The work was supported in part by the Duncan and Nancy MacMillan Faculty Development Chair Endowment Fund (K.Y.K.), Busch Biomedical Research Grant (K.Y.K.) and the Rutgers Faculty Development Grant (K.Y.K.).

1. IMOP細胞における自己再生を維持<ol><li> DMEM / F12、1X B27サプリメント、25 / mlのカルベニシリンおよび20 ng / mlのbFGF：培養培地をiMOP準備します。無菌の試薬を用いてiMOP培養培地50mlをしてください。 37℃の水浴中で50ミリリットルコニカルにDMEM / F12の49ミリリットルをウォームアップ。 <ol><li>解凍50X B27サプリメント、37℃の水浴中で5分間、フィルター滅菌し100 mg / mlのカルベニシリンアリコート。 RTで100μg/ mlのbFGFのアリコートを解凍します。 DMEM / F12には100μg/ mlのbFGFの1ミリリットル50X B27、10μLおよび100 mg / mlのカルベニシリンの12.5μLを追加します。 </li></ol></li><li>培養iMOP細胞を60 mm組織培養皿内のメディアの3ミリリットルを使用してください。メディアの容量を倍にして、一日おきに培養物に新鮮な培地を追加します。 bFGFのその濃度が5 ng / mlで以下に低下しないことを確認してください。 <ol><li> 5の後、5％のCO 2継代細胞と37℃で培養iMOP細胞-培養7日以下の手順に記載されています。 15に転送する文化10 mlピペットチップを用いmLコニカル。 </li></ol></li><li> HBSS 50mlに0.5MのEDTA pH8.0の0.1mlの希釈してHBSS中1mMのEDTAを行います。プレ暖かい1mMのEDTA、37℃の水浴中でHBSSで作ら。 </li><li> 5分間200×gで重力沈降または遠心分離によって細胞を回収。 RTで。 10分 - 重力沈降のために、5、37℃のインキュベーターで培養液を含む15ミリリットルの円錐を配置します。その時間の後、otospheresを観察し、15 mLコニカルの底に集めます。 注：過度の遠心力は、細胞の損傷を引き起こす可能性があります。 </li><li>慎重に吸引細胞ペレットを乱すことなく、ピペットを吸引2ミリリットルを使用してメディアを過ごしました。細胞ペレットに予め温めておいた1mMのEDTAのHBSS溶液0.5mlを追加します。 P1000ピペットを使用して、静かにアップピペット上下2から3回。 37℃の円錐形を置き、単一細胞に解離を促進するために、&lt;5分間インキュベートします。 </li><li>穏やかotospheresが解離しているかどうかを決定するために細胞溶液を渦。おと場合球は、細胞が解離され、円錐形の底に沈殿します。インキュベーションの時間が異なる場合があります。 注：EDTAで長時間のインキュベーションは、過剰な細胞死をもたらすでしょう。 </li><li> 37°Cから細胞を取り出し、EDTAを中和し、希釈するために、メディアの2ミリリットルを追加します。室温で5分間、200×gでの遠心分離によって細胞を収集します。吸引除去が出て2ミリリットル吸引ピペットを用いて、細胞を洗浄するために1×PBSの5ミリリットルを追加し、EDTAを希釈しました。 </li><li>室温で5分間、200×gで細胞をスピンダウンし、2ミリリットル吸引ピペットを用いて1×PBSを吸引除去します。 3回 - 優しくピペッティングと2ダウンによってiMOP培養培地0.5mlにP1000ピペットを用いて細胞を再懸濁します。 </li><li>適切なカセット28とマイクロ流体パーティクルカウンターを用いて細胞を数えます。 