTeSR1培地を追加し、分化を開始する前に、細胞はさらに24時間成長させ。 </li></ol>細胞分化<ol><li>マトリプレートに細胞を再播種した後に、分化の48時間を起動します。 </li><li>プレートから吸引し培地は、PBSで細胞を1回洗浄し、ウェル当たり、以下の分化培地2mlを追加します。10μMのSB431542および100nM LDN-193を補充したKSR分化培地分化のD0として189をマーク、この日。 </li><li> D0からD20まで毎日培地に変更します。 注：一つは、当時の2日間を超えない使用のための培地を調製することができます。 </li><li> 10μMのSB431542、100 nMのLDN-193189、0.25μMのSAG、2100μMのプルモルファミン、および50ng / mlのFGF8bを補充したKSR分化培地：D1とD2のために以下の培地を使用してください。 </li><li> 10μMのSB431542、100 nMのLDN-193189、0.25μMのSAG、2100μMのプルモルファミン、50ng / mlのFGF8b、および3μMのCHIR99021を補充したKSR分化培地：D3とD4のための以下の培地を使用してください。 </li><li> 100 nMのLDN-193189、0.25μMのSAG、2100μMのプルモルファミン、50ng / mlのFGF8b、および3μMCHIR99021を補った75％のKSR分化培地プラス25％、N2分化培地：D5及びD6のために以下の培地を使用してください。 </li><li> 100 nMのLDN-193189および3μMCHIR99021を補った50％KSR分化培地プラス50％、N2分化培地：D7とD8のために以下の培地を使用してください。</li><li> D9とD10のための以下の培地を使用してください：25％KSR分化培地+ 100 nMのLDN-193189および3μMCHIR99021を補った75％のN2分化培地。 </li><li> 3μMのCHIR99021、10ng / mlのBDNF、10ng / mlのGDNF、1 / mlのTGF3、0.2 mMのアスコルビン酸及び0.1mMのcAMPを補足したB27分化培地：D11とD12のために以下の培地を使用してください。 </li><li> 10ng / mlのBDNF、10ng / mlのGDNF、1 / mlのTGF3、0.2 mMのアスコルビン酸及び0.1mMのcAMPを補足したB27の分化培地：分化の残りのために以下の培地を使用してください。 </li><li>分化の約D16には、いくつかのポリ-L-オルニチン/ラミニン/フィブロネクチンプレートを準備します。希は15μg/ mlの冷（4℃）PBSでポリ-L-オルニチン原液を、6ウェルプレートの1ウェルに1ミリリットルを追加し、37℃で一晩プレートをインキュベートする。 </li><li> 、ポリ-L-オルニチンソリューションを吸引滅菌水でプレートを3回洗浄した後、空気のプレートを乾燥。 </li><li>ラミニンストックゾルを希釈1μg/ mlの冷（4℃）PBSでutionは、6ウェルプレートの1ウェルに1ミリリットルを追加し、37℃で一晩プレートをインキュベートする。 </li><li> 、ラミニン液を吸引滅菌水で3回プレートを洗浄した後、空気のプレートを乾燥。 </li><li> 、2 / mlの冷（4℃）PBSでフィブロネクチン原液を希釈し、6ウェルプレートの1ウェルに1ミリリットルを追加し、37℃で一晩培養する。 </li><li>パラフィルムでシールプレート限り2週間、4℃で配置します。 </li><li>分化の20日後、ポリ-L-オルニチン/ラミニン/フィブロネクチンプレートに細胞をreplate。吸引し分化培地は、6ウェルプレートの1ウェルに1ミリリットル温かい（37℃）ACCUTASEを追加し、5分間37℃で細胞をインキュベートする。 </li><li>インキュベーションの間、インキュベーター内で、ポリ-L-オルニチン/ラミニン/フィブロネクチンプレートを配置。 </li><li> 5分間のインキュベーションの後、または細胞がなったときに、上下に数回静かにピペット細胞をセミフローティング単一細胞にそれらを作る。 </li><li> 15ミリリットルコニカルチューブに単一細胞を収集し、拡張に依存して変化する細胞計数のための神経基礎培地の適切な量を追加、（分化の11日後、細胞が15〜20倍に拡大する場合があります）。血球計数器を用いて細胞を数える。 </li><li> 3分間258×gで細胞を遠心。 B27分化培地で上清を吸引除去し、再懸濁細胞を細胞濃度/ mlの培地中1×10 6細胞となるように。 </li><li>吸引プレートからのフィブロネクチン、PBSで2回洗浄し、6ウェルプレートの1ウェルに2ミリリットルの細胞を加える。 注：再プレーティングのための細胞密度は、表面積の2×10 5細胞/ cm 2であるべきである。 </li><li>いずれかのアタッチされていない死細胞を除去するために24時間後に培地に変更します。 </li><li>所定の実験のための所望の時点まで、一日おきに培地を交換。 </li></ol>

