このプロジェクトは、立ち上げ資金のためのロチェスターの大学、GM127652、NIH 助成金 EY025290 で賄われていた。

1. hPSC RPE への分化
<ol><li>維持し、通路 hPSCs 前述した18。 注: (hPSC RPEs、hPSCs など) のすべてのセル成長および微分のプロトコルの期間中 5% CO2 37 ° C で育ちます。</li>
	<li>
分化する前にあらかじめコーティング 6 ウェル プレートに合流 hPSCs を分割します。
	<ol>
<li>コート プレート、約 1 時間の氷の上の基底膜マトリックスを解凍、1:50 で 4 ° C DMEM 培地における液状化・行列を停止に希釈、各ウェルに 800 μ L コーティング混合物を追加し、37 ° C で少なくとも 1 時間インキュベート</li>
		<li>コーティングが完了したら、混合物を吸引し、すぐに井戸に 1.5-2 mL の培地を追加します。</li>
		<li>古い培養皿から hPSCs を持ち上げる、室温で 5 分間 PBS プラス 0.5 ミリメートルの EDTA、細胞を孵化させなさい、ソリューションを吸引、培養培地に小さな塊の細胞を再懸濁します。</li>
		<li>新しい板で 〜 20% の confluency シード細胞。 注:-20 ° C で 100 μ 因数で基底膜マトリックスし 6 ウェル プレートに塗るに 1 つの因数を利用します。コーティングのための時間は、37 ° c、または一晩室温で 4 h まで拡張できます。中断し、単一のセルではなく、3-5 細胞の小さな塊として hPSCs をシードすることが重要です。</li>
	</ol>
</li>
	<li>細胞がフルの confluency に成長できたら、置き換える培分化培地 (4 mL/ウェル) (表 1、アクチビンなし-A)。文化 14 日間 (4 mL/ウェル) 媒体を変更する 3 x/週。 注: hPSC プレート密度ダブルス約すべての 24 h. 重要な細胞死は、微分の手続きの開始後期待されます。死んだ細胞や井戸に破片の集合体は、中程度の変更時に肉眼で見ることができます。</li>
	<li>28 日目に、15 日から 100 ng/mL 人間アクチビン A (表 1アクチビン A の人間) と分化培地を補足します。媒体 (4 mL/も) を変更 3 回/週。</li>
	<li>日 29 (表 1、人間アクチビン A なし) にアクチビン A 補給を停止します。8-10 週間色素 hPSC RPE クラスターが表示される (図 1 aB) まで分化培地で培養を続けます。 注: 色素細胞のクラスターは細胞培養プレート上に小さな暗い点として (図 1 a ・B、トップ)、肉眼で見ることが。署名の玉石の形状と個々 の色素細胞は (図 1 a・ B下) 20 X 顕微鏡下で見ることができます。</li>
</ol>2. 分離と差別化された RPE 細胞の培養
<ol><li>分化培地を取り除き、PBS で 1 回洗浄、0.05% トリプシン プラス 1 U/μ L を各ウェルにコラゲナーゼの 1 mL を加えると, 20-30 分削除トリプシン/コラゲナーゼの 37 ° C で、軽く 6 ウェル プレートの各ウェルに予め温めておいた RPE 媒体の 2 mL を追加 (<c0 > テーブル 2)。 注: 倒立顕微鏡を使用してトリプシン/コラゲナーゼ処理前に多角形である hPSC RPE 細胞の形態を監視するなり充分な消化後ラウンド。</li>
	<li>
プル ガラス パスツール ピペットにブンゼン バーナーを使用します。
	<ol>
<li>両手で両端水平方向だとブンゼン バーナーの上に長い (9 インチ) パスツール ピペットを保持、ピペットの薄い部分の長さを約 2/3。</li>
		<li>ガラスは炎からソフトになると、すぐに引き離し 2 つの端、マイクロのスクレーパーのような先端が新しいピペット (図 2) 末に形成されることなど。</li>