iMOP細胞のコンフルエントプレートが〜6 X10 6個の細胞が含まれています。細胞1希釈：メディアで100を計数するためのカセットに細胞溶液75μlのを追加します。プレートiMOP cultu 6cmの皿で1×10 6個の細胞メディア再（〜1：10希釈）。通路iMOPsごとに5から7日間。 注：大otospheresを形成するか、または組織培養皿の底に付着した細胞は、分化します。培養がコンフルエントを超えている場合に細胞も死んでしまいます。 </li></ol> 2.凍結融解IMOP細胞<ol><li>解凍と凍結前の細胞に4℃の解決策を平衡化することにより合成凍結媒体を準備します。 </li><li> iMOP細胞のコンフルエントに6cm皿から細胞を収集します。 15ミリリットルコニカルに- （8×10 6個の細胞 〜6）10 mlピペットと転送細胞を使用してください。 </li><li>室温で5分間、200×gで重力沈降または遠心分離によって細胞を回収。 otospheresが小さい場合、細胞は重力沈降により決済することはありません。オプション：総細胞数を決定するためにステップ1.9を用いて細胞を数えます。 </li><li>背後に緩い細胞ペレットを残しながら吸引し、ピペットを吸引2ミリリットルを使用してメディアを過ごしました。 </li><li> CEを再懸濁し、合成凍結メディアの0.25ミリリットルを追加約5×10 5〜6×10 3細胞/ mlの密度でLLS。ゆっくりP1000ピペットで細胞をピペット。先端を濾過した1 mlのP1000ピペットを用いて、低温バイアルに細胞懸濁液を転送します。 </li><li>アルコールフリーの冷凍コンテナにバイアルを置き、温度に到達するまで-80℃〜毎分温度1℃を減らすために-80℃で凍結コンテナを配置します。 </li><li>細胞の長期保存のために、液体窒素貯蔵タンクの気相へのバイアルを移します。培養のための細胞を解凍するには、-80℃で凍結した細胞を含む低温バイアルを平衡化。 </li><li> 37℃の水槽で予備iMOP暖かい培養培地。 </li><li> 37℃の水浴中で、バイアルの底に旋回することによって、迅速凍結バイアルを解凍します。最後の氷の結晶が消えたら、解凍した細胞にiMOP予め温めておいた培地の1ミリリットルを追加します。転送15mLの円錐形への細胞とiMOP培養培地の追加の4ミリリットルを追加</li><li> 15ミリリットルの共同スピン5分間nical。 200×gで使用済み培地を吸引し、。 6 10cmディッシュにiMOP培地プレート細胞の2ml中の細胞を再懸濁。拡張のため、5％CO 2で37℃で培養物をインキュベートします。 </li></ol> 3.感覚上皮にIMOP細胞を分化<ol><li> DMEM / F12、1X B27サプリメント、25 / mlのカルベニシリン：iMOP感覚上皮の分化培地50mlのを準備します。 iMOP感覚上皮の分化培地50mlのを行います。 37℃の水浴中で50ミリリットルコニカルにDMEM / F12の49ミリリットルをウォームアップ。解凍50XのB27サプリメントと5分間100 mg / mlのカルベニシリンアリコート。 37℃の水浴中で。 1ミリリットル50X B27およびDMEM / F12に100 mg / mlのカルベニシリンの12.5μLを追加します。 </li><li> 、収穫解離、再懸濁し、細胞をカウントする手順1.4から1.9を繰り返します</li><li> 3日目の培地を使用してiMOPで6センチディッシュでプレート1×10 6個の細胞 。 0日目に、転送培養を15mlコニカルに10 mlのピ​​ペットを用いて。 OTOの収集ステップ1.6で述べたように、重力沈降による球。吸引除去が出ピペットを吸引2ミリリットルを使用してメディアを過ごし、円錐形の底にotospheresを残します。 </li><li>そっと感覚上皮の分化培地2mlに追加します。