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<li>Smidt, M. P., Burbach, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+to+make+a+mesodiencephalic+dopaminergic+neuron%5BTitle%5D)+AND+%22Nature+Reviews.+Neuroscience%22%5BJournal%5D)">How to make a mesodiencephalic dopaminergic neuron.</a> Nature Reviews. Neuroscience. 8, 21-32 (2007).</li>
<li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noggin+and+bFGF+cooperate+to+maintain+the+pluripotency+of+human+embryonic+stem+cells+in+the+absence+of+feeder+layers%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers.</a> Biochemical and Biophysical Research Communications. 330, 934-942 (2005).</li>
<li>Chen, J. K., Taipale, J., Young, K. E., Maiti, T., Beachy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+molecule+modulation+of+Smoothened+activity%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Small molecule modulation of Smoothened activity.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 14071-14076 (2002).</li>
</ol>

The authors declare that they have no competing financial interests.

（ヒトES細胞とhiPSCsの両方を含む）ヒト多能性幹細胞は、大部分を生成するためにインビトロで分化することができないすべての場合、以前の研究19で実証されているDAニューロンを含む私たちの身体の細胞型。ここでは、胚性ドーパミン作動性ニューロンの発生20からの知識や他の研究室14,15から公開されたプロトコルに基づいて、私たちはヒトES細胞とhiPSCsか...

ドーパミン作動性（DA）ニューロンは、中脳、視床下部、網膜、および嗅球を含むいくつかの脳領域、で見つけることができます。前脳における線条体に投影し、錐体外路運動系を形成することにより、黒質緻密部（SNpc）制御挙動および運動A9 DAニューロン。 A9 DAニューロンの変性は、第二の最も一般的なヒト神経変性疾患、現在不治であるパー​​キンソン病（PD）、につながる。細胞置換療法、PD 1の治療のための最も有望な戦略の一つであり、したがって、ヒト胚性幹細胞（hESC）との両方を含む、ヒト多能性幹細胞（hPSCs）からA9 DAニューロンを導出することに大きな関心があった最近のヒト人工多能性幹細胞（hiPSCs）。 多くの研究は、さまざまな方法を使用してhPSCsからA9 DAニューロンを導出しようとしている。すべての神経を介してDAニューロンを生成した最も初期の報告ロゼット前駆段階。外部成長の2-4週間、L.ステューダー要因や同僚に成功神経ロゼットを生成するためにヒトES細胞を誘導した3つの他のグループの有無にかかわらずMS5 2またはPA6 3-5間質細胞との共培養することにより。そして、彼らは機械的切開またはさらなる分化のために酵素消化することにより、これらのロゼットを豊かに。他のレポートでは、研究者は、培養、分化6-9フローティング （EB）胚様体を介して神経ロゼットを生成しました。その後、研究者は、彼らは細胞外マトリックス上でのhESCとhiPSCsメッキ単層ベースの微分法10,11を設置し、in vivoでの胚のDAニューロンの発生を模倣DAニューロンへhPSCsの分化を誘導する培養中の異なる成長因子を追加しました。すべてのこれらの研究は、DAニューロンのいくつかの特徴を有する発現細胞チロシンヒドロキシラーゼ（TH）が得られたが、全体の分化プロセスは、時間と労力がかかり、一般的には非効率的な、そしてより重要なことは、これらのニューロンのエンジンA9アイデンティティはLmx1aの異所性発現12と1を除いてほとんどの研究で実証されていませんでした。近年、新たなフロアプレート（FP）は、プロトコルベースのその後DAニューロン電位を有するFP前駆体が第一の分化の初期段階の間にソニックヘッジホッグおよびカノニカルWntシグナル伝達経路の活性化によって生成された、13-16を開発したこれらのFP細胞をさらにDAニューロンに指定されました。このプロトコルは、より効率的であるが、いくつかの問題が依然として存在する。例えば、全分化プロセスが（少なくとも35日）時間がかかり、15依存性フィーダー細胞であるか、またはEB を16依存性またはA9同一性は14を示していなかったである。 ここでは、in vivoでの胚のDAニューロンの発生やその他の研究者の発表された結果からの知識に基づいて、私たちはEFFのための培養条件を最適化したヒトES細胞とhiPSCs両方からDAニューロンのicient世代。まず、小分子CHIR99021および低分子SAGとプルモルファミンとシグナリングソニックヘッジホッグとカノニカルWntシグナルの活性化により、FP前駆細胞を生成した。これらのFP細胞はFOXA2、Lmx1aの、コリン、OTX2およびネスチンを発現する。次に、 などが BDNF、GDNF、を含む成長因子とDAニューロンにこれらのFPセルを指定しました。カルビンジン17に対して負間、彼らがGIRK2に対して陽性であるように生成されたDAニューロンは、A9細胞型である。このプロトコルは、独立した高効率で再現性のフィーダー細胞またはEBです。このプロトコルを使用すると、人は、またはインビトロ PDのモデル化またはPDのための潜在的治療薬をテストするためのPD患者のhiPSCsからヒトES細胞や細胞移植研究のための正常人のhiPSCsから約4週間でDAニューロンを導出することができる。