		<li>複数「細胞スクレーパー」を作成するために数回をを繰り返します。70% のエタノールの殺菌と引っ張られたピペットをスプレーし、細胞文化フードでそれらを空気乾燥します。</li>
	</ol>
</li>
	<li>
顕微鏡、マイクロ「細胞スクレーパー」を使用して、優しくプレートから色素 hPSC RPE クラスターを分離します。
	<ol>
<li>軽く (こすりしない) 直接色素細胞のある、フロートを培地に一度井戸の底から彼らを切り離して考えます。</li>
		<li>すぐに 20 μ L マイクロ ピペットを使用して、優しく解離 hPSC-網膜色素上皮細胞を吸引、RPE の中の24のプレコート 6 ウェル プレートにそれらを転送するため。</li>
		<li>複数回、必要に応じて 2.3.1–2.3.2 の手順を繰り返すことによって 12 ウェル プレートのプレコート各 5 〜 10 の3セル (~ 10% の confluency 20 X 顕微鏡下で観察) x を収集します。2 mL に媒体の最終巻をもたらします。 注: 解離 hPSC RPE を防ぐために浮遊細胞で動きを避けるためプレートの解離過程の中に解離細胞培地に浮遊しているローカル集中のままが。</li>
	</ol>
</li>
	<li>RPE 中色素分子 (図 1) を形成するための別の 6-8 週間 12 ウェル プレートに培養新たに単離した hPSC 網膜色素上皮細胞 (P0)。</li>
	<li>3 媒体を変更 x/週。メディアを変更すると、各ウェルに古い媒体の約 1/3 量を残すし、新鮮な予め温めておいた RPE 培地 2/3 量を追加します。この段階で 12 ウェル プレートの各ウェルの RPE 培地 2 mL を使用して (したがって、交換中の 1.3 mL たび)。 注: P0 hPSC 網膜色素上皮細胞の単層細胞が積極的に、流体を輸送するいるとタイトジャンクショ25によって固定されます、ドームを形成できます。したがって、ドーム形成は成熟 RPE ステータスのインジケーターです。</li>
</ol>3. イムノブロット RPE 運命検証
<ol><li>カクテルを作る hPSC 網膜色素上皮細胞ライセート プロテアーゼ阻害剤を添加した哺乳類タンパク質抽出バッファーを使用してください。不溶細胞の残骸を破棄する 4 ° C で 15 分間 13,000 × gで遠心分離前に 30 分間氷の上細胞ライセートを孵化させなさい。ライセートの総蛋白濃度を測定します。</li>
	<li>変性 40 μ g 75 ° C で塩化物イオンと SDS サンプルバッファーの総蛋白質の 10 分別の蛋白質のサンプル 15 分、染料のフロントに達するまで 150 V、90 V の定電圧で 10% トリス-グリシンの SDS ページのゲルのゲルの底。</li>
	<li>1 h の 100 V で 10% メタノールを添加した予冷のトリス-グリシン バッファー内の硝酸セルロースの膜に蛋白質を転送します。</li>
	<li>0.1% 非イオン性洗剤 (TBST) と室温で 1 時間 5% 脱脂乾燥したミルクでトリス緩衝生理食塩水の膜をブロックします。</li>
	<li>一晩で 4 ° C ブロック バッファーで希釈した一次抗体と細胞膜を孵化させなさい。網膜色素上皮特異的マーカー蛋白質を次のようにターゲット抗体を希釈: RPE65、1:1, 000CRALBP、1:1, 000BEST1 は、1:1, 000。ローディング コントロールとして β-アクチンを検出する 1: 10,000 で β-アクチン抗体を希釈します。</li>
	<li>TBST の 3 回、各洗浄のため 5 分で膜を洗浄します。</li>
	<li>Fluorophore 共役ヤギ抗マウス IgG 抗体希釈 1: 10,000 室温で 1 時間ブロッキング バッファー内で膜を孵化させなさい。</li>
	<li>TBST 4 回、各洗浄のため 10 分で膜を洗浄します。</li>
	<li>赤外線システム (図 3) を用いたタンパク質を検出します。</li>