大口径10ミリリットルピペットを用いてを6cmディッシュに転送otospheres。 注：過酷なピペッティングから機械的せん断がotospheresから細胞を解離することができます。 </li><li>一日おきに培養物に新鮮な感覚上皮分化培地2 mlを加え。必要に応じて、細胞は、重力沈降によって収集することができ、メディアを交換。 </li><li>円錐15ミリリットルに転送しotospheresが述べたようにピペットを吸引2ミリリットルを使用して、ステップ1.6.Aspirateで使用済み培地を沈降し、邪魔されずotospheresを残すようにすることによって10日目でotospheresを収集します。 </li><li>室温で15分間、1×PBS中の4％ホルムアルデヒド中でインキュベートすることによりotospheresを修正。 、ホルムアルデヒド溶液を除去し、洗浄緩衝液（1×PBS、0.1％トリトンX-10を含有するotospheres洗います0）をブロッキング緩衝液中でインキュベートする前に、（1×PBS、10％正常ヤギ血清で1時間、0.1％トリトンX-100）を含みます。 <ol><li>バッファーを交換し、希釈した抗体をブロッキング緩衝液でサンプルをインキュベートします。 4℃でインキュベートします。 1X PBSで洗浄サンプルは、27の免疫染色を、0.1％トリトンX-100の対象にotosphereを含みます。 P1000ピペットを用いてガラススライド上にメディアをマウントに1×PBSに転送otospheresと場所のotospheres。サンプルの上にカバースリップを置き、取り付けメディアは4℃で乾燥させます。 </li></ol></li><li> 16ビットのCCDカメラを搭載した倒立顕微鏡のセットアップおよび20X 0.75空気または40X 1.3 NA油浸対物レンズのいずれかを使用して、エピ蛍光画像を取得します。ブルー（377±25 nmで/ 447±30 nm）で、グリーン（475±25 nmで/ 540±25 nm）の赤（562±20nmで/ 625：記載されている（励起および発光）の波長を使用して、異なるカラーチャネルからの蛍光を収集20nmの）及び赤外線（628±20nmの/および692±20±NM）。 </li></ol> 4.アッセイするEDU設立<ol><li> 3日目に、プレート6 10cmディッシュ中、1×10 6 iMOP細胞。三日後0日、収穫、解離、再懸濁し、ステップ1.4から1.9を繰り返して細胞をカウントします。 </li><li>プレートiMOP培養培地中2.5×10 5個の細胞と6ウェル皿の異なるウェルにおける感覚上皮の分化培地中の5×10 5細胞 。 </li><li> EDU取り込みアッセイを使用して、3日目にEDU取り込みによってS期の細胞の割合を決定します<ol><li>培地に1μMのEDUの最終濃度を得るためにiMOP培養物に直接EDUの株式を追加します。 </li><li> 2時間iMOP培養でEDUをインキュベートし、5％CO 2で37℃でインキュベートします。収穫は、解離し、収集した細胞は、手順1.4から1.9を繰り返します。 15分間、1X PBS中4％ホルムアルデヒド中で加えます。室温で細胞を固定します。室温で5分間、200×gで遠心分離により細胞を回収。ホルムアルデヒド溶液とproperlを削除yは廃棄してください。 </li><li>ラベルヘキストでの細胞の核とは、製造業者のプロトコルに従って、緑色蛍光色素アジドとEDUを組み込みました。 注：すべての洗浄は1×PBS、二回1×PBSで3％BSAおよび0.1％のTween 20洗浄細胞を用いて行われます。 </li><li>退色防止試薬を用いてスライド上のマウント細胞およびサンプルの上に1.5カバーガラスを配置します。落射蛍光顕微鏡を用いて標識された細胞の蛍光画像を取得します。 </li></ol></li></ol> 5. IMOP由来ニューロンの区別<ol><li>神経基本培地、1X B27サプリメント、2mMのL-グルタミン：神経分化培地50mlのを行います。 5分間37℃の水浴中で50X B27と200mMのL-グルタミンの解凍ボトル。神経基本培地48.5 mlに1ミリリットル50X B27 0.5mlの200mMのL-グルタミンを追加します。 </li><li>滅菌10cmプレートでラウンド12ミリメートル1.5カバーガラスを配置し、滅菌し、カバーガラスをきれいにするためにプレートに70％エタノールを加えることにより、コートカバーガラス。 </li><li>静かな入り江を攪拌彼らはエタノールでカバーされていることを確認するrslips。 RTで10分間プレートを残します。残りのエタノールを洗い流すために、滅菌1X PBSでカバーグラスを3回すすいでください。残りの1×PBSを洗い流すための滅菌H 2 Oで一回カバースリップをすすぎます。 </li><li>ピペットを吸引2ミリリットルを使用して、H 2 Oを吸引除去し、カバーガラスを乾燥させ。 15分間組織培養フード中でUV光にカバースリップを公開。すぐに使用しない場合、Storeは、無菌環境でカバースリップ。 </li><li> 24ウェルディッシュの各ウェルに1 12ミリメートルラウンドカバーガラスを配置します。カバーガラスは、ウェルの底に平らにすることを確実にするために静かにプレートを振ります。 </li><li> 2ミリグラム/ RTでmlのポリ-D-リシンストック溶液を解凍し、1×PBS中10μg/ mlのポリ-D-リジン濃度に希釈します。 10 UG / mlのポリ-D-リジン0.25mlのウェルに追加します。 1時間37℃のインキュベーター内でプレートを残します。 </li><li>滅菌1×PBSでウェルを3回洗浄します。室温で10 mg / mlのラミニンストック溶液を、融解を10μg/ mの希釈1×PBSでリットル。 24マルチウェルディッシュの1ウェルに作業溶液を10μg/ mlのラミニンの0.25ミリリットルを追加します。 37℃のインキュベーターでプレートをインキュベートします。 2ミリリットル吸引ピペットを用いてラミニン液を吸引します。 </li><li> P1000ピペットを用いて1×PBS 1 mlに添加して、ピペットを吸引2mlで吸引することにより、1×PBSを除去することにより、カバーガラスを1×PBSで3回洗浄します。細胞は、めっきされる準備が整うまで、ウェル中の最後の洗浄から1×PBSを残します。解離と3日目に6センチの皿に段階的に1.4-1.9.Plate 1×10 6個の細胞を、細胞をカウント、収穫により神経分化を開始します。 </li><li> 0日、収穫には、解離して1.4から1.9を繰り返して細胞をカウントします。 24マルチウェルディッシュで1.5×10 0.5ミリリットルに5 iMOP細胞ウェルあたり予め温め神経分化メディア-シード1×10 5。吸引し、一日おきに培養物に予熱した神経分化培地を追加します。 </li><li> 15分間4％ホルムアルデヒドでiMOP由来の神経細胞を固定してください。 AT RT 7日目削除ホルムアルデヒド溶液に、洗浄緩衝液でiMOP由来の神経細胞を洗浄し、ブロッキング緩衝液中でインキュベートする前に、（1×PBS、0.1％トリトンX-100を含む）（1×PBS、10％正常ヤギ血清および0.1％トリトンX-100） 1時間。 <ol><li>バッファーを交換し、希釈した抗体をブロッキング緩衝液でサンプルをインキュベートします。 4℃でインキュベートします。 1X PBS、0.1％トリトンX-100を被験者の細胞を含有する洗浄サンプル27を免疫染色します。マウントメディアに配置する前に、1×PBSでカバーガラスを洗ってください。マウントメディアはステップ3.8に記載されているエピ蛍光画像を取得する前に、4℃で乾燥することができます。 </li></ol></li></ol>