<table><tbody><tr><td>DMEM</td><td>Life Technologies</td><td>10569-010</td><td></td></tr><tr><td>FBS</td><td>Life Technologies</td><td>26140</td><td></td></tr><tr><td>penicillin/ampicillin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>10565-018</td><td></td></tr><tr><td>KSR</td><td>Life Technologies</td><td>10828-028</td><td></td></tr><tr><td>Non-essential amino acid</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>b-mercaptoethanol</td><td>Millipore</td><td>ES-007-E</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D Systems</td><td>233-FB-025</td><td></td></tr><tr><td>mTesR1</td><td>STEMCELL Technologies</td><td>5850</td><td></td></tr><tr><td>Collagenase IV</td><td>Life Technologies</td><td>17104-019</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G9391</td><td></td></tr><tr><td>N2 supplement</td><td>Life Technologies</td><td>17502-048</td><td></td></tr><tr><td>B27 supplement</td><td>Life Technologies</td><td>17504-044</td><td></td></tr><tr><td>Neurobasal</td><td>Life Technologies</td><td>21103-049</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Growth factor reduced matrigel</td><td>BD Biosciences</td><td>354230</td><td></td></tr><tr><td>Accutase</td><td>MP Biomedicals</td><td>1000449</td><td></td></tr><tr><td>Thiazovivin</td><td>Santa Cruz Biotechnology</td><td>sc-361380</td><td></td></tr><tr><td>SB431542</td><td>Tocris Bioscience</td><td>1614</td><td></td></tr><tr><td>LDN-193189</td><td>Stemgent</td><td>04-0074</td><td></td></tr><tr><td>SAG</td><td>EMD Millipore</td><td>566660-1MG</td><td></td></tr><tr><td>Purmorphamine</td><td>Santa Cruz Biotechnology</td><td>sc-202785</td><td></td></tr><tr><td>FGF8b</td><td>R&amp;amp;D Systems</td><td>423-F8-025</td><td></td></tr><tr><td>CHIR99021</td><td>Cellagen Technology</td><td>C2447-2s</td><td></td></tr><tr><td>BDNF</td><td>R&amp;amp;D Systems</td><td>248-BD-025</td><td></td></tr><tr><td>GDNF</td><td>R&amp;amp;D Systems</td><td>212-GD-010</td><td></td></tr><tr><td>TGF-beta3</td><td>R&amp;amp;D Systems</td><td>243-B3-002</td><td></td></tr><tr><td>Ascorbic acid</td><td>Sigma-Aldrich</td><td>A4034</td><td></td></tr><tr><td>cAMP</td><td>Sigma-Aldrich</td><td>D0627</td><td></td></tr><tr><td>Mouse anti human NESTIN antibody</td><td>Santa Cruz Biotechnology</td><td>sc-23927</td><td>1/1,000 dilution</td></tr><tr><td>Rabbit anti human OTX2 antibody</td><td>Millipore</td><td>AB9566</td><td>1/2,000 dilutiion</td></tr><tr><td>Goat anti human FOXA2 antibody</td><td>R&amp;amp;D Systems</td><td>AF2400</td><td>1/200 dilution</td></tr><tr><td>rabbit anti human LMX1a antibody</td><td>Millipore</td><td>AB10533</td><td>1/1,000 dilution</td></tr><tr><td>Rabbit anti human TH antibody</td><td>Pel Freez</td><td>P40101</td><td>1/500 dilution</td></tr><tr><td>Chicken anti human TH antibody</td><td>Millipore</td><td>AB9702</td><td>1/500 dilution</td></tr><tr><td>Mouse anti human TUJ1 antibody</td><td>Covance</td><td>MMS-435P</td><td>1/,2000 dilution</td></tr><tr><td>Rabbit anti human GIRK2 antibody</td><td>Abcam</td><td>ab30738</td><td>1/300 dilution</td></tr><tr><td>Rabbit anti human Calbindin antibody</td><td>Abcam</td><td>ab25085</td><td>1/400 dilution</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804</td><td></td></tr></tbody></table>

directed dopaminergic neuron differentiation from human pluripotent stem cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