</ol>4. 免疫染色による細胞内局在 BEST1 をチェックします。
<ol><li>1 回 2 ml の PBS の hPSC 網膜色素上皮細胞を洗浄し、常温で 45 分 4% パラホルムアルデヒドの 2 mL で修正します。</li>
	<li>2 mL の PBS で 2 回洗うし、0.1% 非イオン性界面活性剤と 2% ロバ血清非特異的結合部位をブロックする 45 分の 2 mL の PBS のセルを孵化させなさい。</li>
	<li>室温で 2 h の BEST1 抗体 (希釈 1: 200) 細胞を孵化させなさい。</li>
	<li>2 ml の PBS 3 回の細胞を洗浄します。孵化 fluorophore 共役二次 IgG (1:1, 000) 室温で 1 時間。</li>
	<li>非連結の二次抗体を削除する PBS 3 回 2 mL で洗います。</li>
	<li>共焦点顕微鏡 (図 4) によるステンド グラスの細胞を観察します。</li>
</ol>5. 記録 Ca2 +-hPSC-RPE 細胞パッチク ランプでの Cl-現在の依存
<ol><li>24-72 時間、パッチク ランプ、前に分割 (玉石形や色素)、完全に合流も (12 ウェル プレート) に成熟した P0 hPSC 網膜色素上皮細胞の。、Pbs 中、洗って一度を吸引し、井戸に 0.05% トリプシン プラス 1 U/μ L コラゲナーゼの 1 mL を追加します。 注: 事前使用直前の 37 ° C にトリプシン/コラゲナーゼを温めます。</li>
	<li>8 分 37 ° C で孵化させなさい。 注: この手順後 hPSC 網膜色素上皮細胞がまだ付着性細胞と接続しています。</li>
	<li>水洗いして、プレートからセルに 1 mL ピペットを使用し、消化混合物内のセルを 15 mL の円錐管に転送します。予め温めておいたトリプシン/コラゲナーゼ残留細胞を収集し、それらを組み合わせて 15 mL の円錐管に 1 mL でよく洗ってください。 注: hPSC 網膜色素上皮細胞が大きな塊でまだこの時点で。積極的なピペッティングにより細胞塊を破るしないでください。いくつかの塊が 1 mL 先端の内側の壁に固執します。</li>
	<li>8 分間 37 ° C の乾燥浴で円錐形の管を孵化させなさい。</li>
	<li>1 mL ピペット ピペットをゆっくり上下に 10-15 回消化ミックスを使用します。 注: セルの塊は、まだ目に見えるが、ずっと狭くなった。積極的なピペッティングを避けてください。</li>
	<li>前の 2 つの手順を繰り返します。 注: 細胞の大部分は、ピペッティングした後単一細胞懸濁液にする必要があります。受け入れているいくつかの小さな細胞塊はまだあります。オプション: 穏やかなピペッティングに続いてさらに 5 分間 37 ° C で円錐形の管を孵化させなさい。37 ° C でトリプシン/コラゲナーゼの合計時間 30 分 (8 + 8 + 8 + 5 = 29) を超えてはなりません。</li>
	<li>消化細胞を含む 15 mL コニカル チューブに予め温めておいた RPE 培地 5 mL を追加します。200 x のg細胞を収集するために 5 分で回転します。10-20% の confluency にプレコート 35 mm 料理に再中断と種子の細胞 (例えば100% 10% の confluency 5 35 mm 皿に 12 ウェル プレートにも合流)。 注: すべての回で避けるため積極的なピペッティング、遠心分離後の上清の明白な暗い細胞の残骸によって表される、重要な細胞死をさせることがあります。十分に分離単一セルのシードは、パッチ ・ クランプ記録のために不可欠です。</li>
	<li>全細胞パッチを実行18,26,27,28,29,30,31を前述のようにクランプします。ステップ電位の家族から現在のトレースを取得 (+100、-100 0 の開催可能性から mV mV) (図 5)。 注: 内部と外部のソリューションのレシピそれぞれ表 3 表 4で説明されます。</li>
</ol>