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<li>Kim, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NeuroD-null+mice+are+deaf+due+to+a+severe+loss+of+the+inner+ear+sensory+neurons+during+development%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">NeuroD-null mice are deaf due to a severe loss of the inner ear sensory neurons during development.</a> Development. 128 (3), 417-426 (2001).</li>
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</ol>

The authors have nothing to disclose.

IMOP培養のモニタリング 自己再生を維持し、新規iMOP細胞株の分化を促進するためのプロトコルが記載されており、付加的なめっきフォーマット（表1）に含まれています。日常的拡大とiMOP細胞の分化に役立ついくつかの重要なステップが記載されています。多能性幹細胞培養と同様に、iMOP細胞は、理論的に無限の寿命を有します。?...

The organs of the inner ear, the cochlea, utricle, saccule and three semicircular canals, mediate the ability to hear and balance. Within the cochlea, hair cells convert sounds into electrical signals that are relayed to the spiral ganglion neurons (SGN). The SGNs fire action potentials to propagate neural signals through the auditory circuit. Genetic mutations, ototoxic drugs and exposure to loud sounds contribute to hair cell and SGN death that result in hearing loss1-4. Once lost, these cells are not replaced. Use of iPSC and ESC to generate nascent hair cells or SGNs holds great promise for inner ear cell replacement therapies5-8. A flurry of progress has shown that pluripotent stem cells and inner ear derived progenitors can differentiate into hair cells and SGNs at various stages of maturity. Mammalian embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can be used to generate functional hair cells and SGNs9-11. Stem cells and progenitor cells derived from the mammalian inner ear have also been shown to form hair cells and neurons with properties of their in vivo cellular counterparts12-16. 
Use of iPSC or ESC-derived otic progenitors to replace lost hair cells and SGNs requires efficient differentiation. Improper differentiation or continued proliferation of engrafted stem-derived progenitors in the inner ear can exacerbate inner ear function and pose a tumorigenic risk such as teratomas formation in the inner ear17. There is a clear need for developing culture conditions and understanding differentiation of otic progenitors. One strategy in developing these methods is to recapitulate cell fate decisions made by neurosensory progenitors during inner ear development. Protocols that prevent proliferation and direct otic progenitors into hair cells or SGNs will help improve safety as well as efficacy of replacement therapies. 
During development, the inner ear begins with the thickening of surface ectoderm in a restricted region between rhombomeres 5 and 6 to become the otic placode. As the otic placode invaginates to form an otic cup, a collection of cells in the anterior region of the otic cup gives rise to the neural-sensory-competent domain (NSD), which contains precursors of hair cells and neurons of the inner ear18. Fate mapping studies from mouse, chicken and zebrafish developing inner ear suggest multiple populations of neurosensory progenitors that give rise to the sensory hair cells, surrounding supporting cells and otic neurons19-22. The high mobility group transcription factor, Sox2, has been implicated in sensory cell specification and used as a marker for inner ear progenitors23,24. Hypomorphic mutations that decrease Sox2 expression levels in the inner ear result in the loss of the hair cells, supporting cells and SGNs in the cochlea25,26. 
To study otic progenitor cells undergoing cell fate decisions, a fate restricted immortalized multipotent otic progenitor (iMOP) cell line from Sox2 expressing cochlear progenitors was previously established. iMOP cells were originally derived from embryonic E12.5-13.5 cochlea and infected with a c-Myc retrovirus27. iMOP cells can continually proliferate as colony forming cells known as otospheres and have the capacity to differentiate into hair cells, supporting cells and SGNs27. Understanding the capacity of iMOP cells to differentiate into distinct otic lineages allows application of these findings to efficiently generate iPSC or ESC-derived hair cells and SGNs. Efficient differentiation protocols will open new avenues for cell replacement therapies of inner ear diseases that are recalcitrant to conventional treatments. A crucial issue in generating otic cells by in vitro cell culture is to have differentiation markers that help determine if cells are undergoing differentiating. Cdkn1b (p27KIP) has been extensively used as an early marker for differentiation in developing inner ear, however, expression of Cdkn1b in iMOP cells and how it correlates to differentiation has not been addressed. In this study, the current culture conditions and how Cdkn1b expression correlates to other markers of iMOP differentiation are described.

