我々 は RPE 細胞の準備のため手術とスーザン ボーデンに彼女の援助のためのパテ レダーマンを感謝したいです。我々 はまた、このプロジェクトのための資金の NYSTEM C028504 を認めます。ジャスティン D. ミラーは NIH によってサポートされて F32EY025931 を付与します。

動物を含むすべてのプロシージャは、機関動物ケアおよび使用委員会 (IACUC)、アルバニーのニューヨークの州立大学、によって承認されています。
1. 事前注入準備
<ol><li>
HRPESC RPE の細胞懸濁液の調製 注: 滅菌のティッシュ文化フードのすべての次の手順を実行し、基本的な生殖不能の技術と知識が必要です。<ol>
<li>主な hRPE 細胞を分離および 24 ウェル プレート12に培養細胞を 50 から 90 年間人間のドナーから目が高齢者します。通路 1 細胞を凍結、融解必要に応じて文化通路 2 (P2) 細胞の注入の 4-5 週間 (図 1 a) の。</li>
		<li>培地12を外し、軽く 500 μ L で 2 回リンス井戸で加温ダルベッコ リン酸緩衝生理食塩水カルシウム & マグネシウムなし X 1 (1 DPBS CMF x) 1 を追加することによって X DPBS-CMF 井戸 1,000 μ L ピペットを使用し、真空を使用してそれを削除することに。</li>
		<li>各ウェルに 300 μ L トリプシン/DNAse を追加します。トリプシン/dn (0.25% トリプシン-EDTA の 1 mL あたり 4 区 DNase) セルを分離する 37 ° C で 4 分で hRPESC 網膜色素上皮細胞を孵化させなさい。</li>
		<li>彼らを切り上げているかどうかを顕微鏡下で細胞を確認します。まだ切り上げ彼らない場合さらに 2 分間トリプシン/DNase のセルをインキュベートし続けます。</li>
		<li>セルが切り上げられます、一度カップ刻んだ井戸から剥離細胞とトリプシン/DNase を不活化する、予め温めておいた培の等量と 15 mL の円錐管に剥離細胞を含むトリプシン/DNase を転送する 1,000 μ L ピペットを使用します。</li>
		<li>予め温めておいた 1 と井戸をリンス優しく特に; 井戸の縁の周りを上下、ピペッティングにより X DPBS CMF以前の円錐管にこれらの細胞を追加します。</li>
		<li>細胞のペレットへの 4 ° C で 5 分間 286 x g で円錐形の管を遠心します。</li>
		<li>上澄みを除去し、培地 1 mL の細胞を再懸濁します。</li>
		<li>診断を使用してセルをカウントします。</li>
		<li>細胞のペレットへの 4 ° C で 5 分間 286 × g で遠心分離機します。</li>
		<li>上澄みを除去し、細胞に滅菌バランスの取れた塩ソリューション (BSS) 50,000 細胞/μ L で (網膜注入中に 1 μ L のボリュームの 50,000 セルを配信) に再懸濁します。</li>
		<li>1.7 mL 遠心チューブに氷-水混合物の注入に使用されるまで最終的な細胞懸濁液 (CS) を転送します。</li>
	</ol>
</li>
	<li>
セル インジェクターの準備
	<ol>
<li>注射器とインジェクターを組み立てるにはしっかりとネジに滅菌 33 ゲージ ベベル針を挿入します。</li>
		<li>5-6 回 100% エタノールでインジェクターをフラッシュします。</li>
		<li>5-6 回の 70% エタノール噴射装置をフラッシュします。</li>
