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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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 border="0" cellpadding="0" cellspacing="0" width="778">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Name of Material/ Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DMEM</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10569-010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">FBS</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">26140</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">penicillin/ampicillin</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DMEM/F12</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10565-018</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">KSR</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Non-essential amino acid</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">b-mercaptoethanol</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">ES-007-E</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">bFGF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">233-FB-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">mTesR1</td>
			<td style="width:184px;">STEMCELL Technologies</td>
			<td style="width:119px;">5850</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Collagenase IV</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17104-019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Gelatin</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">G9391</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">N2 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">B27 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17504-044</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Neurobasal</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">21103-049</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Glutamax</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">35050-061</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">PBS</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10010-023</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Growth factor reduced matrigel</td>
			<td style="width:184px;">BD Biosciences</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:308px;">Accutase</td>
			<td style="width:184px;">MP Biomedicals</td>
			<td style="width:119px;">1000449</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Thiazovivin</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-361380</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SB431542</td>
			<td style="width:184px;">Tocris Bioscience</td>
			<td style="width:119px;">1614</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">LDN-193189</td>
			<td style="width:184px;">Stemgent</td>
			<td style="width:119px;">04-0074</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SAG</td>
			<td style="width:184px;">EMD Millipore</td>
			<td style="width:119px;">566660-1MG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Purmorphamine</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-202785</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">FGF8b</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">423-F8-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">CHIR99021</td>
			<td style="width:184px;">Cellagen Technology</td>
			<td style="width:119px;">C2447-2s</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">BDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">248-BD-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">GDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">212-GD-010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">TGF-beta3</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">243-B3-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Ascorbic acid</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4034</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">cAMP</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0627</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Mouse anti human NESTIN antibody</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-23927</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human OTX2 antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9566</td>
			<td style="width:167px;">1/2000 dilutiion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Goat anti human FOXA2 antibody</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2400</td>
			<td style="width:167px;">1/200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">rabbit anti human LMX1a antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB10533</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human TH antibody</td>
			<td style="width:184px;">Pel Freez</td>
			<td style="width:119px;">P40101</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Chicken anti human TH antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9702</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Mouse anti human TUJ1 antibody</td>
			<td style="width:184px;">Covance</td>
			<td style="width:119px;">MMS-435P</td>
			<td style="width:167px;">1/2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human GIRK2 antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab30738</td>
			<td style="width:167px;">1/300 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human Calbindin antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab25085</td>
			<td style="width:167px;">1/400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Centrifuge</td>
			<td style="width:184px;">Eppendorf</td>
			<td style="width:119px;">5804</td>
			<td style="width:167px;"></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

16.4K 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

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

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

ヒト多能性幹細胞から終脳のグルタミン酸作動性ニューロンの仕様

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

ヒト神経の集合体のためのcGMP適用可能な展開法は、幹細胞および前駆細胞は、多能性幹細胞や胎児の脳組織由来

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

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

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

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

ヒト多能性幹細胞からの神経堤前駆細胞のフィーダーフリー導出

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

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

ニューウトジェネシスとシナプス成熟の高含有量スクリーニングに対するヒト多能性幹細胞由来ニューロンの分化後再形成

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

インビトロにおけるマウス胚性幹細胞からの神経細胞分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

ヒト人工多能性幹細胞由来脳微小血管内皮細胞の誘導、拡大、凍結保存および特性評価

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

多能性幹細胞からのヒト神経細胞および希突起膠細胞の作製によるニューロン-希突起膠細胞相互作用のモデル化

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

ヒトミクログリア様細胞:人工多能性幹細胞からの分化とヒトシナプトソームを用いた in vitro 生細胞貪食アッセイ

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

ヒト幹細胞移植および検証のための ex vivo モデルとしての成体ヒト皮質の有機型培養

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...