分化プロトコルの概要を図1に示されている。ここに提示分化プロトコルの効率は、出発細胞の状態に依存する。このため、第1の分化のために単一細胞にhPSCsを解離する前にすべての差別化コロニーを除去し、そして第二に、一つはゼラチンでコーティングしたプレート上で細胞を培養することによって、ほとんどの、すべてではない、MEFフィーダー細胞を減少させる、ことを確?...

Watch this Scientific Journal Video about Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells at JoVE.com

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

ヒト多能性幹細胞からドーパミン作動性ニューロンの分化を監督

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

directed-dopaminergic-neuron-differentiation-from-human-pluripotent

Research

JoVE Journal

Neuroscience

16.5K ビュー.  Stanford University School of Medicine. We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson’s disease.

ビデオ: Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

マイクロ電極アレイ上のネットワーク・アクティビティを測定するための人工多能性幹細胞の急速な神経分化

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

発生生物学

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト神経前駆細胞と神経細胞の効率的な導出

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

神経科学

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

末梢単核細胞からの誘導神経幹細胞の生成と移植研究におけるドーパミン作動性ニューロン前駆体への分化

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

フィーダーフリーおよび化学的に定義された培養条件下でのヒト多能性幹細胞を用いた後神経節系交感神経ニューロンの効率的な分化

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

ヒト多能性幹細胞からの血原性内皮細胞の指向性分化

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Multititreプレートフォーマットでヒト多能性幹細胞から神経細胞の迅速かつ効率的な生成

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

生物学

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

ヒト人工多能性幹細胞由来皮質およびドーパミン作動性ニューロンの統合されたライブ細胞モニタリングによる自動生産

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

Source: Cosset, E. et al. <a href="https://app.jove.com/v/59682">Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases.</a> J. Vis. Exp. (2019)
This video demonstrates the differentiation of human embryonic stem cells (hESCs) into dopaminergic neurons, to provide a model for studying Parkinson&#39;s disease. The hESCs are cultured in a neural induction medium in a microwell plate to form 3D neurospheres. These are then cultured in a neural induction medium to promote the differentiation into dopaminergic progenitor neurons. The final maturation of the neurons occurs in a culture insert under air-liquid interface conditions, resulting in 3D organoids consisting of mature dopaminergic neurons.

ヒト胚性幹細胞からのドーパミン作動性ニューロンオルガノイドの作製

1. hESC-derived dopaminergic organoids for Parkinson&#39;s Disease (PD) studies

	Day 0: Amplify hESCs in 2D culture up to 60% confluency (day 0), then ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

Differentiating Non-Transgenic and Transgenic Human Pluripotent Stem Cells into Neural Progenitor Cells

Source: Cvetkovic, C., et al. <a href="https://app.jove.com/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video demonstrates the generation of neural progenitor cells from non-transgenic and transgenic human pluripotent stem cells (hPSCs). hPSCs are differentiated into neural progenitor cells using a neuronal differentiation medium. While differentiation is induced in non-transgenic cells using small molecules, the transgenic hPSCs are differentiated by adding an antibiotic that induces the promoter for a neuronal differentiation-specific transcription factor.

非トランスジェニックおよびトランスジェニックヒト多能性幹細胞の神経前駆細胞への分化

Ethic approvals were obtained when performing the experiments.
1. Cell Culture and Reagent Preparation

	Prepare coated plates for cell culture.
	
		...

神経細胞培養技術

高度な神経細胞モデル

Human Pluripotent Stem Cell Differentiation in Neurons Using Engineered Lentiviruses

Source: Assetta, B., et al., <a href="https://app.jove.com/v/61778">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions.</a> J. Vis. Exp. (2020).
This video demonstrates the process of inducing human pluripotent stem cells to differentiate into neurons by transfecting them with engineered lentiviruses carrying a neurogenic transcription factor gene under the control of&#160; tetracycline-responsive element, followed by treatment with a tetracycline derivative and maturation medium.

人工レンチウイルスを用いたニューロンにおけるヒト多能性幹細胞分化

1. Human neuron induction from human pluripotent stem cells

	Lentivirus preparation (~5 days, detailed protocol as described previously)
	
		Plate ~1 ...