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+light+peak+of+the+electroretinogram+is+dependent+on+voltage-gated+calcium+channels+and+antagonized+by+bestrophin+(best-1).">The light peak of the electroretinogram is dependent on voltage-gated calcium channels and antagonized by bestrophin (best-1).</a> J Gen Physiol. 127 (5), 577-589 (2006).</li><li>Volland, S., Esteve-Rudd, J., Hoo, J., Yee, C., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+some+organizational+characteristics+of+the+mouse+central+retina+and+the+human+macula.">A comparison of some organizational characteristics of the mouse central retina and the human macula.</a> PLoS One. 10 (4), e0125631 (2015).</li><li>Kamao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+induced+pluripotent+stem+cell-derived+retinal+pigment+epithelium+cell+sheets+aiming+for+clinical+application.">Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application.</a> Stem Cell Reports. 2 (2), 205-218 (2014).</li><li>Idelson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+embryonic+stem+cells+into+functional+retinal+pigment+epithelium+cells.">Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells.</a> Cell Stem Cell. 5 (4), 396-408 (2009).</li><li>Mandai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+Induced+Stem-Cell-Derived+Retinal+Cells+for+Macular+Degeneration.">Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration.</a> N Engl J Med. 376 (11), 1038-1046 (2017).</li><li>Maminishkis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confluent+monolayers+of+cultured+human+fetal+retinal+pigment+epithelium+exhibit+morphology+and+physiology+of+native+tissue.">Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.</a> Invest Ophthalmol Vis Sci. 47 (8), 3612-3624 (2006).</li><li>Maruotti, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+scalable+process+for+the+differentiation+of+retinal+pigment+epithelium+from+human+pluripotent+stem+cells.">A simple and scalable process for the differentiation of retinal pigment epithelium from human pluripotent stem cells.</a> Stem Cells Transl Med. 2 (5), 341-354 (2013).</li><li>Yang, T., He, L. L., Chen, M., Fang, K., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-inspired+voltage-dependent+calcium+channel+blockers.">Bio-inspired voltage-dependent calcium channel blockers.</a> Nat Commun. 4, 2540 (2013).</li><li>Yang, T., Hendrickson, W. A., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preassociated+apocalmodulin+mediates+Ca2+-dependent+sensitization+of+activation+and+inactivation+of+TMEM16A/16B+Ca2+-gated+Cl-+channels.">Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl- channels.</a> Proc Natl Acad Sci U S A. 111 (51), 18213-18218 (2014).</li><li>Yang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+selectivity+in+bestrophin+ion+channels.">Structure and selectivity in bestrophin ion channels.</a> Science. 346 (6207), 355-359 (2014).</li><li>Yang, T., Puckerin, A., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+RGK+GTPases+differentially+use+alpha1-+and+auxiliary+beta-binding-dependent+mechanisms+to+inhibit+CaV1.2/CaV2.2+channels.">Distinct RGK GTPases differentially use alpha1- and auxiliary beta-binding-dependent mechanisms to inhibit CaV1.2/CaV2.2 channels.</a> PLoS One. 7 (5), e37079 (2012).</li><li>Yang, T., Suhail, Y., Dalton, S., Kernan, T., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+molecules+for+inducibly+inactivating+CaV+channels.">Genetically encoded molecules for inducibly inactivating CaV channels.</a> Nat Chem Biol. 3 (12), 795-804 (2007).</li><li>Yang, T., Xu, X., Kernan, T., Wu, V., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rem,+a+member+of+the+RGK+GTPases,+inhibits+recombinant+CaV1.2+channels+using+multiple+mechanisms+that+require+distinct+conformations+of+the+GTPase.">Rem, a member of the RGK GTPases, inhibits recombinant CaV1.2 channels using multiple mechanisms that require distinct conformations of the GTPase.</a> J Physiol. 588 (Pt 10), 1665-1681 (2010).</li><li>Gong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+Human+Protein-Induced+Pluripotent+Stem+Cells+toward+a+Retinal+Pigment+Epithelial+Cell+Fate.">Differentiation of Human Protein-Induced Pluripotent Stem Cells toward a Retinal Pigment Epithelial Cell Fate.</a> PLoS One. 10 (11), e0143272 (2015).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+activates+bestrophin+ion+channels+through+direct+interaction.">ATP activates bestrophin ion channels through direct interaction.</a> Nat Commun. 9 (1), 3126 (2018).</li></ol>

