Name: differentiation, maintenance, and analysis of human retinal pigment epithelium cells: a disease-in-a-dish model for best1 mutations
Uploaded: 2018-08-24T12:30:00.000+00:00
Duration: 6 min 39 s
Description: 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

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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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 ...

differentiation-maintenance-analysis-human-retinal-pigment-epithelium

Research

JoVE Journal

Genetics

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

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

幹細胞から網膜色素上皮細胞の効率的な導出

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

発生生物学

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

網膜色素上皮生理学と病態の研究のための実験的モデル

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

神経科学

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

Xeno 無料の定義された、人間の世代の iPSC 由来網膜細胞モデル フィーダー フリー培養条件

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority of RP patients have progressive photoreceptor degeneration leading to peripheral vision loss, night blindness, and finally, total blindness. To date, thousands of mutations in more than 90 genes have been reported to be associated with RP. Currently, there are few animal models available for all the affected genes and different types of mutations, which largely hampers the deciphering of the mechanisms underlying the gene/mutation pathology and limits treatment and drug development. Patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids (ROs) have provided a better system to model the human early-onset disease than cells and animals. In order to study RP, those patient-derived 3D retinal organoids were utilized to recapitulate the clinical phenotypes of RP. In the RP patient-derived ROs, Rhodopsin mislocalization was clearly displayed. Compared with other animal models, patient iPSC-derived retinal organoid models more closely recapitulated RP features and represent an ideal approach for investigating the disease pathogenesis and for drug development.

In this protocol, retinitis pigmentosa patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids were generated. Those organoids successfully recapitulated some clinical phenotypes of the retinitis pigmentosa disease.

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority ...

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

健常および網膜疾患特異的ヒト誘起多能性幹細胞からの網膜オルガノイドの作製

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).
We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina.
The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

創薬のためのヒト網膜オルガノイドの毒性スクリーニング

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and ...

生物工学

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

新しいヒトの言語性の高いレティナル文化技術の特徴付け

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

医学

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

非ウイルス遺伝子治療のための哺乳動物一次色素上皮細胞の単離、培養、遺伝子工学

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

生物学

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

一次マウス網膜色素上皮細胞の効率的解離と培養

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

<meta charset="utf8"></head><body>げっ歯類の眼球における網膜の厚さと神経節細胞層の数の評価

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

<meta charset="utf8"></head><body>網膜を評価するためのラットおよびマウス眼球の組織学的スライドの調製と分析

This manuscript presents a protocol for the histological preparation of slides from rodent eyeballs. With the proposed method, the inner retina can be easily visualized and assessed under a light microscope. The procedure is presented for the eyeballs of mice and rats.

網膜を評価するためのラットおよびマウス眼球の組織学的スライドの調製と分析

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many ...

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

要約

さらに動画を探す

この動画の章

幹細胞から網膜色素上皮細胞の効率的な導出

網膜色素上皮生理学と病態の研究のための実験的モデル

Xeno 無料の定義された、人間の世代の iPSC 由来網膜細胞モデルフィーダーフリー培養条件

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

健常および網膜疾患特異的ヒト誘起多能性幹細胞からの網膜オルガノイドの作製

創薬のためのヒト網膜オルガノイドの毒性スクリーニング

新しいヒトの言語性の高いレティナル文化技術の特徴付け

非ウイルス遺伝子治療のための哺乳動物一次色素上皮細胞の単離、培養、遺伝子工学

一次マウス網膜色素上皮細胞の効率的解離と培養

網膜を評価するためのラットおよびマウス眼球の組織学的スライドの調製と分析

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

要約

さらに動画を探す

この動画の章

幹細胞から網膜色素上皮細胞の効率的な導出

網膜色素上皮生理学と病態の研究のための実験的モデル

Xeno 無料の定義された、人間の世代の iPSC 由来網膜細胞モデル フィーダー フリー培養条件

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

健常および網膜疾患特異的ヒト誘起多能性幹細胞からの網膜オルガノイドの作製

創薬のためのヒト網膜オルガノイドの毒性スクリーニング

新しいヒトの言語性の高いレティナル文化技術の特徴付け

非ウイルス遺伝子治療のための哺乳動物一次色素上皮細胞の単離、培養、遺伝子工学

一次マウス網膜色素上皮細胞の効率的解離と培養

網膜を評価するためのラットおよびマウス眼球の組織学的スライドの調製と分析

Xeno 無料の定義された、人間の世代の iPSC 由来網膜細胞モデルフィーダーフリー培養条件