<table><tbody><tr><td>CoolCell LX Alcohol-Free Cell Freezing Containers</td><td>BioCision</td><td>BCS-405</td><td></td></tr><tr><td>Cryogenic Vials (2 ml)&amp;nbsp;</td><td>Corning</td><td>430654</td><td></td></tr><tr><td>1.5 Thickness Glass Coverslip (Round 12 mm)</td><td>Electron Microscopy Sciences</td><td>72230-01</td><td></td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>11320-082</td><td></td></tr><tr><td>Neurobasal Medium</td><td>Life Technologies</td><td>21103</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) pH 7.4</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Life Technologies</td><td>14025-092</td><td></td></tr><tr><td>B27 Supplement (50X) Serum Free</td><td>Life Technologies</td><td>17504-044</td><td>Stored as 1 ml aliquots</td></tr><tr><td>L-Glutamine(200 mM)&amp;nbsp;</td><td>Life Technologies</td><td>25030-081</td><td>Stored as 5 ml aliquots</td></tr><tr><td>Natural Mouse Laminin</td><td>Life Technologies</td><td>23017-015</td><td>Stored as 1 mg/ml aliquots</td></tr><tr><td>Click-iT EdU Alexa Fluor 488&amp;nbsp;</td><td>Life Technologies</td><td>C10337</td><td></td></tr><tr><td>Synth-A-Freeze Cryopreservation Media</td><td>Life Technologies</td><td>A12542-01</td><td></td></tr><tr><td>Prolong Gold Antifade Mountant</td><td>Life Technologies</td><td>47743-736</td><td>Stored as 10 mg/ml 100 &amp;micro;l aliquots</td></tr><tr><td>Moxi Z Mini Automated Cell Counter</td><td>Orflo&amp;nbsp;</td><td>MXZ001</td><td></td></tr><tr><td>Moxi Z Cassette Type S</td><td>Orflo&amp;nbsp;</td><td>MXC002</td><td></td></tr><tr><td>Recombinant Murine Fibroblast Growth Factor, basic (bFGF)</td><td>Peprotech</td><td>450-33</td><td>Resuspended in 0.1% BSA in H20 and stored as 20 mg/ml aliquots</td></tr><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>P7886</td><td>Resuspended in 1X PBS and stored as 10 mg/ml 100 &amp;micro;l aliquots</td></tr><tr><td>Carbenicillin, Disodium Salt</td><td>Thermo Fisher Scientific</td><td>BP2648-1</td><td>Resuspended in 10 mM Hepes pH 7.4 and stored as 100 mg/ml aliquots</td></tr><tr><td>5 ml pipet individually wrapped paperback (200/case)</td><td>Thermo Fisher Scientific</td><td>1367811D</td><td></td></tr><tr><td>10 ml pipet individually wrapped paperback (200/case)</td><td>Thermo Fisher Scientific</td><td>1367811E</td><td></td></tr><tr><td>Tissue Culture Treated Biolite 24-Well Plate</td><td>Thermo Fisher Scientific</td><td>130188</td><td></td></tr><tr><td>Tissue Culture Treated Biolite 6-Well Plate</td><td>Thermo Fisher Scientific</td><td>130184&amp;nbsp;</td><td></td></tr><tr><td>Tissue Culture Treated 6 cm Dish</td><td>Thermo Fisher Scientific</td><td>130181&amp;nbsp;</td><td></td></tr><tr><td>EMD Millipore Millex Sterile Syringe PVDF Filter Pore size: 0.22 &amp;mu;m</td><td>Thermo Fisher Scientific</td><td>SLGV033RS</td><td></td></tr><tr><td>TipOne filter pipet tips 0.1 - 10 &amp;mu;l elongated filter tip</td><td>USA Scientific</td><td>1120-3810</td><td></td></tr><tr><td>TipOne filter pipet tips 1 - 20 &amp;mu;l filter tip</td><td>USA Scientific</td><td>1120-1810</td><td></td></tr><tr><td>TipOne filter pipet tips 1 - 200 &amp;mu;l&amp;nbsp; filter tip</td><td>USA Scientific</td><td>1120-8810</td><td></td></tr><tr><td>TipOne filter pipet tips 101 - 1000 &amp;mu;l filter tip</td><td>USA Scientific</td><td>1126-7810</td><td></td></tr><tr><td>15 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)</td><td>USA Scientific</td><td>1475-0511</td><td></td></tr><tr><td>50 ml conical tubes sterile 20 bags of 25 tubes (500 tubes)</td><td>USA Scientific</td><td>1500-1211</td><td></td></tr></tbody></table>

initiating differentiation in immortalized multipotent otic progenitor cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

IMOP細胞におけるbFGFの撤退下げます増殖 iMOP細胞の増殖能を減少させ、iMOP細胞の分化を開始するには、bFGFを培地から取り出しました。成長因子の離脱が増殖を減少させることを確認するために、EDUの取り込みを増殖アッセイとして使用しました。 （bFGFを含まない）iMOP培地（bFGFを含有）と感覚上皮培地で培?...

Watch this Scientific Journal Video about Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells at JoVE.com

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

不死化多能耳の前駆細胞に分化を開始

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

initiating-differentiation-immortalized-multipotent-otic-progenitor

Research

JoVE Journal

Developmental Biology

8.5K ビュー.  Rutgers University. The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

ビデオ: Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

ヒト胎児脳から由来オリゴデンドロサイト前駆細胞培養システム

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

神経科学

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

オリゴデンドロサイト前駆体に胚性幹細胞の分化

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

生物学

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

ヒト人工多能性幹細胞からの破骨細胞の分化と特性評価

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

発生生物学

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

生殖様細胞に新生マウス皮膚由来幹細胞の分化<em&gt;インビトロ</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

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 ...