著者が明らかに何もありません。

病気の料理方法の最も重要な手順は、 BEST1の RPE の正しいセル血統に病気の原因となる変異を有する hPSCs を区別することです。したがって、各分化実験後結果の hPSC 網膜色素上皮細胞慎重によって検証されるべきの成熟状態の RPE 固有の形態とタンパク質マーカー16,17,18。クローン アーチファクトを最小限に抑える?...

それは、少なくとも 5 つの網膜変性疾患がBEST1遺伝子1,2,3,4,5,6遺伝子の突然変異によって引き起こされるを記載されています。,7,8、200 と依然として増加を既に報告された突然変異の数。これらのBEST1-準の病気として知られている bestrophinopathies、進歩的な視野の損失と、さらには失明を引き起こすし、効果的な治療法は現在ありません。BEST1、すなわち BESTROPHIN1 の蛋白質の製品 (BEST1) は Ca2 +-アクティブ Cl-チャネル (CaCC) 目5,6、網膜色素上皮 (RPE) に特異的に発現 8,9。BEST1の重要な臨床表現型-関連疾患は眼球電図10,11; 測定光ピーク (LP) と呼ばれる、光の刺激に減らされた視覚反応LP は RPE12,13,14CaCC によって媒介されると考えられています。BEST1の突然変異の病理メカニズムをよりよく理解するために、潜在的な治療に向けてに取り組む、内生人間の網膜色素上皮細胞に発現変異の BEST1 チャンネルの勉強は必須です。
ただし、取得 RPE ライブ患者から直接細胞はない非常に実用的です。ネイティブの網膜色素上皮細胞は、人間の死体と胎児のバイオプシーから収穫されることが、これらの情報源に困難なアクセシビリティは大幅に、科学的な研究を制限しています。したがって、代替の RPE ソース以外の人間の目を持つことが重要です。この呼び出しは、幹細胞技術の最近の進歩によって回答されている網膜色素上皮細胞の機能が今ひと多能性幹細胞 (hPSCs) を含む萌芽期の幹細胞 (hESCs) から区別することができ、誘導多能性幹細胞 (hiPSCs)、後者リプログラミング ドナー16,17,18からプライマリ皮膚線維芽細胞によって生成されています。重要なは、自己更新と hPSCs の多能性確保 hiPSCs の患者特異性と hESCs (例えば、CRISPR によって) のゲノムの変更可能性の多彩な料理での疾患モデルを提供しています RPEs を生成する信頼性の高いソース目的のBEST1の突然変異。
hPSC RPE マウス RPE モデル上のいくつかの利点がある: 1) BEST1ノックアウト マウスの網膜異常19マウスとヒトの間 RPE でBEST1のさまざまな遺伝的要件の可能性を高めるを表示しません。2) 人間の網膜色素上皮細胞の 3% がマウス20; 35% と対照をなして、嬢だけ3) による網膜色素上皮移植の臨床を促進する網膜の治療疾患21。しかし、動物モデルがまだライブ システムに RPE の生理学と病理学を勉強するために不可欠である、ことの発癌性を見落すことができません。
ここでの手順では、研究と臨床目的のため使用することができます網膜色素上皮分化プロトコルに役に立つと適度に単純な hPSC について説明します。このプロトコルでは、ナイアシン (ビタミン B3) を使用して、hPSCs、RPE への分化誘導アクチビン A と治療に起因するさらに神経組織への分化を増強セル22を差別化のアポトーシス活性を減衰させることによってして、ニコチンアミド治療を色素細胞 (RPE への分化の記号) の数を増やす可能性が示されています。結果の hPSC 網膜色素上皮細胞は、ネイティブひと網膜色素上皮細胞22として同じキー マーカー、石畳の形態と細胞機能を表示します。したがって、研究の設定で、結果の hPSC 網膜色素上皮細胞は、法、免疫染色と細胞パッチクの詳細な実験手順は提供されていますを含む下流の機能解析に適しています。臨床的に、幹細胞由来網膜色素上皮細胞は、動物試験とひと試験23の黄斑変性症の移植治療のための大きな可能性を示しています。