		<li>5-6 回 BSS とインジェクターをフラッシュします。</li>
		<li>(図 1 b) 解剖顕微鏡下で針の先端から 600 μ m の位置にある黒のマーカーペン滅菌とインジェクター針をマークします。</li>
		<li>注射用マイクロマニピュレーターにインジェクターを配置します。</li>
	</ol>
</li>
</ol>2. 網膜注入
<ol><li>
外科領域と動物の準備
	<ol>
<li>4-5 週齢 RCS ラット (60-100 g) の重量を量るし、イソフルラン蒸気の配信システムを使用してそれを麻酔します。 注:誘発するには、麻酔は 5% にイソフルランの流量を維持します。足を押すことによって麻酔の深さを確認し、手術中に麻酔維持のための 2-3%、流量を減らします。</li>
		<li>滅菌手術ドレープ、滅菌手術領域を設定する解剖顕微鏡のステージの下に、加熱パッドを配置します。</li>
		<li>外科領域にラットを転送し、ラットを麻酔を維持するためにイソフルラン システムに接続されている鼻の円錐形に配置します。</li>
		<li>ガーゼでラットの体をカバーしてください。完全麻酔を確認するラットのつま先をピンチします。</li>
	</ol>
</li>
	<li>
顕微鏡下強膜網膜注入
	<ol>
<li>ラットの unoperated 目に目の潤滑剤の滴を適用します。</li>
		<li>注射、外科医の右手に向かって頭とその外科医の方のための天井に直面して左眼の右側にラットを配置します。</li>
		<li>小さなハサミで目をカバーするひげをトリムします。</li>
		<li>左眼の時間的側面から目の洗浄量を点滴し、目を洗うに綿棒で鼻側に過剰を収集します。</li>
		<li>それぞれの滴を適用することによって投与後 10 月受験 (たて 10% フェニレフリンから手術の日にそれ 0.9% 生理食塩水で希釈することによって作られる) 1% 2.5% とトロピカミド フェニレフリンで瞳孔を散大させます。</li>
		<li>4-6 回目の周囲の皮膚を軽く引いてするまぶたを開き、目は若干れ角膜輪部の後方の領域にアクセスするため、突出しています。</li>
		<li>目の洗浄の適用ドロップ、目の潤いを保ちます。</li>
		<li>優しく (1.1. 12 のステップで準備) CS をカップ刻んだし、1.2 μ L CS とインジェクターをロードします。余分な 0.2 μ L が注入逆流を補うために使用されます。 注:について私たちの測定に基づく 5,000-8,000 セルはセル損失の約 10-16% に相当する 50,000 の細胞/μ の注射で逆流で失われ、0.2 μ L CS の余分をこのセル損失を補償するために注入されました。</li>
		<li>満ちている CS マイクロマニピュレーターにインジェクターを配置 (またはそれを保持するアシスタント) 垂直として RPE 細胞傾向にある懸濁液で簡単に沈みます。</li>
		<li>目にプロパラカイン 0.5% (ローカル局所麻酔薬) のドロップを適用し、綿アプリケーター付き過剰を削除します。 注:この手順は、角膜反射を抑制するし、目が後続の手順の実行中に点滅するを防ぐ必要があります。</li>
		<li>結膜、角膜の後方をグリップし、鼻、目を回転させる「テント」結膜を持ち上げて鉗子を使用します。</li>
		<li>結膜に小さな開口部を確認し、基になる強膜を公開「テント」上部をカットするはさみを使用します。</li>