Differentiating Human Embryonic Stem Cells into Neural Progenitors

Source: Jeon, K. et al., <a href="https://app.jove.com/v/60571">Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells.</a> J. Vis. Exp.&#160;(2020)
This video demonstrates the differentiation of human embryonic stem cells, or hESCs, directly into neural progenitor cells without using feeder cells or any intermediate steps. The selective inhibition of bone morphogenetic protein or BMP and transforming growth factor-beta, or TGF-&#946;, signaling pathways leads to hESCs differentiation into neural progenitor cells, or NPCs.

ヒト胚性幹細胞の神経前駆細胞への分化

1. Preparation of human embryonic stem cells (hESC)-qualified Basement Membrane Matrix-coated Plates

	Slowly thaw the hESC-qualified basement membrane ...

JoVE Encyclopedia of Experiments

神経細胞培養技術

高度な神経細胞モデル

A Co-culture Method to Model Neuron-Oligodendrocyte Interactions

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 establishment of a co-culture of human induced neurons (iNs) and induced oligodendrocyte precursor cells (iOPCs). iNs are mixed with iOPCs in a co-culture and then incubated to support the maturation of both iNs into neurons and iOPCs into oligodendrocytes. Neurons form synaptic connections with other neurons and oligodendrocytes, while oligodendrocytes develop myelin sheaths around the neurons&#39; axons.

ニューロン-オリゴデンドロサイト相互作用をモデル化するための共培養法

1. Human neuron induction from human pluripotent stem cells

	Lentivirus preparation (~5 days)
	
		Plate ~1 million Human embryonic kidney 293T (HEK293T) ...

共培養と相互作用の研究

Differentiation of Neural Progenitor Cells into Neurons

Source: Mao, X., et al. <a href="https://app.jove.com/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
The video showcased a cell culture method for differentiating neural progenitor cells into neurons. By incubating cells in a nutrient-rich medium supplemented with growth factors, the progenitor cells matured into neurons with developed axons and dendrites. A stabilizing agent ensured cell viability and protected against oxidative damage throughout the differentiation process.

神経前駆細胞のニューロンへの分化

1. Differentiation from NPCs to neurons (Phase II)

	Seed about 5 x 105 mouse embryonic stem cells or mESC derivatives within 2 mL of basal ...

Differentiating Otic Progenitor Cells Into Sensory Epithelial Cells Followed by Immunostaining

Source: Azadeh, J. et al., <a href="https://app.jove.com/v/53692">Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells.</a>&#160;J. Vis. Exp. (2016).
This video demonstrates the differentiation of immortalized multipotent otic progenitor (iMOP) cells into sensory epithelial cells. It outlines the steps involved in culturing iMOP cells, forming otospheres, inducing differentiation, and visualizing cellular components and differentiation markers through fluorescence microscopy to confirm successful cell differentiation.

耳前駆細胞の感覚上皮細胞への分化とそれに続く免疫染色

1. Maintaining Self-renewal in IMOP Cells

	Prepare iMOP culture media: DMEM/F12, 1X B27 supplement, 25 &#181;g/ml carbenicillin and 20 ng/ml basic ...

Differentiating Immortalized Multipotent Otic Progenitors into Spiral Ganglion Neurons and Evaluating the Differentiation

Source: Azadeh, J., et al. <a href="https://app.jove.com/v/53692">Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells.</a> J. Vis. Exp. (2016).
The video demonstrates the differentiation of immortalized multipotent otic progenitors (iMOPs) into spiral ganglion neurons (SGNs) and immunofluorescence-based confirmation of the differentiation. The iMOPs are plated onto culture substrate-coated coverslips in a neuronal differentiation medium for differentiation into SGNs. The differentiated cells are labeled with antibodies specific for neuronal differentiation markers and analyzed under a microscope to confirm differentiation.

不死化多能性耳前駆細胞の螺旋神経節ニューロンへの分化と分化の評価

1. Differentiating iMOP-Derived Neurons 

	Make 50 ml of neuronal differentiation media: Neurobasal media, 1X B27 supplement, 2 mM L-Glutamine. Thaw ...