<table>
	<tbody>
		<tr>
			<td>Knock-Out (KO) DMEM</td>
			<td>ThermoFisher</td>
			<td>10829018</td>
			<td></td>
		</tr>
		<tr>
			<td>KO serum replacement</td>
			<td>ThermoFisher</td>
			<td>10829028</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids</td>
			<td>ThermoFisher</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>ThermoFisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>ThermoFisher</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Human activin-A</td>
			<td>PeproTech</td>
			<td>120-14</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM (a modification)</td>
			<td>Sigma-Aldrich</td>
			<td>M4526</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td></td>
		</tr>
		<tr>
			<td>N1 supplement</td>
			<td>Sigma-Aldrich</td>
			<td>N6530</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine-penicillin-streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>G1146</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M7156</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0386</td>
			<td></td>
		</tr>
		<tr>
			<td>Triiodo-thyronin</td>
			<td>Sigma-Aldrich</td>
			<td>T5516</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR-1 medium</td>
			<td>Stemcell Technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356230</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Gibco</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>VWR</td>
			<td>45000-664</td>
			<td></td>
		</tr>
		<tr>
			<td>M-PER mammalian protein extraction reagent</td>
			<td>Pierce</td>
			<td>78501</td>
			<td></td>
		</tr>
		<tr>
			<td>proteinase inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>4693159001</td>
			<td></td>
		</tr>
		<tr>
			<td>RPE65 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB100-355</td>
			<td></td>
		</tr>
		<tr>
			<td>CRALBP antibody</td>
			<td>Abcam</td>
			<td>ab15051</td>
			<td></td>
		</tr>
		<tr>
			<td>BEST1 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB300-164</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta Actin antibody</td>
			<td>ThermoFisher</td>
			<td>MA5-15739</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated donkey anti-mouse IgG</td>
			<td>ThermoFisher</td>
			<td>A-21202</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG</td>
			<td>ThermoFisher</td>
			<td>SA5-35521</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG</td>
			<td>LI-COR Biosciences</td>
			<td>926-68071</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>HEKA EPC10 patch clamp amplifier</td>
			<td>Warner Instruments</td>
			<td>895000</td>
			<td></td>
		</tr>
		<tr>
			<td>Patchmaster</td>
			<td>Warner Instruments</td>
			<td>895040</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation, maintenance, and analysis of human retinal pigment epithelium cells: a disease-in-a-dish model for best1 mutations

人間BEST1遺伝子の 200 以上の遺伝的変異を特定し、網膜変性疾患にリンクされている、病理学的メカニズムままBEST1を研究するためのモデル良い体内の不足のために主にとらえどころのないです。生理学的な条件の下でその変異体。BEST1は、イオン チャネル、すなわち BESTROPHIN1 をエンコード (BEST1)、網膜色素上皮 (RPE) の機能ただし、ネイティブのひと網膜色素上皮細胞に非常に限定されたユーザー補助は、科学研究の主要な課題を表します。このプロトコルでは、ひと多能性幹細胞 (hPSCs) から誘導される分化によってBEST1病気を引き起こす突然変異をつける人間の RPEs を生成する方法について説明します。HPSCs は自己再生可能、このアプローチにより、hPSC-RPEs 法、蛍光抗体法、パッチク ランプなどの様々 な実験的解析のための着実なソースを持っている研究者の非常に強力な皿での疾患モデルを提供しますBEST1-網膜の条件に関連付けられています。特に、この戦略は、RPE (病態) 生理と RPE でネイティブ表現の興味の他の遺伝子研究に適用できます。