		<li>鉗子を使用して、残り結膜角膜マージンの端をつかんで、瞳孔軸テーブル トップ (図 1) を基準にして約 30 度の角度で、鼻、目を回転します。針挿入時反力を提供するために、最適な角度で目を維持するために、結膜縁の継続的な把握が必要です。</li>
		<li>細胞の注入のための下穴をするためには、滅菌ベベル 31 ゲージ インスリンの針の先端位置は 1,200-1,500 μ m 直面している先端の開口縁の後方で。</li>
		<li>10-15 度 (目的の注射部位の虚数平面に接線) 強膜上にあるインスリンの針の角度を調整します。約 500 μ m の針の深さに強膜・脈絡膜複合体にゆっくりと浸透します。色素性のラットで針の端が「消滅」色素性の脈絡膜の下にあります。ここで使用されるインスリンの針のブランド、針の先端から面取りまでの距離は 500 μ m です。</li>
		<li>慎重に撤回するインスリンの針 (血の非常に小さい胸水が見られるかもしれない)。</li>
		<li>過度の出血が認められる場合、目槍は、必要に応じて、穴をオフに適用できます。継続的な出血後槍アプリケーション容器が破損しています。</li>
		<li>上下強膜の局所的な面を基準にして約 10-15 度の角度に直面して開口をパイロット穴に挿入、RPE 細胞ロード インジェクター針をガイドします。</li>
		<li>インジェクター針を網膜の領域にアクセスする約 500 μ m の深さにパイロット穴にそっと挿入します。黒のペンのマークの端と針が (図 1と1 D) 色素性の脈絡膜で覆われているポイントの間 100 μ m のマージンについて必要があります。</li>
		<li>優しく細胞 (約 1.2 μ L) の適切な量を注入する注入器注射器のプランジャーを押し下げるにアシスタントを頼みます。プランジャーを押すとアシスタントとしていくつかの反力を提供する準備ができてください。 注:アシスタントの前に模擬練習は外科医とこのステップに必要な経験を持つアシスタントの両方を提供できます。</li>
		<li>ペンで視覚的に焦点を当てながらエッジをマーク、25-30 秒の場所にインジェクターを保持、インジェクターをゆっくりと上げます。少量の逆流がよく観察されます。 注:逆流を認められなかった場合で、ケナコルト硝子体内注入をされてありますいる可能性があります。逆流シールや、強膜の下に充填セルを介して場合は、注入が浅すぎていた。</li>
		<li>3 回滅菌目洗うと注射部位から細胞排出を洗うし、綿アプリケーター付き過剰を収集します。</li>
		<li>運営の目に目の潤滑剤の滴を適用し、ラットを移植細胞の場所と網膜の胞のサイズを検討する 10 月駅に転送します。</li>
	</ol>
</li>
</ol>3. 後注入治療
<ol><li>
抗炎症、痛みの緩和治療
	<ol>
<li>痛みを軽減する皮下生理食塩水で 0.1 mg/kg 体重でブプレノルフィンを注入します。</li>
		<li>腹腔内注入 (i. p.) 炎症制御による生理食塩水で 1.6 mg/kg 体重でデキサメタゾンを注入します。</li>
	</ol>
</li>
	<li>
動物の回復
	<ol>
<li>ラットを体温を維持する熱ランプの下で回復ケージに戻ります。</li>
		<li>運営の目には出血の兆候を確認します。</li>
		<li>麻酔が出てくるようにラットを観察します。</li>
		<li>回復した動物を新鮮なケージに戻る手術カードでケージをフラグし、毎日の苦痛、眼出血、角膜混濁の兆候を監視します。懸念がある場合は、すぐに獣医師を通知します。</li>
	</ol>
</li>
</ol>