差別化された P0 hPSC RPE 人口の高純度を達成するためにめざす手動分離の最も技術的に挑戦的なステップです。成功した分離後 > P0人口の 90% の細胞が成長し、成熟署名 RPE 形態 (図 1) を表示します。RPE 非または P0人口の未熟な網膜色素上皮細胞のマイナーな部分の存在はほぼ避けられないが、下流の実験と干渉しない限?...

Watch this Scientific Journal Video about Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations at JoVE.com

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

差別化、保守、および人間の網膜色素上皮細胞の解析: BEST1変異の皿での疾患モデル

患者由来の変異ひと多能性幹細胞からの網膜色素上皮 (RPE) を区別するためにプロトコルをご紹介します。変異細胞は、免疫ブロット、蛍光抗体法、パッチク ランプなど機能の分析に使用可能性があります。この病気の料理方法は、ネイティブのひと網膜色素上皮細胞を得ることが困難を回避できます。

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

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Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

6.7K ビュー.  University of Rochester, School of Medicine and Dentistry. 患者由来の変異ひと多能性幹細胞からの網膜色素上皮 (RPE) を区別するためにプロトコルをご紹介します。変異細胞は、免疫ブロット、蛍光抗体法、パッチク ランプなど機能の分析に使用可能性があります。この病気の料理方法は、ネイティブのひと網膜色素上皮細胞を得ることが困難を回避できます。

ビデオ: Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

生理的酸素条件に曝露されたヒト細胞におけるキャップ結合タンパク質の解析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

遺伝学

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

ヒト膵臓の開発にリネージュの決定要因を問い合わせるためのゲノム編集とhPSCsの指向性分化

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA 変更染色: 共焦点画像の数値解析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

CRISPR/Cas9 によるマウスおよび人間の造血前駆細胞の高効率遺伝子破壊

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

ホット スポット領域の変異の包括的かつ同時検出のための単一液滴デジタル ポリメラーゼの連鎖反応

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

人間でタグ内因性のタンパク質誘導多能性幹細胞 CRISPR/Cas9 を使用して

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

CRISPR を介したゲノム人間病原菌カンジダの編集

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

サルモネラ菌におけるフィットネスのハイスループット分析に関するデジタルPCRベースの競争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

ヒト膵島の単一細胞RNAシーケンシングと解析

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the TILLING system has less applicability for insertion/deletion (Indel) detection and traditional TILLING needs more complex steps, like CEL I nuclease digestion and gel electrophoresis. To improve the throughput and selection efficiency, and to make the screening of both Indels and single base substitions (SBSs) possible, a new high-resolution melting (HRM)-based TILLING system is developed. Here, we present a detailed HRM-TILLING protocol and show its application in mutation screening. This method can analyze the mutations of PCR amplicons by measuring the denaturation of double-stranded DNA at high temperatures. HRM analysis is directly performed post-PCR without additional processing. Moreover, a simple, safe and fast (SSF) DNA extraction method is integrated with HRM-TILLING to identify both Indels and SBSs. Its simplicity, robustness and high throughput make it potentially useful for mutation scanning in rice and other crops.

In this article, we present the protocol that is described as high-resolution melting analysis (HRM)-based Target Induced Local Lesions In Genomes (TILLING). This method utilizes fluorescence changes during the melting of the DNA duplex and is suitable for high-throughput screening of both insertion/deletion (Indel) and single base substition (SBS).

米の耕作集団における高解像度溶融による変異の同定

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the ...