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著者が明らかに何もありません。

この資料に示されている網膜注入法はインジェクター針が神経網膜を傷つけるまたは硝子体腔を乱すことがなく目の壁の外側の層 (強膜・脈絡膜・網膜色素複合体) を貫通強膜の経路です。代替トランス vitreal アプローチには、レンズの損傷を齧歯動物のレンズが硝子体腔の大半を占めているので、白内障につながるの潜在的なリスクがあります。このメソッドと比較して、本手法はリスクが...

人間の網膜は光感覚組織として目関数の後ろに位置し、視覚認知に重要な役割を果たしています。網膜細胞の機能不全や細胞死したがってビジョンの問題や永久的な盲目を引き起こします。変性や網膜の異なる層の細胞の機能不全を伴う疾患はその中で AMD は最も一般的なタイプとの先進国で高齢者に回復不能な失明の主要な原因、網膜変性疾患として知られています。1,2。AMD の病理学的プロセスが網膜色素上皮層と順番視細胞の生理学、神経網膜の萎縮およびビジョンの損失3につながるの RPE サポートを損なう基になるブルッフ膜の間「ドルーゼン」蓄積関連付け 4,5。これまでの治療がない高度な (非血管新生) AMD を乾燥します。幹細胞療法として再生医療の新しいパラダイムの出現は、健康な細胞の幹細胞の機能不全や死者の網膜色素上皮細胞を交換の希望をもたらします。確かに、広範な臨床研究移植の幹細胞 (例えば、ヒト胚性幹細胞)-RPE 変性動物モデルに派生の網膜色素上皮細胞が実行6、7にいくつかの進歩をされています。臨床試験8,9 (NCT01344993、ClinicalTrials.gov)。最近では、幹細胞が人間の網膜色素上皮層、人間の網膜色素上皮幹細胞 (hRPESCs) の居住者の代替ソース研究室によって識別され、現在 amd hRPESC 派生した網膜色素上皮細胞 (RPE-hRPESC) 移植療法の臨床研究で使用されて10,11,12,13。
網膜下注入の技術は、上記当社グループを含む複数のグループによる前臨床試験に適用されます。動物の網膜注入の 2 つの一般的なアプローチ: トランス vitreal と強膜。トランス vitreal 方法の利点はレンズに全体 vitreal 腔を越える前眼に浸透、浸透、subretinal に到達する目に後ろに網膜と針の端を直接観察することが外科医のスペース14,,1516。ただし、それは (前部と後部)、2 つの場所で網膜を中断すること、レンズを傷つけるリスクを運ぶし、針が取り消されるとき硝子体に細胞の逆流の可能性があります必要があります。対照的に、トランス強膜方法は、原則として、網膜と硝子体の関与を回避し、逆流が目を終了します。色素性の齧歯動物の外科医は当初、強膜の浸透を観察できるが、色素性の脈絡膜に通過後針の端が表示されなくなります。直接観察することがなく網膜に違反は一般的と網膜剥離、硝子体に細胞や血液の配信になります。また、眼の表面が湾曲しているために、強膜注射のため最も効果的などの針の角度と深さを知ることは非常に困難です。
この可視化の記事で光コヒーレンストモグラフィ (OCT)、注射部位の詳細な検査ができると手術後の評価を使用して通知強膜網膜注入法を紹介します。私たちの強膜注入法は、定義済みの場所、角度、および非常に低い外科的外傷と高信頼性を生成する注射針の深さを利用しています。ここでは、人間の AMD の前臨床モデル RCS ラットの網膜注入 hRPESC 網膜色素上皮細胞を具体的に示す.この注入法で正常にかつ一貫して救わ hRPESC 網膜色素上皮細胞に非常に高い成功率で RCS ラットの目の網膜。細胞の注射以前が認め RCS 視細胞の保全に注入13後少なくとも 2 ヶ月。この手順は、解剖顕微鏡の下で行われ、学ぶことは簡単です。(外科医と助手) 注射を実行する二人必要で、それぞれの動物の注入の平均時間は 5 分未満。定義された角度と注射針の深さは 10 月が正常網膜注入を達成するために利用可能な研究所を可能にします。それは再現性の高い網膜にアクセスできます、細胞移植のみならず、医薬品配送と遺伝子治療のために使用できます。

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			<td style="width:231px;">Please refer to &#34;Reference #12&#34; for cell isolation and mainteinance.</td>
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development of a refined protocol for trans-scleral subretinal transplantation of human retinal pigment epithelial cells into rat eyes

加齢に伴う黄斑変性症 (AMD) などの網膜変性疾患は、世界的な不可逆的な視力喪失の主要な原因です。AMD は、機能的にサポートおよび解剖学的神経網膜の周りを囲むセルの単分子膜は、網膜色素上皮 (RPE) 細胞の退化によって特徴付けられます。非血管新生 AMD (AMD) の現在の薬理学的治療法は、病気の進行を遅くしか新規治療戦略の特定を目的とした研究を施行したビジョンを復元できません。変性の網膜色素上皮細胞を乾燥 AMD を将来的に治療するために健康な細胞の将来性に置き換えます。AMD のための幹細胞補充療法の広範な前臨床研究は、幹細胞由来網膜色素上皮細胞の網膜注入の技術を適用する動物モデルの網膜への移植を含みます。これらの前臨床動物研究で最も頻繁に使用されるアプローチは、針の端の直接可視化の欠乏によって困難になされる、網膜の損傷で起因できるしばしば強膜ルートを通じてです。針終了位置の直接観察が可能硝子体を通して別のアプローチがより目の組織が邪魔と手術のトラウマ高いリスクを運ぶ。我々 は正常にし一貫してラット網膜下腔網膜色素上皮細胞を提供し、過剰な網膜の損傷を避けるために定義された針の角度と深さを使用するリスクが少ないと再現性のある変更された強膜注入手法を開発しました。この方法で配信される細胞は、最低 2 ヶ月は王立外科医師 (RCS) ラットで結構効果があります以前実証されています。細胞移植のみならず小分子や遺伝子治療の配信、この手法を使用することができます。

この資料で説明する手法を使用すると、一貫して納品 hRPESC 網膜色素上皮細胞 RCS ラットの網膜下腔の場所、角度、および組織 (図 1 b-D にインジェクター針挿入の深さを正確に制御することにより).すぐに次の移植は、OCT 検査を行った注射部位及び移植の細胞によって作成された網膜下の水疱を観察します。手術後 10 月評価は注射や網膜の損?...

Watch this Scientific Journal Video about Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes at JoVE.com

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

網膜の注射は、加齢黄斑変性症に対する幹細胞補充療法の臨床研究で広く適用されています。この可視化の記事でラットの目に細胞を提供する強膜によるリスクの少ない、再現性と正確に変更された網膜注入手法について述べる。

強膜網膜移植ひと網膜色素上皮細胞のラットの目の洗練されたプロトコルの開発

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

development-refined-protocol-for-trans-scleral-subretinal

Research

JoVE Journal

Neuroscience

9.4K ビュー.  Neural Stem Cell Institute. 網膜の注射は、加齢黄斑変性症に対する幹細胞補充療法の臨床研究で広く適用されています。この可視化の記事でラットの目に細胞を提供する強膜によるリスクの少ない、再現性と正確に変更された網膜注入手法について述べる。

ビデオ: Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

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

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

網膜色素上皮の全マウントするためのマウスの眼の解剖

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

ラット胚神経細胞の分離と文化：クイックプロトコル

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

マウス網膜色素上皮における遺伝子治療用ウイルスベクターの縁アプローチ、網膜下注射

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Enrichment of Bruch's Membrane from Human Donor Eyes

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of the center&#160;of the retina, called the macula, resulting in the loss of central vision. Early AMD is characterised by the presence of small, yellowish lesions called soft drusen that can progress onto late AMD such as geographic atrophy (dry AMD) or neovascularisation (wet AMD). Although the clinical changes are well described, and the understanding of genetic influences on conferring AMD risk are getting ever more detailed, one area lacking major progress is an understanding of the biochemical consequences of genetic risk. This is partly due to difficulties in understanding the biochemistry of Bruch&#8217;s membrane, a very thin extracellular matrix that acts as a biological filter of material from the blood supply and a scaffold on which the retinal pigment epithelial (RPE) cell monolayer resides. Drusen form within Bruch&#8217;s membrane and their presence disrupts nutrient flow to the RPE cells. Only by investigating the protein composition of Bruch&#8217;s membrane, and indeed how other proteins interact with it, can researchers hope to unravel the biochemical mechanisms underpinning drusen formation, development of AMD and subsequent vision loss. This paper details methodologies for enriching either whole Bruch&#8217;s membrane, or just from the macula region, so that it can be used for downstream biochemical analysis, and provide examples of how this is already changing the understanding of Bruch&#8217;s membrane biochemistry.

Identifying proteins specifically associated with Bruch&#8217;s membrane in human eyes is an important step in understanding the biochemical mechanisms behind eye diseases such as age-related macular degeneration. This protocol describes how to enrich this sheet of extracellular matrix for down-stream biochemical analysis.

ヒトドナー目からブルッフ膜の濃縮

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

網膜神経節細胞の定義されたサブセットの単一細胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

早期の生後早期のマウス脳に介在ニューロン前駆体の Homochronic 移植

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

化学遺伝子解析で作られた皮質間ニューロン前駆体を早期産後マウス脳に移植

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神経クラッシュ損傷のラットモデルにおける機能的評価のための3D運動学的解析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

ネコ大型動物モデルにおけるヒト胚性幹細胞由来網膜組織の網膜下移植

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...